Chemistry

Modern Analytical Chemistry
David Harvey
DePauw University

Boston

Burr Ridge, IL Dubuque, IA Madison, WI New York San Francisco St. Louis Bangkok Bogotá Caracas Lisbon London Madrid Mexico City Milan New Delhi Seoul Singapore Sydney Taipei Toronto

McGraw-Hill Higher Education
A Division of The McGraw-Hill Companies
MODERN ANALYTICAL CHEMISTRY Copyright © 2000 by The McGraw-Hill Companies, Inc. All rights reserved. Printed in the United States of America. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a data base or retrieval system, without the prior written permission of the publisher. This book is printed on acid-free paper. 1 2 3 4 5 6 7 8 9 0 KGP/KGP 0 9 8 7 6 5 4 3 2 1 0 ISBN 0–07–237547–7 Vice president and editorial director: Kevin T. Kane Publisher: James M. Smith Sponsoring editor: Kent A. Peterson Editorial assistant: Jennifer L. Bensink Developmental editor: Shirley R. Oberbroeckling Senior marketing manager: Martin J. Lange Senior project manager: Jayne Klein Production supervisor: Laura Fuller Coordinator of freelance design: Michelle D. Whitaker Senior photo research coordinator: Lori Hancock Senior supplement coordinator: Audrey A. Reiter Compositor: Shepherd, Inc. Typeface: 10/12 Minion Printer: Quebecor Printing Book Group/Kingsport Freelance cover/interior designer: Elise Lansdon Cover image: © George Diebold/The Stock Market Photo research: Roberta Spieckerman Associates Colorplates: Colorplates 1–6, 8, 10: © David Harvey/Marilyn E. Culler, photographer; Colorplate 7: Richard Megna/Fundamental Photographs; Colorplate 9: © Alfred Pasieka/Science Photo Library/Photo Researchers, Inc.; Colorplate 11: From H. Black, Environ. Sci. Technol., 1996, 30, 124A. Photos courtesy D. Pesiri and W. Tumas, Los Alamos National Laboratory; Colorplate 12: Courtesy of Hewlett-Packard Company; Colorplate 13: © David Harvey. Library of Congress Cataloging-in-Publication Data Harvey, David, 1956– Modern analytical chemistry / David Harvey. — 1st ed. p. cm. Includes bibliographical references and index. ISBN 0–07–237547–7 1. Chemistry, Analytic. I. Title. QD75.2.H374 2000 543—dc21 99–15120 CIP INTERNATIONAL EDITION ISBN 0–07–116953–9 Copyright © 2000. Exclusive rights by The McGraw-Hill Companies, Inc. for manufacture and export. This book cannot be re-exported from the country to which it is consigned by McGraw-Hill. The International Edition is not available in North America. www.mhhe.com

Contents

Contents
Conservation of Electrons 23 Using Conservation Principles in Stoichiometry Problems 23 2D Basic Equipment and Instrumentation 25 2D.1 Instrumentation for Measuring Mass 25 2D.2 Equipment for Measuring Volume 26 2D.3 Equipment for Drying Samples 29 2E Preparing Solutions 30 2E.1 Preparing Stock Solutions 30 2E.2 Preparing Solutions by Dilution 31 2F The Laboratory Notebook 32 2G Key Terms 32 2H Summary 33 2I Problems 33 2J Suggested Readings 34 2K References 34 2C.5 2C.6

Preface

xii

Chapter 1
Introduction
1A 1B 1C 1D 1E 1F 1G 1H

1

What is Analytical Chemistry? 2 The Analytical Perspective 5 Common Analytical Problems 8 Key Terms 9 Summary 9 Problems 9 Suggested Readings 10 References 10

Chapter 2
Basic Tools of Analytical Chemistry 11
2A Numbers in Analytical Chemistry 12 2A.1 Fundamental Units of Measure 12 2A.2 Significant Figures 13 2B Units for Expressing Concentration 15 2B.1 Molarity and Formality 15 2B.2 Normality 16 2B.3 Molality 18 2B.4 Weight, Volume, and Weight-to-Volume Ratios 18 2B.5 Converting Between Concentration Units 2B.6 p-Functions 19 2C Stoichiometric Calculations 20 2C.1 Conservation of Mass 22 2C.2 Conservation of Charge 22 2C.3 Conservation of Protons 22 2C.4 Conservation of Electron Pairs 23

Chapter 3
The Language of Analytical Chemistry 35
3A Analysis, Determination, and Measurement 3B Techniques, Methods, Procedures, and Protocols 36 3C Classifying Analytical Techniques 37 3D Selecting an Analytical Method 38 3D.1 Accuracy 38 3D.2 Precision 39 3D.3 Sensitivity 39 3D.4 Selectivity 40 3D.5 Robustness and Ruggedness 42 3D.6 Scale of Operation 42 3D.7 Equipment, Time, and Cost 44 3D.8 Making the Final Choice 44 36

18

iii

iv
3E

Modern Analytical Chemistry

Developing the Procedure 45 3E.1 Compensating for Interferences 45 3E.2 Calibration and Standardization 47 3E.3 Sampling 47 3E.4 Validation 47 3F Protocols 48 3G The Importance of Analytical Methodology 3H Key Terms 50 3I Summary 50 3J Problems 51 3K Suggested Readings 52 3L References 52

48

Chapter 4
Evaluating Analytical Data 53
4A Characterizing Measurements and Results 54 4A.1 Measures of Central Tendency 54 4A.2 Measures of Spread 55 4B Characterizing Experimental Errors 57 4B.1 Accuracy 57 4B.2 Precision 62 4B.3 Error and Uncertainty 64 4C Propagation of Uncertainty 64 4C.1 A Few Symbols 65 4C.2 Uncertainty When Adding or Subtracting 65 4C.3 Uncertainty When Multiplying or Dividing 66 4C.4 Uncertainty for Mixed Operations 66 4C.5 Uncertainty for Other Mathematical Functions 67 4C.6 Is Calculating Uncertainty Actually Useful? 68 4D The Distribution of Measurements and Results 70 4D.1 Populations and Samples 71 4D.2 Probability Distributions for Populations 71 4D.3 Confidence Intervals for Populations 75 4D.4 Probability Distributions for Samples 77 4D.5 Confidence Intervals for Samples 80 4D.6 A Cautionary Statement 81 4E Statistical Analysis of Data 82 4E.1 Significance Testing 82 4E.2 Constructing a Significance Test 83 4E.3 One-Tailed and Two-Tailed Significance Tests 84

4E.4 Errors in Significance Testing 84 4F Statistical Methods for Normal Distributions – 4F.1 Comparing X to µ 85 4F.2 Comparing s2 to σ2 87 4F.3 Comparing Two Sample Variances 88 4F.4 Comparing Two Sample Means 88 4F.5 Outliers 93 4G Detection Limits 95 4H Key Terms 96 4I Summary 96 4J Suggested Experiments 97 4K Problems 98 4L Suggested Readings 102 4M References 102

85

Chapter 5
Calibrations, Standardizations, and Blank Corrections 104
5A Calibrating Signals 105 5B Standardizing Methods 106 5B.1 Reagents Used as Standards 106 5B.2 Single-Point versus Multiple-Point Standardizations 108 5B.3 External Standards 109 5B.4 Standard Additions 110 5B.5 Internal Standards 115 5C Linear Regression and Calibration Curves 117 5C.1 Linear Regression of Straight-Line Calibration Curves 118 5C.2 Unweighted Linear Regression with Errors in y 119 5C.3 Weighted Linear Regression with Errors in y 124 5C.4 Weighted Linear Regression with Errors in Both x and y 127 5C.5 Curvilinear and Multivariate Regression 127 5D Blank Corrections 128 5E Key Terms 130 5F Summary 130 5G Suggested Experiments 130 5H Problems 131 5I Suggested Readings 133 5J References 134

Contents

v

Chapter 6
Equilibrium Chemistry 135
Reversible Reactions and Chemical Equilibria 136 6B Thermodynamics and Equilibrium Chemistry 136 6C Manipulating Equilibrium Constants 138 6D Equilibrium Constants for Chemical Reactions 139 6D.1 Precipitation Reactions 139 6D.2 Acid–Base Reactions 140 6D.3 Complexation Reactions 144 6D.4 Oxidation–Reduction Reactions 145 6E Le Châtelier’s Principle 148 6F Ladder Diagrams 150 6F.1 Ladder Diagrams for Acid–Base Equilibria 150 6F.2 Ladder Diagrams for Complexation Equilibria 153 6F.3 Ladder Diagrams for Oxidation–Reduction Equilibria 155 6G Solving Equilibrium Problems 156 6G.1 A Simple Problem: Solubility of Pb(IO3)2 in Water 156 6G.2 A More Complex Problem: The Common Ion Effect 157 6G.3 Systematic Approach to Solving Equilibrium Problems 159 6G.4 pH of a Monoprotic Weak Acid 160 6G.5 pH of a Polyprotic Acid or Base 163 6G.6 Effect of Complexation on Solubility 165 6H Buffer Solutions 167 6H.1 Systematic Solution to Buffer Problems 168 6H.2 Representing Buffer Solutions with Ladder Diagrams 170 6I Activity Effects 171 6J Two Final Thoughts About Equilibrium Chemistry 175 6K Key Terms 175 6L Summary 175 6M Suggested Experiments 176 6N Problems 176 6O Suggested Readings 178 6P References 178 6A

Chapter

7

Obtaining and Preparing Samples for Analysis 179
7A The Importance of Sampling 180 7B Designing a Sampling Plan 182 7B.1 Where to Sample the Target Population 182 7B.2 What Type of Sample to Collect 185 7B.3 How Much Sample to Collect 187 7B.4 How Many Samples to Collect 191 7B.5 Minimizing the Overall Variance 192 7C Implementing the Sampling Plan 193 7C.1 Solutions 193 7C.2 Gases 195 7C.3 Solids 196 7D Separating the Analyte from Interferents 201 7E General Theory of Separation Efficiency 202 7F Classifying Separation Techniques 205 7F.1 Separations Based on Size 205 7F.2 Separations Based on Mass or Density 206 7F.3 Separations Based on Complexation Reactions (Masking) 207 7F.4 Separations Based on a Change of State 209 7F.5 Separations Based on a Partitioning Between Phases 211 7G Liquid–Liquid Extractions 215 7G.1 Partition Coefficients and Distribution Ratios 216 7G.2 Liquid–Liquid Extraction with No Secondary Reactions 216 7G.3 Liquid–Liquid Extractions Involving Acid–Base Equilibria 219 7G.4 Liquid–Liquid Extractions Involving Metal Chelators 221 7H Separation versus Preconcentration 223 7I Key Terms 224 7J Summary 224 7K Suggested Experiments 225 7L Problems 226 7M Suggested Readings 230 7N References 231

vi

Modern Analytical Chemistry

Chapter 8
Gravimetric Methods of Analysis
8A

232

Overview of Gravimetry 233 8A.1 Using Mass as a Signal 233 8A.2 Types of Gravimetric Methods 234 8A.3 Conservation of Mass 234 8A.4 Why Gravimetry Is Important 235 8B Precipitation Gravimetry 235 8B.1 Theory and Practice 235 8B.2 Quantitative Applications 247 8B.3 Qualitative Applications 254 8B.4 Evaluating Precipitation Gravimetry 254 8C Volatilization Gravimetry 255 8C.1 Theory and Practice 255 8C.2 Quantitative Applications 259 8C.3 Evaluating Volatilization Gravimetry 262 8D Particulate Gravimetry 262 8D.1 Theory and Practice 263 8D.2 Quantitative Applications 264 8D.3 Evaluating Precipitation Gravimetry 265 8E Key Terms 265 8F Summary 266 8G Suggested Experiments 266 8H Problems 267 8I Suggested Readings 271 8J References 272

9B.7 Characterization Applications 309 9B.8 Evaluation of Acid–Base Titrimetry 311 9C Titrations Based on Complexation Reactions 314 9C.1 Chemistry and Properties of EDTA 315 9C.2 Complexometric EDTA Titration Curves 317 9C.3 Selecting and Evaluating the End Point 322 9C.4 Representative Method 324 9C.5 Quantitative Applications 327 9C.6 Evaluation of Complexation Titrimetry 331 9D Titrations Based on Redox Reactions 331 9D.1 Redox Titration Curves 332 9D.2 Selecting and Evaluating the End Point 337 9D.3 Representative Method 340 9D.4 Quantitative Applications 341 9D.5 Evaluation of Redox Titrimetry 350 9E Precipitation Titrations 350 9E.1 Titration Curves 350 9E.2 Selecting and Evaluating the End Point 354 9E.3 Quantitative Applications 354 9E.4 Evaluation of Precipitation Titrimetry 357 9F Key Terms 357 9G Summary 357 9H Suggested Experiments 358 9I Problems 360 9J Suggested Readings 366 9K References 367

Chapter 9
Titrimetric Methods of Analysis 273
9A Overview of Titrimetry 274 9A.1 Equivalence Points and End Points 274 9A.2 Volume as a Signal 274 9A.3 Titration Curves 275 9A.4 The Buret 277 9B Titrations Based on Acid–Base Reactions 278 9B.1 Acid–Base Titration Curves 279 9B.2 Selecting and Evaluating the End Point 287 9B.3 Titrations in Nonaqueous Solvents 295 9B.4 Representative Method 296 9B.5 Quantitative Applications 298 9B.6 Qualitative Applications 308

Chapter

10

Spectroscopic Methods of Analysis 368
10A Overview of Spectroscopy 369 10A.1 What Is Electromagnetic Radiation 369 10A.2 Measuring Photons as a Signal 372 10B Basic Components of Spectroscopic Instrumentation 374 10B.1 Sources of Energy 375 10B.2 Wavelength Selection 376 10B.3 Detectors 379 10B.4 Signal Processors 380 10C Spectroscopy Based on Absorption 380 10C.1 Absorbance of Electromagnetic Radiation 380 10C.2 Transmittance and Absorbance 384 10C.3 Absorbance and Concentration: Beer’s Law 385

Contents

vii

Beer’s Law and Multicomponent Samples 386 10C.5 Limitations to Beer’s Law 386 10D Ultraviolet-Visible and Infrared Spectrophotometry 388 10D.1 Instrumentation 388 10D.2 Quantitative Applications 394 10D.3 Qualitative Applications 402 10D.4 Characterization Applications 403 10D.5 Evaluation 409 10E Atomic Absorption Spectroscopy 412 10E.1 Instrumentation 412 10E.2 Quantitative Applications 415 10E.3 Evaluation 422 10F Spectroscopy Based on Emission 423 10G Molecular Photoluminescence Spectroscopy 423 10G.1 Molecular Fluorescence and Phosphorescence Spectra 424 10G.2 Instrumentation 427 10G.3 Quantitative Applications Using Molecular Luminescence 429 10G.4 Evaluation 432 10H Atomic Emission Spectroscopy 434 10H.1 Atomic Emission Spectra 434 10H.2 Equipment 435 10H.3 Quantitative Applications 437 10H.4 Evaluation 440 10I Spectroscopy Based on Scattering 441 10I.1 Origin of Scattering 441 10I.2 Turbidimetry and Nephelometry 441 10J Key Terms 446 10K Summary 446 10L Suggested Experiments 447 10M Problems 450 10N Suggested Readings 458 10O References 459

10C.4

11B Potentiometric Methods of Analysis 465 11B.1 Potentiometric Measurements 466 11B.2 Reference Electrodes 471 11B.3 Metallic Indicator Electrodes 473 11B.4 Membrane Electrodes 475 11B.5 Quantitative Applications 485 11B.6 Evaluation 494 11C Coulometric Methods of Analysis 496 11C.1 Controlled-Potential Coulometry 497 11C.2 Controlled-Current Coulometry 499 11C.3 Quantitative Applications 501 11C.4 Characterization Applications 506 11C.5 Evaluation 507 11D Voltammetric Methods of Analysis 508 11D.1 Voltammetric Measurements 509 11D.2 Current in Voltammetry 510 11D.3 Shape of Voltammograms 513 11D.4 Quantitative and Qualitative Aspects of Voltammetry 514 11D.5 Voltammetric Techniques 515 11D.6 Quantitative Applications 520 11D.7 Characterization Applications 527 11D.8 Evaluation 531 11E Key Terms 532 11F Summary 532 11G Suggested Experiments 533 11H Problems 535 11I Suggested Readings 540 11J References 541

Chapter 12
Chromatographic and Electrophoretic Methods 543
12A Overview of Analytical Separations 544 12A.1 The Problem with Simple Separations 544 12A.2 A Better Way to Separate Mixtures 544 12A.3 Classifying Analytical Separations 546 12B General Theory of Column Chromatography 547 12B.1 Chromatographic Resolution 549 12B.2 Capacity Factor 550 12B.3 Column Selectivity 552 12B.4 Column Efficiency 552

Chapter 11
Electrochemical Methods of Analysis 461
11A Classification of Electrochemical Methods 462 11A.1 Interfacial Electrochemical Methods 462 11A.2 Controlling and Measuring Current and Potential 462

viii

Modern Analytical Chemistry

12B.5 Peak Capacity 554 12B.6 Nonideal Behavior 555 12C Optimizing Chromatographic Separations 556 12C.1 Using the Capacity Factor to Optimize Resolution 556 12C.2 Using Column Selectivity to Optimize Resolution 558 12C.3 Using Column Efficiency to Optimize Resolution 559 12D Gas Chromatography 563 12D.1 Mobile Phase 563 12D.2 Chromatographic Columns 564 12D.3 Stationary Phases 565 12D.4 Sample Introduction 567 12D.5 Temperature Control 568 12D.6 Detectors for Gas Chromatography 569 12D.7 Quantitative Applications 571 12D.8 Qualitative Applications 575 12D.9 Representative Method 576 12D.10 Evaluation 577 12E High-Performance Liquid Chromatography 578 12E.1 HPLC Columns 578 12E.2 Stationary Phases 579 12E.3 Mobile Phases 580 12E.4 HPLC Plumbing 583 12E.5 Sample Introduction 584 12E.6 Detectors for HPLC 584 12E.7 Quantitative Applications 586 12E.8 Representative Method 588 12E.9 Evaluation 589 12F Liquid–Solid Adsorption Chromatography 590 12G Ion-Exchange Chromatography 590 12H Size-Exclusion Chromatography 593 12I Supercritical Fluid Chromatography 596 12J Electrophoresis 597 12J.1 Theory of Capillary Electrophoresis 598 12J.2 Instrumentation 601 12J.3 Capillary Electrophoresis Methods 604 12J.4 Representative Method 607 12J.5 Evaluation 609 12K Key Terms 609 12L Summary 610 12M Suggested Experiments 610 12N Problems 615

12O Suggested Readings 12P References 620

620

Chapter 13
Kinetic Methods of Analysis 622
13A Methods Based on Chemical Kinetics 623 13A.1 Theory and Practice 624 13A.2 Instrumentation 634 13A.3 Quantitative Applications 636 13A.4 Characterization Applications 638 13A.5 Evaluation of Chemical Kinetic Methods 639 13B Radiochemical Methods of Analysis 642 13B.1 Theory and Practice 643 13B.2 Instrumentation 643 13B.3 Quantitative Applications 644 13B.4 Characterization Applications 647 13B.5 Evaluation 648 13C Flow Injection Analysis 649 13C.1 Theory and Practice 649 13C.2 Instrumentation 651 13C.3 Quantitative Applications 655 13C.4 Evaluation 658 13D Key Terms 658 13E Summary 659 13F Suggested Experiments 659 13G Problems 661 13H Suggested Readings 664 13I References 665

Chapter 14
Developing a Standard Method 666
14A Optimizing the Experimental Procedure 667 14A.1 Response Surfaces 667 14A.2 Searching Algorithms for Response Surfaces 668 14A.3 Mathematical Models of Response Surfaces 674 14B Verifying the Method 683 14B.1 Single-Operator Characteristics 683 14B.2 Blind Analysis of Standard Samples 683 14B.3 Ruggedness Testing 684 14B.4 Equivalency Testing 687

Contents

ix

14C

Validating the Method as a Standard Method 687 14C.1 Two-Sample Collaborative Testing 688 14C.2 Collaborative Testing and Analysis of Variance 693 14C.3 What Is a Reasonable Result for a Collaborative Study? 698 14D Key Terms 699 14E Summary 699 14F Suggested Experiments 699 14G Problems 700 14H Suggested Readings 704 14I References 704

15D 15E 15F 15G 15H 15I

Key Terms 721 Summary 722 Suggested Experiments 722 Problems 722 Suggested Readings 724 References 724

Appendixes
Appendix 1A Appendix 1B Appendix 1C Appendix 1D Appendix 1E Appendix 2 Appendix 3A Appendix 3B Appendix 3C Appendix 3D Appendix 3E Appendix 4 Appendix 5 Appendix 6 Appendix 7 Single-Sided Normal Distribution 725 t-Table 726 F-Table 727 Critical Values for Q-Test 728 Random Number Table 728 Recommended Reagents for Preparing Primary Standards 729 Solubility Products 731 Acid Dissociation Constants 732 Metal–Ligand Formation Constants 739 Standard Reduction Potentials 743 Selected Polarographic Half-Wave Potentials 747 Balancing Redox Reactions 748 Review of Chemical Kinetics 750 Countercurrent Separations 755 Answers to Selected Problems 762

Chapter 15
Quality Assurance 705
15A Quality Control 706 15B Quality Assessment 708 15B.1 Internal Methods of Quality Assessment 708 15B.2 External Methods of Quality Assessment 711 15C Evaluating Quality Assurance Data 712 15C.1 Prescriptive Approach 712 15C.2 Performance-Based Approach 714

Glossary Index

769

781

x

Modern Analytical Chemistry

A Guide to Using This Text
. . . in Chapter
Representative Methods
Annotated methods of typical analytical procedures link theory with practice. The format encourages students to think about the design of the procedure and why it works.
246
Modern Analytical Chemistry An additional problem is encountered when the isolated solid is nonstoichiometric. For example, precipitating Mn2+ as Mn(OH)2, followed by heating to produce the oxide, frequently produces a solid with a stoichiometry of MnOx , where x varies between 1 and 2. In this case the nonstoichiometric product results from the formation of a mixture of several oxides that differ in the oxidation state of manganese. Other nonstoichiometric compounds form as a result of lattice defects in the crystal structure.6 Representative Method The best way to appreciate the importance of the theoretical and practical details discussed in the previous section is to carefully examine the procedure for a typical precipitation gravimetric method. Although each method has its own unique considerations, the determination of Mg2+ in water and wastewater by precipitating MgNH4PO4 ⋅ 6H2O and isolating Mg2P2O7 provides an instructive example of a typical procedure.

Representative Methods

Margin Notes
Margin notes direct students to colorplates located toward the middle of the book

Method 8.1

Determination of Mg2+ in Water and Wastewater7

Description of Method. Magnesium is precipitated as MgNH4PO4 ⋅ 6H2O using
(NH4)2HPO4 as the precipitant. The precipitate’s solubility in neutral solutions (0.0065 g/100 mL in pure water at 10 °C) is relatively high, but it is much less soluble in the presence of dilute ammonia (0.0003 g/100 mL in 0.6 M NH3). The precipitant is not very selective, so a preliminary separation of Mg2+ from potential interferents is necessary. Calcium, which is the most significant interferent, is usually removed by its prior precipitation as the oxalate. The presence of excess ammonium salts from the precipitant or the addition of too much ammonia can lead to the formation of Mg(NH4)4(PO4)2, which is subsequently isolated as Mg(PO3)2 after drying. The precipitate is isolated by filtration using a rinse solution of dilute ammonia. After filtering, the precipitate is converted to Mg2P2O7 and weighed.

110

Modern Analytical Chemistry either case, the calibration curve provides a means for relating Ssamp to the analyte’s concentration. EXAMPLE 5.3

Procedure. Transfer a sample containing no more than 60 mg of Mg2+ into a
600-mL beaker. Add 2–3 drops of methyl red indicator, and, if necessary, adjust the volume to 150 mL. Acidify the solution with 6 M HCl, and add 10 mL of 30% w/v (NH4)2HPO4. After cooling, add concentrated NH3 dropwise, and while constantly stirring, until the methyl red indicator turns yellow (pH > 6.3). After stirring for 5 min, add 5 mL of concentrated NH3, and continue stirring for an additional 10 min. Allow the resulting solution and precipitate to stand overnight. Isolate the precipitate by filtration, rinsing with 5% v/v NH3. Dissolve the precipitate in 50 mL of 10% v/v HCl, and precipitate a second time following the same procedure. After filtering, carefully remove the filter paper by charring. Heat the precipitate at 500 °C until the residue is white, and then bring the precipitate to constant weight at 1100 °C.

Color plate 1 shows an example of a set of external standards and their corresponding normal calibration curve.

A second spectrophotometric method for the quantitative determination of Pb2+ levels in blood gives a linear normal calibration curve for which Sstand = (0.296 ppb–1) × CS + 0.003 What is the Pb2+ level (in ppb) in a sample of blood if Ssamp is 0.397? SOLUTION To determine the concentration of Pb2+ in the sample of blood, we replace Sstand in the calibration equation with Ssamp and solve for CA CA = Ssamp – 0.003 0.296 ppb –1 = 0.397 – 0.003 = 1.33 ppb 0.296 ppb –1 q

Questions
1. Why does the procedure call for a sample containing no more than 60 mg of

It is worth noting that the calibration equation in this problem includes an extra term that is not in equation 5.3. Ideally, we expect the calibration curve to give a signal of zero when CS is zero. This is the purpose of using a reagent blank to correct the measured signal. The extra term of +0.003 in our calibration equation results from uncertainty in measuring the signal for the reagent blank and the standards.

An external standardization allows a related series of samples to be analyzed using a single calibration curve. This is an important advantage in laboratories where many samples are to be analyzed or when the need for a rapid throughput of l i iti l t ii l f th t l t d

Examples of Typical Problems
Each example problem includes a detailed solution that helps students in applying the chapter’s material to practical problems. matrix matching Adjusting the matrix of an external standard so that it is the same as the matrix of the samples to be analyzed. method of standard additions A standardization in which aliquots of a standard solution are added to the sample.

y There is a serious limitation, however, to an external standardization. The relationship between Sstand and CS in equation 5.3 is determined when the analyte is present in the external standard’s matrix. In using an external standardization, we assume that any difference between the matrix of the standards and the sample’s matrix has no effect on the value of k. A proportional determinate error is introduced when differences between the two matrices cannot be ignored. This is shown in Figure 5.4, where the relationship between the signal and the amount of analyte is shown for both the sample’s matrix and the standard’s matrix. In this example, using a normal calibration curve results in a negative determinate error. When matrix problems are expected, an effort is made to match the matrix of the standards to that of the sample. This is known as matrix matching. When the sample’s matrix is unknown, the matrix effect must be shown to be negligible, or an alternative method of standardization must be used. Both approaches are discussed in the following sections.

5B.4 Standard Additions
The complication of matching the matrix of the standards to that of the sample can be avoided by conducting the standardization in the sample. This is known as the method of standard additions. The simplest version of a standard addition is shown in Figure 5.5. A volume, Vo, of sample is diluted to a final volume, Vf, and the signal, Ssamp is measured. A second identical aliquot of sample is

Bold-faced Key Terms with Margin Definitions
Key words appear in boldface when they are introduced within the text. The term and its definition appear in the margin for quick review by the student. All key words are also defined in the glossary.

x

. . . End of Chapter y y

5E KEY TERMS aliquot (p. 111) external standard (p. 109) internal standard (p. 116) linear regression (p. 118) matrix matching (p. 110) method of standard additions multiple-point standardization (p. 109) normal calibration curve (p. 109) primary reagent (p. 106) reagent grade (p. 107) residual error (p. 118) (p. 110) secondary reagent (p. 107) single-point standardization (p. 108) standard deviation about the regression (p. 121) total Youden blank (p. 129)

List of Key Terms
The key terms introduced within the chapter are listed at the end of each chapter. Page references direct the student to the definitions in the text.

5F SUMMARY
In a quantitative analysis, we measure a signal and calculate the amount of analyte using one of the following equations. Smeas = knA + Sreag Smeas = kCA + Sreag To obtain accurate results we must eliminate determinate errors affecting the measured signal, Smeas, the method’s sensitivity, k, and any signal due to the reagents, Sreag. To ensure that Smeas is determined accurately, we calibrate the equipment or instrument used to obtain the signal. Balances are calibrated using standard weights. When necessary, we can also correct for the buoyancy of air. Volumetric glassware can be calibrated by measuring the mass of water contained or delivered and using the density of water to calculate the true volume. Most instruments have calibration standards suggested by the manufacturer. An analytical method is standardized by determining its sensitivity. There are several approaches to standardization, including the use of external standards, the method of standard addition, and the use of an internal standard. The most desirable standardization strategy is an external standardization. The method of standard additions, in which known amounts of analyte are added to the sample, is used when the sample’s matrix complicates the analysis. An internal standard, which is a species (not analyte) added to all samples and standards, is used when the procedure does not allow for the reproducible handling of samples and standards. Standardizations using a single standard are common, but also are subject to greater uncertainty. Whenever possible, a multiplepoint standardization is preferred. The results of a multiple-point standardization are graphed as a calibration curve. A linear regression analysis can provide an equation for the standardization. A reagent blank corrects the measured signal for signals due to reagents other than the sample that are used in an analysis. The most common reagent blank is prepared by omitting the sample. When a simple reagent blank does not compensate for all constant sources of determinate error, other types of blanks, such as the total Youden blank, can be used.

Summary
The summary provides the student with a brief review of the important concepts within the chapter.

Suggested Experiments
An annotated list of representative experiments is provided from the Journal of Chemical Education.

5G Suggested EXPERIMENTS

Suggested Readings
Standardization—External standards, standard additions, and internal standards are a common feature of many quantitative analyses. Suggested experiments using these standardization methods are found in later chapters. A good project experiment for introducing external standardization, standard additions, and the importance of the sample’s y y matrix is to explore the effect of pH on the quantitative analysis of an acid–base indicator. Using bromothymol blue as an example, external standards can be prepared in a pH 9 1G SUGGESTED READINGS buffer and used to analyze samples buffered to different pHs in the range of 6–10. Results can be compared with those The role of analytical chemistry within the broader discipline of obtained using a standard addition. chemistry has been discussed by many prominent analytical chemists. Several notable examples follow. Baiulescu, G. E.; Patroescu, C.; Chalmers, R. A. Education and Teaching in Analytical Chemistry. Ellis Horwood: Chichester, 1982. Hieftje, G. M. “The Two Sides of Analytical Chemistry,” Anal. Chem. 1985, 57, 256A–267A. Kissinger, P. T. “Analytical Chemistry—What is It? Who Needs It? Why Teach It?” Trends Anal. Chem. 1992, 11, 54–57.

Experiments

The following exercises and experiments help connect the material in this chapter to the analytical laboratory.

Calibration—Volumetric glassware (burets, pipets, and volumetric flasks) can be calibrated in the manner described in Example 5.1. Most instruments have a calibration sample that can be prepared to verify the instrument’s accuracy and precision. For example, as described in this chapter, a solution of 60.06 ppm K2Cr2O7 in 0.0050 M H2SO4 should give an absorbance of 0.640 ± 0.010 at a wavelength of 350.0 nm when using 0.0050 M H2SO4 as a reagent blank. These exercises also provide practice with using volumetric glassware, weighing samples, and preparing solutions.

Suggested readings give the student access to more comprehensive discussion of the topics introduced within the chapter.

References
The references cited in the chapter are provided so the student can access them for further information.

Laitinen, H. A. “Analytical Chemistry in a Changing World,” Anal. Chem. 1980, 52, 605A–609A. Laitinen, H. A. “History of Analytical Chemistry in the U.S.A.,” Talanta 1989, 36, 1–9. Laitinen, H. A.; Ewing, G. (eds). A History of Analytical Chemistry. The Division of Analytical Chemistry of the American Chemical Society: Washington, D.C., 1972. McLafferty, F. W. “Analytical Chemistry: Historic and Modern,” Acc. Chem. Res. 1990, 23, 63–64.

1H REFERENCES
1. Ravey, M. Spectroscopy 1990, 5(7), 11. 2. de Haseth, J. Spectroscopy 1990, 5(7), 11. 3. Fresenius, C. R. A System of Instruction in Quantitative Chemical Analysis. John Wiley and Sons: New York, 1881. 4. Hillebrand, W. F.; Lundell, G. E. F. Applied Inorganic Analysis, John Wiley and Sons: New York, 1953. 5. Van Loon, J. C. Analytical Atomic Absorption Spectroscopy. Academic Press: New York, 1980. 6. Murray, R. W. Anal. Chem. 1991, 63, 271A. 7. For several different viewpoints see (a) Beilby, A. L. J. Chem. Educ. 1970, 47, 237–238; (b) Lucchesi, C. A. Am. Lab. 1980, October, 113–119; (c) Atkinson, G. F. J. Chem. Educ. 1982, 59, 201–202; (d) Pardue, H. L.; Woo, J. J. Chem. Educ. 1984, 61, 409–412; (e) Guarnieri, M. J. Chem. Educ. 1988, 65, 201–203; (f) de Haseth, J. Spectroscopy 1990, 5, 20–21; (g) Strobel, H. A. Am. Lab. 1990, October, 17–24. 8. Hieftje, G. M. Am. Lab. 1993, October, 53–61. 9. See, for example, the following laboratory texts: (a) Sorum, C. H.; Lagowski, J. J. Introduction to Semimicro Qualitative Analysis, 5th ed. Prentice-Hall: Englewood Cliffs, NJ, 1977.; (b) Shriner, R. L.; Fuson, R. C.; Curtin, D. Y. The Systematic Identification of Organic Compounds, 5th ed. John Wiley and Sons: New York, 1964.

3J PROBLEMS
1. When working with a solid sample, it often is necessary to bring the analyte into solution by dissolving the sample in a suitable solvent. Any solid impurities that remain are removed by filtration before continuing with the analysis. In a typical total analysis method, the procedure might read After dissolving the sample in a beaker, remove any solid impurities by passing the solution containing the analyte through filter paper, collecting the solution in a clean Erlenmeyer flask. Rinse the beaker with several small portions of solvent, passing these rinsings through the filter paper, and collecting them in the same Erlenmeyer flask. Finally, rinse the filter paper with several portions of solvent, collecting the rinsings in the same Erlenmeyer flask. For a typical concentration method, however, the procedure might state

4. A sample was analyzed to determine the concentration of an analyte. Under the conditions of the analysis, the sensitivity is 17.2 ppm–1. What is the analyte’s concentration if Smeas is 35.2 and Sreag is 0.6? 5. A method for the analysis of Ca2+ in water suffers from an interference in the presence of Zn2+. When the concentration of Ca2+ is 50 times greater than that of Zn2+, an analysis for Ca2+ gives a relative error of –2.0%. What is the value of the selectivity coefficient for this method? 6. The quantitative analysis for reduced glutathione in blood is complicated by the presence of many potential interferents. In one study, when analyzing a solution of 10-ppb glutathione and 1.5-ppb ascorbic acid, the signal was 5.43 times greater than that obtained for the analysis of 10-ppb glutathione.12 What is the selectivity coefficient for this analysis? The same study found that when analyzing a solution of 350-ppb methionine and 10-ppb glutathione the signal was 0 906 times less than that obtained for the analysis

Problems
A variety of problems, many based on data from the analytical literature, provide the student with practical examples of current research.

xi

Preface
A

Preface

s currently taught, the introductory course in analytical chemistry emphasizes quantitative (and sometimes qualitative) methods of analysis coupled with a heavy dose of equilibrium chemistry. Analytical chemistry, however, is more than equilibrium chemistry and a collection of analytical methods; it is an approach to solving chemical problems. Although discussing different methods is important, that discussion should not come at the expense of other equally important topics. The introductory analytical course is the ideal place in the chemistry curriculum to explore topics such as experimental design, sampling, calibration strategies, standardization, optimization, statistics, and the validation of experimental results. These topics are important in developing good experimental protocols, and in interpreting experimental results. If chemistry is truly an experimental science, then it is essential that all chemistry students understand how these topics relate to the experiments they conduct in other chemistry courses. Currently available textbooks do a good job of covering the diverse range of wet and instrumental analysis techniques available to chemists. Although there is some disagreement about the proper balance between wet analytical techniques, such as gravimetry and titrimetry, and instrumental analysis techniques, such as spectrophotometry, all currently available textbooks cover a reasonable variety of techniques. These textbooks, however, neglect, or give only brief consideration to, obtaining representative samples, handling interferents, optimizing methods, analyzing data, validating data, and ensuring that data are collected under a state of statistical control. In preparing this textbook, I have tried to find a more appropriate balance between theory and practice, between “classical” and “modern” methods of analysis, between analyzing samples and collecting and preparing samples for analysis, and between analytical methods and data analysis. Clearly, the amount of material in this textbook exceeds what can be covered in a single semester; it’s my hope, however, that the diversity of topics will meet the needs of different instructors, while, perhaps, suggesting some new topics to cover. The anticipated audience for this textbook includes students majoring in chemistry, and students majoring in other science disciplines (biology, biochemistry, environmental science, engineering, and geology, to name a few), interested in obtaining a stronger background in chemical analysis. It is particularly appropriate for chemistry majors who are not planning to attend graduate school, and who often do not enroll in those advanced courses in analytical chemistry that require physical chemistry as a pre-requisite. Prior coursework of a year of general chemistry is assumed. Competence in algebra is essential; calculus is used on occasion, however, its presence is not essential to the material’s treatment.

xii

Preface

xiii

Key Features of This Textbook
Key features set this textbook apart from others currently available. • A stronger emphasis on the evaluation of data. Methods for characterizing chemical measurements, results, and errors (including the propagation of errors) are included. Both the binomial distribution and normal distribution are presented, and the idea of a confidence interval is developed. Statistical methods for evaluating data include the t-test (both for paired and unpaired data), the F-test, and the treatment of outliers. Detection limits also are discussed from a statistical perspective. Other statistical methods, such as ANOVA and ruggedness testing, are presented in later chapters. • Standardizations and calibrations are treated in a single chapter. Selecting the most appropriate calibration method is important and, for this reason, the methods of external standards, standard additions, and internal standards are gathered together in a single chapter. A discussion of curve-fitting, including the statistical basis for linear regression (with and without weighting) also is included in this chapter. • More attention to selecting and obtaining a representative sample. The design of a statistically based sampling plan and its implementation are discussed earlier, and in more detail than in other textbooks. Topics that are covered include how to obtain a representative sample, how much sample to collect, how many samples to collect, how to minimize the overall variance for an analytical method, tools for collecting samples, and sample preservation. • The importance of minimizing interferents is emphasized. Commonly used methods for separating interferents from analytes, such as distillation, masking, and solvent extraction, are gathered together in a single chapter. • Balanced coverage of analytical techniques. The six areas of analytical techniques—gravimetry, titrimetry, spectroscopy, electrochemistry, chromatography, and kinetics—receive roughly equivalent coverage, meeting the needs of instructors wishing to emphasize wet methods and those emphasizing instrumental methods. Related methods are gathered together in a single chapter encouraging students to see the similarities between methods, rather than focusing on their differences. • An emphasis on practical applications. Throughout the text applications from organic chemistry, inorganic chemistry, environmental chemistry, clinical chemistry, and biochemistry are used in worked examples, representative methods, and end-of-chapter problems. • Representative methods link theory with practice. An important feature of this text is the presentation of representative methods. These boxed features present typical analytical procedures in a format that encourages students to think about why the procedure is designed as it is. • Separate chapters on developing a standard method and quality assurance. Two chapters provide coverage of methods used in developing a standard method of analysis, and quality assurance. The chapter on developing a standard method includes topics such as optimizing experimental conditions using response surfaces, verifying the method through the blind analysis of standard samples and ruggedness testing, and collaborative testing using Youden’s two-sample approach and ANOVA. The chapter on quality assurance covers quality control and internal and external techniques for quality assessment, including the use of duplicate samples, blanks, spike recoveries, and control charts.

xiv

Preface

• Problems adapted from the literature. Many of the in-chapter examples and endof-chapter problems are based on data from the analytical literature, providing students with practical examples of current research in analytical chemistry. • An emphasis on critical thinking. Critical thinking is encouraged through problems in which students are asked to explain why certain steps in an analytical procedure are included, or to determine the effect of an experimental error on the results of an analysis. • Suggested experiments from the Journal of Chemical Education. Rather than including a short collection of experiments emphasizing the analysis of standard unknowns, an annotated list of representative experiments from the Journal of Chemical Education is included at the conclusion of most chapters. These experiments may serve as stand alone experiments, or as starting points for individual or group projects.

The Role of Equilibrium Chemistry in Analytical Chemistry
Equilibrium chemistry often receives a significant emphasis in the introductory analytical chemistry course. While an important topic, its overemphasis can cause students to confuse analytical chemistry with equilibrium chemistry. Although attention to solving equilibrium problems is important, it is equally important for students to recognize when such calculations are impractical, or when a simpler, more qualitative approach is all that is needed. For example, in discussing the gravimetric analysis of Ag+ as AgCl, there is little point in calculating the equilibrium solubility of AgCl since the concentration of Cl– at equilibrium is rarely known. It is important, however, to qualitatively understand that a large excess of Cl– increases the solubility of AgCl due to the formation of soluble silver-chloro complexes. Balancing the presentation of a rigorous approach to solving equilibrium problems, this text also introduces the use of ladder diagrams as a means for providing a qualitative picture of a system at equilibrium. Students are encouraged to use the approach best suited to the problem at hand.

Computer Software
Many of the topics covered in analytical chemistry benefit from the availability of appropriate computer software. In preparing this text, however, I made a conscious decision to avoid a presentation tied to a single computer platform or software package. Students and faculty are increasingly experienced in the use of computers, spreadsheets, and data analysis software; their use is, I think, best left to the personal choice of each student and instructor.

Organization
The textbook’s organization can be divided into four parts. Chapters 1–3 serve as an introduction, providing an overview of analytical chemistry (Chapter 1); a review of the basic tools of analytical chemistry, including significant figures, units, and stoichiometry (Chapter 2); and an introduction to the terminology used by analytical chemists (Chapter 3). Familiarity with the material in these chapters is assumed throughout the remainder of the text. Chapters 4–7 cover a number of topics that are important in understanding how a particular analytical method works. Later chapters are mostly independent of the material in these chapters. Instructors may pick and choose from among the topics

Preface

xv

of these chapters, as needed, to support individual course goals. The statistical analysis of data is covered in Chapter 4 at a level that is more complete than that found in other introductory analytical textbooks. Methods for calibrating equipment, standardizing methods, and linear regression are gathered together in Chapter 5. Chapter 6 provides an introduction to equilibrium chemistry, stressing both the rigorous solution to equilibrium problems, and the use of semi-quantitative approaches, such as ladder diagrams. The importance of collecting the right sample, and methods for separating analytes and interferents are covered in Chapter 7. Chapters 8–13 cover the major areas of analysis, including gravimetry (Chapter 8), titrimetry (Chapter 9), spectroscopy (Chapter 10), electrochemistry (Chapter 11), chromatography and electrophoresis (Chapter 12), and kinetic methods (Chapter 13). Related techniques, such as acid–base titrimetry and redox titrimetry, or potentiometry and voltammetry, are gathered together in single chapters. Combining related techniques together encourages students to see the similarities between methods, rather than focusing on their differences. The first technique presented in each chapter is generally that which is most commonly covered in the introductory course. Finally, the textbook concludes with two chapters discussing the design and maintenance of analytical methods, two topics of importance to analytical chemists. Chapter 14 considers the development of an analytical method, including its optimization, verification, and validation. Quality control and quality assessment are discussed in Chapter 15.

Acknowledgments
Before beginning an academic career I was, of course, a student. My interest in chemistry and teaching was nurtured by many fine teachers at Westtown Friends School, Knox College, and the University of North Carolina at Chapel Hill; their collective influence continues to bear fruit. In particular, I wish to recognize David MacInnes, Alan Hiebert, Robert Kooser, and Richard Linton. I have been fortunate to work with many fine colleagues during my nearly 17 years of teaching undergraduate chemistry at Stockton State College and DePauw University. I am particularly grateful for the friendship and guidance provided by Jon Griffiths and Ed Paul during my four years at Stockton State College. At DePauw University, Jim George and Bryan Hanson have willingly shared their ideas about teaching, while patiently listening to mine. Approximately 300 students have joined me in thinking and learning about analytical chemistry; their questions and comments helped guide the development of this textbook. I realize that working without a formal textbook has been frustrating and awkward; all the more reason why I appreciate their effort and hard work. The following individuals reviewed portions of this textbook at various stages during its development. David Ballantine Northern Illinois University John E. Bauer Illinois State University Ali Bazzi University of Michigan–Dearborn Steven D. Brown University of Delaware Wendy Clevenger University of Tennessee–Chattanooga Cathy Cobb Augusta State University Paul Flowers University of North Carolina–Pembroke Nancy Gordon University of Southern Maine

xvi

Preface

Virginia M. Indivero Swarthmore College Michael Janusa Nicholls State University J. David Jenkins Georgia Southern University Richard S. Mitchell Arkansas State University George A. Pearse, Jr. Le Moyne College Gary Rayson New Mexico State University David Redfield NW Nazarene University

Vincent Remcho West Virginia University Jeanette K. Rice Georgia Southern University Martin W. Rowe Texas A&M University Alexander Scheeline University of Illinois James D. Stuart University of Connecticut Thomas J. Wenzel Bates College David Zax Cornell University

I am particularly grateful for their detailed written comments and suggestions for improving the manuscript. Much of what is good in the final manuscript is the result of their interest and ideas. George Foy (York College of Pennsylvania), John McBride (Hofstra University), and David Karpovich (Saginaw Valley State University) checked the accuracy of problems in the textbook. Gary Kinsel (University of Texas at Arlington) reviewed the page proofs and provided additional suggestions. This project began in the summer of 1992 with the support of a course development grant from DePauw University’s Faculty Development Fund. Additional financial support from DePauw University’s Presidential Discretionary Fund also is acknowledged. Portions of the first draft were written during a sabbatical leave in the Fall semester of the 1993/94 academic year. A Fisher Fellowship provided release time during the Fall 1995 semester to complete the manuscript’s second draft. Alltech and Associates (Deerfield, IL) graciously provided permission to use the chromatograms in Chapter 12; the assistance of Jim Anderson, Vice-President, and Julia Poncher, Publications Director, is greatly appreciated. Fred Soster and Marilyn Culler, both of DePauw University, provided assistance with some of the photographs. The editorial staff at McGraw-Hill has helped guide a novice through the process of developing this text. I am particularly thankful for the encouragement and confidence shown by Jim Smith, Publisher for Chemistry, and Kent Peterson, Sponsoring Editor for Chemistry. Shirley Oberbroeckling, Developmental Editor for Chemistry, and Jayne Klein, Senior Project Manager, patiently answered my questions and successfully guided me through the publishing process. Finally, I would be remiss if I did not recognize the importance of my family’s support and encouragement, particularly that of my parents. A very special thanks to my daughter, Devon, for gifts too numerous to detail.

How to Contact the Author
Writing this textbook has been an interesting (and exhausting) challenge. Despite my efforts, I am sure there are a few glitches, better examples, more interesting endof-chapter problems, and better ways to think about some of the topics. I welcome your comments, suggestions, and data for interesting problems, which may be addressed to me at DePauw University, 602 S. College St., Greencastle, IN 46135, or electronically at harvey@depauw.edu.

Chapter 1
Introduction

C

hemistry is the study of matter, including its composition, structure, physical properties, and reactivity. There are many approaches to studying chemistry, but, for convenience, we traditionally divide it into five fields: organic, inorganic, physical, biochemical, and analytical. Although this division is historical and arbitrary, as witnessed by the current interest in interdisciplinary areas such as bioanalytical and organometallic chemistry, these five fields remain the simplest division spanning the discipline of chemistry. Training in each of these fields provides a unique perspective to the study of chemistry. Undergraduate chemistry courses and textbooks are more than a collection of facts; they are a kind of apprenticeship. In keeping with this spirit, this text introduces the field of analytical chemistry and the unique perspectives that analytical chemists bring to the study of chemistry.

1

2

Modern Analytical Chemistry

1A What Is Analytical Chemistry?
“Analytical chemistry is what analytical chemists do.”* We begin this section with a deceptively simple question. What is analytical chemistry? Like all fields of chemistry, analytical chemistry is too broad and active a discipline for us to easily or completely define in an introductory textbook. Instead, we will try to say a little about what analytical chemistry is, as well as a little about what analytical chemistry is not. Analytical chemistry is often described as the area of chemistry responsible for characterizing the composition of matter, both qualitatively (what is present) and quantitatively (how much is present). This description is misleading. After all, almost all chemists routinely make qualitative or quantitative measurements. The argument has been made that analytical chemistry is not a separate branch of chemistry, but simply the application of chemical knowledge.1 In fact, you probably have performed quantitative and qualitative analyses in other chemistry courses. For example, many introductory courses in chemistry include qualitative schemes for identifying inorganic ions and quantitative analyses involving titrations. Unfortunately, this description ignores the unique perspective that analytical chemists bring to the study of chemistry. The craft of analytical chemistry is not in performing a routine analysis on a routine sample (which is more appropriately called chemical analysis), but in improving established methods, extending existing methods to new types of samples, and developing new methods for measuring chemical phenomena.2 Here’s one example of this distinction between analytical chemistry and chemical analysis. Mining engineers evaluate the economic feasibility of extracting an ore by comparing the cost of removing the ore with the value of its contents. To estimate its value they analyze a sample of the ore. The challenge of developing and validating the method providing this information is the analytical chemist’s responsibility. Once developed, the routine, daily application of the method becomes the job of the chemical analyst. Another distinction between analytical chemistry and chemical analysis is that analytical chemists work to improve established methods. For example, several factors complicate the quantitative analysis of Ni2+ in ores, including the presence of a complex heterogeneous mixture of silicates and oxides, the low concentration of Ni2+ in ores, and the presence of other metals that may interfere in the analysis. Figure 1.1 is a schematic outline of one standard method in use during the late nineteenth century.3 After dissolving a sample of the ore in a mixture of H2SO4 and HNO3, trace metals that interfere with the analysis, such as Pb2+, Cu2+ and Fe3+, are removed by precipitation. Any cobalt and nickel in the sample are reduced to Co and Ni, isolated by filtration and weighed (point A). After dissolving the mixed solid, Co is isolated and weighed (point B). The amount of nickel in the ore sample is determined from the difference in the masses at points A and B. %Ni = mass point A – mass point B × 100 mass sample

*Attributed to C. N. Reilley (1925–1981) on receipt of the 1965 Fisher Award in Analytical Chemistry. Reilley, who was a professor of chemistry at the University of North Carolina at Chapel Hill, was one of the most influential analytical chemists of the last half of the twentieth century.

Chapter 1 Introduction
Original Sample
1:3 H2SO4/HNO3 100°C (8–10 h) dilute w/H2O, digest 2–4 h

3

PbSO4 Sand

Cu2+, Fe3+ Co2+, Ni2+ dilute bubble H2S(g)

Fe3+, Co2+, Ni2+ cool, add NH3 digest 50°–70°, 30 min

CuS

Fe(OH)3 HCl Fe3+ neutralize w/ NH3 Na2CO3, CH3COOH

Co2+, Ni2+ slightly acidify w/ HCl heat, bubble H2S (g)

Waste

CoS, NiS aqua regia heat, add HCl until strongly acidic bubble H2S (g)

Basic ferric acetate Co2+, Ni2+ heat add Na2CO3 until alkaline NaOH

CuS, PbS

Co(OH)2, Ni(OH)2 heat Waste

CoO, NiO Solid heat, H2 (g)

Key A Solution

Co, Ni
HNO3 K2CO3, KNO3 CH3COOH digest 24 h

Ni2+

K3Co(NO3)5
H2O, HCl

Co2+ as above

Waste

Co

B

Figure 1.1
Analytical scheme outlined by Fresenius3 for the gravimetric analysis of Ni in ores.

4

Modern Analytical Chemistry
Original sample
HNO3, HCl, heat

Residue

Solution
20% NH4Cl 10% tartaric acid take alkaline with 1:1 NH3

take acid with HCl 10% tartaric acid take alkaline with 1:1 NH3

Yes

Is solid present?

Solid Key Solution No take acid with HCl 1% alcoholic DMG take alkaline with 1:1 NH3

Figure 1.2
Analytical scheme outlined by Hillebrand and Lundell4 for the gravimetric analysis of Ni in ores (DMG = dimethylgloxime). The factor of 0.2031 in the equation for %Ni accounts for the difference in the formula weights of Ni(DMG)2 and Ni; see Chapter 8 for more details.

A

Ni(DMG)2(s)

%Ni =

mass A × 0.2031 × 100 g sample

The combination of determining the mass of Ni2+ by difference, coupled with the need for many reactions and filtrations makes this procedure both time-consuming and difficult to perform accurately. The development, in 1905, of dimethylgloxime (DMG), a reagent that selectively precipitates Ni2+ and Pd2+, led to an improved analytical method for determining Ni2+ in ores.4 As shown in Figure 1.2, the mass of Ni2+ is measured directly, requiring fewer manipulations and less time. By the 1970s, the standard method for the analysis of Ni 2+ in ores progressed from precipitating Ni(DMG) 2 to flame atomic absorption spectrophotometry,5 resulting in an even more rapid analysis. Current interest is directed toward using inductively coupled plasmas for determining trace metals in ores. In summary, a more appropriate description of analytical chemistry is “. . . the science of inventing and applying the concepts, principles, and . . . strategies for measuring the characteristics of chemical systems and species.”6 Analytical chemists typically operate at the extreme edges of analysis, extending and improving the ability of all chemists to make meaningful measurements on smaller samples, on more complex samples, on shorter time scales, and on species present at lower concentrations. Throughout its history, analytical chemistry has provided many of the tools and methods necessary for research in the other four traditional areas of chemistry, as well as fostering multidisciplinary research in, to name a few, medicinal chemistry, clinical chemistry, toxicology, forensic chemistry, material science, geochemistry, and environmental chemistry.

Chapter 1 Introduction You will come across numerous examples of qualitative and quantitative methods in this text, most of which are routine examples of chemical analysis. It is important to remember, however, that nonroutine problems prompted analytical chemists to develop these methods. Whenever possible, we will try to place these methods in their appropriate historical context. In addition, examples of current research problems in analytical chemistry are scattered throughout the text. The next time you are in the library, look through a recent issue of an analytically oriented journal, such as Analytical Chemistry. Focus on the titles and abstracts of the research articles. Although you will not recognize all the terms and methods, you will begin to answer for yourself the question “What is analytical chemistry”?

5

1B The Analytical Perspective
Having noted that each field of chemistry brings a unique perspective to the study of chemistry, we now ask a second deceptively simple question. What is the “analytical perspective”? Many analytical chemists describe this perspective as an analytical approach to solving problems.7 Although there are probably as many descriptions of the analytical approach as there are analytical chemists, it is convenient for our purposes to treat it as a five-step process: 1. 2. 3. 4. 5. Identify and define the problem. Design the experimental procedure. Conduct an experiment, and gather data. Analyze the experimental data. Propose a solution to the problem.

Figure 1.3 shows an outline of the analytical approach along with some important considerations at each step. Three general features of this approach deserve attention. First, steps 1 and 5 provide opportunities for analytical chemists to collaborate with individuals outside the realm of analytical chemistry. In fact, many problems on which analytical chemists work originate in other fields. Second, the analytical approach is not linear, but incorporates a “feedback loop” consisting of steps 2, 3, and 4, in which the outcome of one step may cause a reevaluation of the other two steps. Finally, the solution to one problem often suggests a new problem. Analytical chemistry begins with a problem, examples of which include evaluating the amount of dust and soil ingested by children as an indicator of environmental exposure to particulate based pollutants, resolving contradictory evidence regarding the toxicity of perfluoro polymers during combustion, or developing rapid and sensitive detectors for chemical warfare agents.* At this point the analytical approach involves a collaboration between the analytical chemist and the individuals responsible for the problem. Together they decide what information is needed. It is also necessary for the analytical chemist to understand how the problem relates to broader research goals. The type of information needed and the problem’s context are essential to designing an appropriate experimental procedure. Designing an experimental procedure involves selecting an appropriate method of analysis based on established criteria, such as accuracy, precision, sensitivity, and detection limit; the urgency with which results are needed; the cost of a single analysis; the number of samples to be analyzed; and the amount of sample available for
*These examples are taken from a series of articles, entitled the “Analytical Approach,” which has appeared as a regular feature in the journal Analytical Chemistry since 1974.

6

Modern Analytical Chemistry
1. Identify the problem Determine type of information needed (qualitative, quantitative, characterization, or fundamental) Identify context of the problem 5. Propose a solution Conduct external evaluation

2. Design the experimental procedure Establish design criteria (accuracy, precision, scale of operation, sensitivity, selectivity, cost, speed) Identify interferents Select method Establish validation criteria Establish sampling strategy Feedback loop 4. Analyze the experimental data Reduce or transform data Analyze statistics Verify results Interpret results

3. Conduct an experiment Calibrate instruments and equipment Standardize reagents

Figure 1.3
Flow diagram for the analytical approach to solving problems; modified after Atkinson.7c

Gather data

analysis. Finding an appropriate balance between these parameters is frequently complicated by their interdependence. For example, improving the precision of an analysis may require a larger sample. Consideration is also given to collecting, storing, and preparing samples, and to whether chemical or physical interferences will affect the analysis. Finally, a good experimental procedure may still yield useless information if there is no method for validating the results. The most visible part of the analytical approach occurs in the laboratory. As part of the validation process, appropriate chemical or physical standards are used to calibrate any equipment being used and any solutions whose concentrations must be known. The selected samples are then analyzed and the raw data recorded. The raw data collected during the experiment are then analyzed. Frequently the data must be reduced or transformed to a more readily analyzable form. A statistical treatment of the data is used to evaluate the accuracy and precision of the analysis and to validate the procedure. These results are compared with the criteria established during the design of the experiment, and then the design is reconsidered, additional experimental trials are run, or a solution to the problem is proposed. When a solution is proposed, the results are subject to an external evaluation that may result in a new problem and the beginning of a new analytical cycle.

Chapter 1 Introduction As an exercise, let’s adapt this model of the analytical approach to a real problem. For our example, we will use the determination of the sources of airborne pollutant particles. A description of the problem can be found in the following article: “Tracing Aerosol Pollutants with Rare Earth Isotopes” by Ondov, J. M.; Kelly, W. R. Anal. Chem. 1991, 63, 691A–697A. Before continuing, take some time to read the article, locating the discussions pertaining to each of the five steps outlined in Figure 1.3. In addition, consider the following questions: 1. 2. 3. 4. 5. 6. 7. 8. 9. What is the analytical problem? What type of information is needed to solve the problem? How will the solution to this problem be used? What criteria were considered in designing the experimental procedure? Were there any potential interferences that had to be eliminated? If so, how were they treated? Is there a plan for validating the experimental method? How were the samples collected? Is there evidence that steps 2, 3, and 4 of the analytical approach are repeated more than once? Was there a successful conclusion to the problem?

7

According to our model, the analytical approach begins with a problem. The motivation for this research was to develop a method for monitoring the transport of solid aerosol particulates following their release from a high-temperature combustion source. Because these particulates contain significant concentrations of toxic heavy metals and carcinogenic organic compounds, they represent a significant environmental hazard. An aerosol is a suspension of either a solid or a liquid in a gas. Fog, for example, is a suspension of small liquid water droplets in air, and smoke is a suspension of small solid particulates in combustion gases. In both cases the liquid or solid particulates must be small enough to remain suspended in the gas for an extended time. Solid aerosol particulates, which are the focus of this problem, usually have micrometer or submicrometer diameters. Over time, solid particulates settle out from the gas, falling to the Earth’s surface as dry deposition. Existing methods for monitoring the transport of gases were inadequate for studying aerosols. To solve the problem, qualitative and quantitative information were needed to determine the sources of pollutants and their net contribution to the total dry deposition at a given location. Eventually the methods developed in this study could be used to evaluate models that estimate the contributions of point sources of pollution to the level of pollution at designated locations. Following the movement of airborne pollutants requires a natural or artificial tracer (a species specific to the source of the airborne pollutants) that can be experimentally measured at sites distant from the source. Limitations placed on the tracer, therefore, governed the design of the experimental procedure. These limitations included cost, the need to detect small quantities of the tracer, and the absence of the tracer from other natural sources. In addition, aerosols are emitted from high-temperature combustion sources that produce an abundance of very reactive species. The tracer, therefore, had to be both thermally and chemically stable. On the basis of these criteria, rare earth isotopes, such as those of Nd, were selected as tracers. The choice of tracer, in turn, dictated the analytical method (thermal ionization mass spectrometry, or TIMS) for measuring the isotopic abundances of

8

Modern Analytical Chemistry Nd in samples. Unfortunately, mass spectrometry is not a selective technique. A mass spectrum provides information about the abundance of ions with a given mass. It cannot distinguish, however, between different ions with the same mass. Consequently, the choice of TIMS required developing a procedure for separating the tracer from the aerosol particulates. Validating the final experimental protocol was accomplished by running a model study in which 148Nd was released into the atmosphere from a 100-MW coal utility boiler. Samples were collected at 13 locations, all of which were 20 km from the source. Experimental results were compared with predictions determined by the rate at which the tracer was released and the known dispersion of the emissions. Finally, the development of this procedure did not occur in a single, linear pass through the analytical approach. As research progressed, problems were encountered and modifications made, representing a cycle through steps 2, 3, and 4 of the analytical approach. Others have pointed out, with justification, that the analytical approach outlined here is not unique to analytical chemistry, but is common to any aspect of science involving analysis.8 Here, again, it helps to distinguish between a chemical analysis and analytical chemistry. For other analytically oriented scientists, such as physical chemists and physical organic chemists, the primary emphasis is on the problem, with the results of an analysis supporting larger research goals involving fundamental studies of chemical or physical processes. The essence of analytical chemistry, however, is in the second, third, and fourth steps of the analytical approach. Besides supporting broader research goals by developing and validating analytical methods, these methods also define the type and quality of information available to other research scientists. In some cases, the success of an analytical method may even suggest new research problems.

1C Common Analytical Problems
In Section 1A we indicated that analytical chemistry is more than a collection of qualitative and quantitative methods of analysis. Nevertheless, many problems on which analytical chemists work ultimately involve either a qualitative or quantitative measurement. Other problems may involve characterizing a sample’s chemical or physical properties. Finally, many analytical chemists engage in fundamental studies of analytical methods. In this section we briefly discuss each of these four areas of analysis. Many problems in analytical chemistry begin with the need to identify what is present in a sample. This is the scope of a qualitative analysis, examples of which include identifying the products of a chemical reaction, screening an athlete’s urine for the presence of a performance-enhancing drug, or determining the spatial distribution of Pb on the surface of an airborne particulate. Much of the early work in analytical chemistry involved the development of simple chemical tests to identify the presence of inorganic ions and organic functional groups. The classical laboratory courses in inorganic and organic qualitative analysis,9 still taught at some schools, are based on this work. Currently, most qualitative analyses use methods such as infrared spectroscopy, nuclear magnetic resonance, and mass spectrometry. These qualitative applications of identifying organic and inorganic compounds are covered adequately elsewhere in the undergraduate curriculum and, so, will receive no further consideration in this text.

qualitative analysis An analysis in which we determine the identity of the constituent species in a sample.

Chapter 1 Introduction Perhaps the most common type of problem encountered in the analytical lab is a quantitative analysis. Examples of typical quantitative analyses include the elemental analysis of a newly synthesized compound, measuring the concentration of glucose in blood, or determining the difference between the bulk and surface concentrations of Cr in steel. Much of the analytical work in clinical, pharmaceutical, environmental, and industrial labs involves developing new methods for determining the concentration of targeted species in complex samples. Most of the examples in this text come from the area of quantitative analysis. Another important area of analytical chemistry, which receives some attention in this text, is the development of new methods for characterizing physical and chemical properties. Determinations of chemical structure, equilibrium constants, particle size, and surface structure are examples of a characterization analysis. The purpose of a qualitative, quantitative, and characterization analysis is to solve a problem associated with a sample. A fundamental analysis, on the other hand, is directed toward improving the experimental methods used in the other areas of analytical chemistry. Extending and improving the theory on which a method is based, studying a method’s limitations, and designing new and modifying old methods are examples of fundamental studies in analytical chemistry.

9

quantitative analysis An analysis in which we determine how much of a constituent species is present in a sample.

characterization analysis An analysis in which we evaluate a sample’s chemical or physical properties. fundamental analysis An analysis whose purpose is to improve an analytical method’s capabilities.

1D KEY TERMS characterization analysis (p. 9) fundamental analysis (p. 9) qualitative analysis (p. 8) quantitative analysis (p. 9)

1E SUMMARY
Analytical chemists work to improve the ability of all chemists to make meaningful measurements. Chemists working in medicinal chemistry, clinical chemistry, forensic chemistry, and environmental chemistry, as well as the more traditional areas of chemistry, need better tools for analyzing materials. The need to work with smaller quantities of material, with more complex materials, with processes occurring on shorter time scales, and with species present at lower concentrations challenges analytical chemists to improve existing analytical methods and to develop new analytical techniques. Typical problems on which analytical chemists work include qualitative analyses (what is present?), quantitative analyses (how much is present?), characterization analyses (what are the material’s chemical and physical properties?), and fundamental analyses (how does this method work and how can it be improved?).

1F PROBLEMS
1. For each of the following problems indicate whether its solution requires a qualitative, quantitative, characterization, or fundamental study. More than one type of analysis may be appropriate for some problems. a. A hazardous-waste disposal site is believed to be leaking contaminants into the local groundwater. b. An art museum is concerned that a recent acquisition is a forgery. c. A more reliable method is needed by airport security for detecting the presence of explosive materials in luggage. d. The structure of a newly discovered virus needs to be determined. e. A new visual indicator is needed for an acid–base titration. f. A new law requires a method for evaluating whether automobiles are emitting too much carbon monoxide. 2. Read a recent article from the column “Analytical Approach,” published in Analytical Chemistry, or an article assigned by your instructor, and write an essay summarizing the nature of the problem and how it was solved. As a guide, refer back to Figure 1.3 for one model of the analytical approach.

10

Modern Analytical Chemistry

1G SUGGESTED READINGS
The role of analytical chemistry within the broader discipline of chemistry has been discussed by many prominent analytical chemists. Several notable examples follow. Baiulescu, G. E.; Patroescu, C.; Chalmers, R. A. Education and Teaching in Analytical Chemistry. Ellis Horwood: Chichester, 1982. Hieftje, G. M. “The Two Sides of Analytical Chemistry,” Anal. Chem. 1985, 57, 256A–267A. Kissinger, P. T. “Analytical Chemistry—What is It? Who Needs It? Why Teach It?” Trends Anal. Chem. 1992, 11, 54–57. Laitinen, H. A. “Analytical Chemistry in a Changing World,” Anal. Chem. 1980, 52, 605A–609A. Laitinen, H. A. “History of Analytical Chemistry in the U.S.A.,” Talanta 1989, 36, 1–9. Laitinen, H. A.; Ewing, G. (eds). A History of Analytical Chemistry. The Division of Analytical Chemistry of the American Chemical Society: Washington, D.C., 1972. McLafferty, F. W. “Analytical Chemistry: Historic and Modern,” Acc. Chem. Res. 1990, 23, 63–64. Mottola, H. A. “The Interdisciplinary and Multidisciplinary Nature of Contemporary Analytical Chemistry and Its Core Components,” Anal. Chim. Acta 1991, 242, 1–3. Tyson, J. Analysis: What Analytical Chemists Do. Royal Society of Chemistry: Cambridge, England, 1988. Several journals are dedicated to publishing broadly in the field of analytical chemistry, including Analytical Chemistry, Analytica Chimica Acta, Analyst, and Talanta. Other journals, too numerous to list, are dedicated to single areas of analytical chemistry. Current research in the areas of quantitative analysis, qualitative analysis, and characterization analysis are reviewed biannually (odd-numbered years) in Analytical Chemistry’s “Application Reviews.” Current research on fundamental developments in analytical chemistry are reviewed biannually (even-numbered years) in Analytical Chemistry’s “Fundamental Reviews.”

1H REFERENCES
1. Ravey, M. Spectroscopy 1990, 5(7), 11. 2. de Haseth, J. Spectroscopy 1990, 5(7), 11. 3. Fresenius, C. R. A System of Instruction in Quantitative Chemical Analysis. John Wiley and Sons: New York, 1881. 4. Hillebrand, W. F.; Lundell, G. E. F. Applied Inorganic Analysis, John Wiley and Sons: New York, 1953. 5. Van Loon, J. C. Analytical Atomic Absorption Spectroscopy. Academic Press: New York, 1980. 6. Murray, R. W. Anal. Chem. 1991, 63, 271A. 7. For several different viewpoints see (a) Beilby, A. L. J. Chem. Educ. 1970, 47, 237–238; (b) Lucchesi, C. A. Am. Lab. 1980, October, 113–119; (c) Atkinson, G. F. J. Chem. Educ. 1982, 59, 201–202; (d) Pardue, H. L.; Woo, J. J. Chem. Educ. 1984, 61, 409–412; (e) Guarnieri, M. J. Chem. Educ. 1988, 65, 201–203; (f) de Haseth, J. Spectroscopy 1990, 5, 20–21; (g) Strobel, H. A. Am. Lab. 1990, October, 17–24. 8. Hieftje, G. M. Am. Lab. 1993, October, 53–61. 9. See, for example, the following laboratory texts: (a) Sorum, C. H.; Lagowski, J. J. Introduction to Semimicro Qualitative Analysis, 5th ed. Prentice-Hall: Englewood Cliffs, NJ, 1977.; (b) Shriner, R. L.; Fuson, R. C.; Curtin, D. Y. The Systematic Identification of Organic Compounds, 5th ed. John Wiley and Sons: New York, 1964.

Chapter 2
Basic Tools of Analytical Chemistry

I

n the chapters that follow we will learn about the specifics of analytical chemistry. In the process we will ask and answer questions such as “How do we treat experimental data?” “How do we ensure that our results are accurate?” “How do we obtain a representative sample?” and “How do we select an appropriate analytical technique?” Before we look more closely at these and other questions, we will first review some basic numerical and experimental tools of importance to analytical chemists.

11

12

Modern Analytical Chemistry

2A Numbers in Analytical Chemistry
Analytical chemistry is inherently a quantitative science. Whether determining the concentration of a species in a solution, evaluating an equilibrium constant, measuring a reaction rate, or drawing a correlation between a compound’s structure and its reactivity, analytical chemists make measurements and perform calculations. In this section we briefly review several important topics involving the use of numbers in analytical chemistry.

2A.1 Fundamental Units of Measure
Imagine that you find the following instructions in a laboratory procedure: “Transfer 1.5 of your sample to a 100 volumetric flask, and dilute to volume.” How do you do this? Clearly these instructions are incomplete since the units of measurement are not stated. Compare this with a complete instruction: “Transfer 1.5 g of your sample to a 100-mL volumetric flask, and dilute to volume.” This is an instruction that you can easily follow. Measurements usually consist of a unit and a number expressing the quantity of that unit. Unfortunately, many different units may be used to express the same physical measurement. For example, the mass of a sample weighing 1.5 g also may be expressed as 0.0033 lb or 0.053 oz. For consistency, and to avoid confusion, scientists use a common set of fundamental units, several of which are listed in Table 2.1. These units are called SI units after the Système International d’Unités. Other measurements are defined using these fundamental SI units. For example, we measure the quantity of heat produced during a chemical reaction in joules, (J), where 1J =1 m2 kg s2

SI units Stands for Système International d’Unités. These are the internationally agreed on units for measurements.

scientific notation A shorthand method for expressing very large or very small numbers by indicating powers of ten; for example, 1000 is 1 × 103.

Table 2.2 provides a list of other important derived SI units, as well as a few commonly used non-SI units. Chemists frequently work with measurements that are very large or very small. A mole, for example, contains 602,213,670,000,000,000,000,000 particles, and some analytical techniques can detect as little as 0.000000000000001 g of a compound. For simplicity, we express these measurements using scientific notation; thus, a mole contains 6.0221367 × 1023 particles, and the stated mass is 1 × 10–15 g. Sometimes it is preferable to express measurements without the exponential term, replacing it with a prefix. A mass of 1 × 10–15 g is the same as 1 femtogram. Table 2.3 lists other common prefixes.

Table 2.1
Measurement

Fundamental SI Units
Unit
kilogram liter meter kelvin second ampere mole

Symbol kg L m K s A mol

mass volume distance temperature time current amount of substance

Chapter 2 Basic Tools of Analytical Chemistry

13

Table 2.2
Measurement
length force pressure

Other SI and Non-SI Units
Unit
angstrom newton pascal atmosphere joule watt coulomb volt degree Celsius degree Fahrenheit

Symbol
Å N Pa atm J W C V °C °F

Equivalent SI units
1 Å = 1 × 10–10 m 1 N = 1 m kg/s2 1 Pa = 1 N/m2 = 1 kg/(m s2) 1 atm = 101,325 Pa 1 J = 1 N m = 1 m2 kg/s2 1 W = 1 J/s = 1 m2 kg/s3 1C=1A s 1 V = 1 W/A = 1 m2 kg/(s3 A) °C = K – 273.15 °F = 1.8(K – 273.15) + 32

⋅

energy, work, heat power charge potential temperature

⋅ ⋅

⋅ ⋅ ⋅ ⋅

⋅

Table 2.3
Exponential
1012 109 106 103 10–1 10–2 10–3 10–6 10–9 10–12 10–15 10–18

Common Prefixes for Exponential Notation
Prefix
tera giga mega kilo deci centi milli micro nano pico femto atto

Symbol
T G M k d c m µ n p f a

2A.2 Significant Figures
Recording a measurement provides information about both its magnitude and uncertainty. For example, if we weigh a sample on a balance and record its mass as 1.2637 g, we assume that all digits, except the last, are known exactly. We assume that the last digit has an uncertainty of at least ±1, giving an absolute uncertainty of at least ±0.0001 g, or a relative uncertainty of at least ±0.0001 g × 100 = ±0.0079% 1.2637 g Significant figures are a reflection of a measurement’s uncertainty. The number of significant figures is equal to the number of digits in the measurement, with the exception that a zero (0) used to fix the location of a decimal point is not considered significant. This definition can be ambiguous. For example, how many significant figures are in the number 100? If measured to the nearest hundred, then there is one significant figure. If measured to the nearest ten, however, then two significant figures The digits in a measured quantity, including all digits known exactly and one digit (the last) whose quantity is uncertain.

14

Modern Analytical Chemistry significant figures are included. To avoid ambiguity we use scientific notation. Thus, 1 × 102 has one significant figure, whereas 1.0 × 102 has two significant figures. For measurements using logarithms, such as pH, the number of significant figures is equal to the number of digits to the right of the decimal, including all zeros. Digits to the left of the decimal are not included as significant figures since they only indicate the power of 10. A pH of 2.45, therefore, contains two significant figures. Exact numbers, such as the stoichiometric coefficients in a chemical formula or reaction, and unit conversion factors, have an infinite number of significant figures. A mole of CaCl2, for example, contains exactly two moles of chloride and one mole of calcium. In the equality 1000 mL = 1 L both numbers have an infinite number of significant figures. Recording a measurement to the correct number of significant figures is important because it tells others about how precisely you made your measurement. For example, suppose you weigh an object on a balance capable of measuring mass to the nearest ±0.1 mg, but record its mass as 1.762 g instead of 1.7620 g. By failing to record the trailing zero, which is a significant figure, you suggest to others that the mass was determined using a balance capable of weighing to only the nearest ±1 mg. Similarly, a buret with scale markings every 0.1 mL can be read to the nearest ±0.01 mL. The digit in the hundredth’s place is the least significant figure since we must estimate its value. Reporting a volume of 12.241 mL implies that your buret’s scale is more precise than it actually is, with divisions every 0.01 mL. Significant figures are also important because they guide us in reporting the result of an analysis. When using a measurement in a calculation, the result of that calculation can never be more certain than that measurement’s uncertainty. Simply put, the result of an analysis can never be more certain than the least certain measurement included in the analysis. As a general rule, mathematical operations involving addition and subtraction are carried out to the last digit that is significant for all numbers included in the calculation. Thus, the sum of 135.621, 0.33, and 21.2163 is 157.17 since the last digit that is significant for all three numbers is in the hundredth’s place. 135.621 + 0.33 + 21.2163 = 157.1673 = 157.17 When multiplying and dividing, the general rule is that the answer contains the same number of significant figures as that number in the calculation having the fewest significant figures. Thus, 22.91 × 0.152 = 0.21361 = 0.214 16.302 It is important to remember, however, that these rules are generalizations. What is conserved is not the number of significant figures, but absolute uncertainty when adding or subtracting, and relative uncertainty when multiplying or dividing. For example, the following calculation reports the answer to the correct number of significant figures, even though it violates the general rules outlined earlier. 101 = 1.02 99

Chapter 2 Basic Tools of Analytical Chemistry Since the relative uncertainty in both measurements is roughly 1% (101 ±1, 99 ±1), the relative uncertainty in the final answer also must be roughly 1%. Reporting the answer to only two significant figures (1.0), as required by the general rules, implies a relative uncertainty of 10%. The correct answer, with three significant figures, yields the expected relative uncertainty. Chapter 4 presents a more thorough treatment of uncertainty and its importance in reporting the results of an analysis. Finally, to avoid “round-off ” errors in calculations, it is a good idea to retain at least one extra significant figure throughout the calculation. This is the practice adopted in this textbook. Better yet, invest in a good scientific calculator that allows you to perform lengthy calculations without recording intermediate values. When the calculation is complete, the final answer can be rounded to the correct number of significant figures using the following simple rules. 1. Retain the least significant figure if it and the digits that follow are less than halfway to the next higher digit; thus, rounding 12.442 to the nearest tenth gives 12.4 since 0.442 is less than halfway between 0.400 and 0.500. 2. Increase the least significant figure by 1 if it and the digits that follow are more than halfway to the next higher digit; thus, rounding 12.476 to the nearest tenth gives 12.5 since 0.476 is more than halfway between 0.400 and 0.500. 3. If the least significant figure and the digits that follow are exactly halfway to the next higher digit, then round the least significant figure to the nearest even number; thus, rounding 12.450 to the nearest tenth gives 12.4, but rounding 12.550 to the nearest tenth gives 12.6. Rounding in this manner prevents us from introducing a bias by always rounding up or down.

15

2B Units for Expressing Concentration
Concentration is a general measurement unit stating the amount of solute present in a known amount of solution Concentration = amount of solute amount of solution 2.1 concentration An expression stating the relative amount of solute per unit volume or unit mass of solution.

Although the terms “solute” and “solution” are often associated with liquid samples, they can be extended to gas-phase and solid-phase samples as well. The actual units for reporting concentration depend on how the amounts of solute and solution are measured. Table 2.4 lists the most common units of concentration.

2B.1 Molarity and Formality
Both molarity and formality express concentration as moles of solute per liter of solution. There is, however, a subtle difference between molarity and formality. Molarity is the concentration of a particular chemical species in solution. Formality, on the other hand, is a substance’s total concentration in solution without regard to its specific chemical form. There is no difference between a substance’s molarity and formality if it dissolves without dissociating into ions. The molar concentration of a solution of glucose, for example, is the same as its formality. For substances that ionize in solution, such as NaCl, molarity and formality are different. For example, dissolving 0.1 mol of NaCl in 1 L of water gives a solution containing 0.1 mol of Na+ and 0.1 mol of Cl–. The molarity of NaCl, therefore, is zero since there is essentially no undissociated NaCl in solution. The solution, molarity The number of moles of solute per liter of solution (M). formality The number of moles of solute, regardless of chemical form, per liter of solution (F).

16

Modern Analytical Chemistry

Table 2.4
Name
molarity formality normality molality weight % volume %

Common Units for Reporting Concentration
Unitsa
moles solute liters solution number FWs solute liters solution number EWs solute liters solution moles solute kg solvent g solute 100 g solution mL solute 100 mL solution g solute 100 mL solution g solute 10 6 g solution g solute 10 9 g solution

Symbol
M F N m % w/w % v/v % w/v ppm ppb

weight-to-volume % parts per million parts per billion

aFW

= formula weight; EW = equivalent weight.

instead, is 0.1 M in Na+ and 0.1 M in Cl–. The formality of NaCl, however, is 0.1 F because it represents the total amount of NaCl in solution. The rigorous definition of molarity, for better or worse, is largely ignored in the current literature, as it is in this text. When we state that a solution is 0.1 M NaCl we understand it to consist of Na+ and Cl– ions. The unit of formality is used only when it provides a clearer description of solution chemistry. Molar concentrations are used so frequently that a symbolic notation is often used to simplify its expression in equations and writing. The use of square brackets around a species indicates that we are referring to that species’ molar concentration. Thus, [Na+] is read as the “molar concentration of sodium ions.”

2B.2 Normality
Normality is an older unit of concentration that, although once commonly used, is frequently ignored in today’s laboratories. Normality is still used in some handbooks of analytical methods, and, for this reason, it is helpful to understand its meaning. For example, normality is the concentration unit used in Standard Methods for the Examination of Water and Wastewater,1 a commonly used source of analytical methods for environmental laboratories. Normality makes use of the chemical equivalent, which is the amount of one chemical species reacting stoichiometrically with another chemical species. Note that this definition makes an equivalent, and thus normality, a function of the chemical reaction in which the species participates. Although a solution of H2SO4 has a fixed molarity, its normality depends on how it reacts.

normality The number of equivalents of solute per liter of solution (N).

Chapter 2 Basic Tools of Analytical Chemistry The number of equivalents, n, is based on a reaction unit, which is that part of a chemical species involved in a reaction. In a precipitation reaction, for example, the reaction unit is the charge of the cation or anion involved in the reaction; thus for the reaction Pb2+(aq) + 2I–(aq) Pb2+ I–. equivalent The moles of a species that can donate one reaction unit.

17

t PbI2(s)

n = 2 for and n = 1 for In an acid–base reaction, the reaction unit is the number of H+ ions donated by an acid or accepted by a base. For the reaction between sulfuric acid and ammonia H2SO4(aq) + 2NH3(aq)

t 2NH4+(aq) + SO42–(aq)

we find that n = 2 for H2SO4 and n = 1 for NH3. For a complexation reaction, the reaction unit is the number of electron pairs that can be accepted by the metal or donated by the ligand. In the reaction between Ag+ and NH3 Ag+(aq) + 2NH3(aq)

t Ag(NH3)2+(aq)

the value of n for Ag+ is 2 and that for NH3 is 1. Finally, in an oxidation–reduction reaction the reaction unit is the number of electrons released by the reducing agent or accepted by the oxidizing agent; thus, for the reaction 2Fe3+(aq) + Sn2+(aq)

t Sn4+(aq) + 2Fe2+(aq) equivalent weight The mass of a compound containing one equivalent (EW). formula weight The mass of a compound containing one mole (FW).

n = 1 for Fe3+ and n = 2 for Sn2+. Clearly, determining the number of equivalents for a chemical species requires an understanding of how it reacts. Normality is the number of equivalent weights (EW) per unit volume and, like formality, is independent of speciation. An equivalent weight is defined as the ratio of a chemical species’ formula weight (FW) to the number of its equivalents EW = FW n

Consequently, the following simple relationship exists between normality and molarity. N=n×M Example 2.1 illustrates the relationship among chemical reactivity, equivalent weight, and normality. EXAMPLE

2.1

Calculate the equivalent weight and normality for a solution of 6.0 M H3PO4 given the following reactions: (a) H3PO4(aq) + 3OH–(aq) (b) (c)

t PO43–(aq) + 3H2O(l) H3PO4(aq) + 2NH3(aq) t HPO42–(aq) + 2NH4+(aq) H3PO4(aq) + F–(aq) t H2PO4–(aq) + HF(aq)

SOLUTION For phosphoric acid, the number of equivalents is the number of H+ ions donated to the base. For the reactions in (a), (b), and (c) the number of equivalents are 3, 2, and 1, respectively. Thus, the calculated equivalent weights and normalities are

18

Modern Analytical Chemistry FW 97.994 = = 32.665 n 3 FW 97.994 (b) EW = = = 48.997 n 2 FW 97.994 (c) EW = = = 97.994 n 1 (a) EW = N = n × M = 3 × 6.0 = 18 N N = n × M = 2 × 6.0 = 12 N N = n × M = 1 × 6.0 = 6.0 N

2B.3 Molality molality The number of moles of solute per kilogram of solvent (m).

Molality is used in thermodynamic calculations where a temperature independent unit of concentration is needed. Molarity, formality and normality are based on the volume of solution in which the solute is dissolved. Since density is a temperature dependent property a solution’s volume, and thus its molar, formal and normal concentrations, will change as a function of its temperature. By using the solvent’s mass in place of its volume, the resulting concentration becomes independent of temperature.

2B.4 Weight, Volume, and Weight-to-Volume Ratios weight percent Grams of solute per 100 g of solution. (% w/w). volume percent Milliliters of solute per 100 mL of solution (% v/v). weight-to-volume percent Grams of solute per 100 mL of solution (% w/v). parts per million Micrograms of solute per gram of solution; for aqueous solutions the units are often expressed as milligrams of solute per liter of solution (ppm). parts per billion Nanograms of solute per gram of solution; for aqueous solutions the units are often expressed as micrograms of solute per liter of solution (ppb).

Weight percent (% w/w), volume percent (% v/v) and weight-to-volume percent (% w/v) express concentration as units of solute per 100 units of sample. A solution in which a solute has a concentration of 23% w/v contains 23 g of solute per 100 mL of solution. Parts per million (ppm) and parts per billion (ppb) are mass ratios of grams of solute to one million or one billion grams of sample, respectively. For example, a steel that is 450 ppm in Mn contains 450 µg of Mn for every gram of steel. If we approximate the density of an aqueous solution as 1.00 g/mL, then solution concentrations can be expressed in parts per million or parts per billion using the following relationships. ppm = mg µg = liter mL µg ng ppb = = liter mL

For gases a part per million usually is a volume ratio. Thus, a helium concentration of 6.3 ppm means that one liter of air contains 6.3 µL of He.

2B.5 Converting Between Concentration Units
The units of concentration most frequently encountered in analytical chemistry are molarity, weight percent, volume percent, weight-to-volume percent, parts per million, and parts per billion. By recognizing the general definition of concentration given in equation 2.1, it is easy to convert between concentration units. EXAMPLE 2.2 A concentrated solution of aqueous ammonia is 28.0% w/w NH3 and has a density of 0.899 g/mL. What is the molar concentration of NH3 in this solution? SOLUTION 28.0 g NH 3 0.899 g solution 1 mole NH 3 1000 mL × × × = 14.8 M 100 g solution mL solution 17.04 g NH 3 liter

Chapter 2 Basic Tools of Analytical Chemistry EXAMPLE 2.3 The maximum allowed concentration of chloride in a municipal drinking water supply is 2.50 × 102 ppm Cl–. When the supply of water exceeds this limit, it often has a distinctive salty taste. What is this concentration in moles Cl–/liter? SOLUTION 2.50 × 102 mg Cl – 1g 1 mole Cl – × × = 7.05 × 10 –3 M L 1000 mg 35.453 g Cl –

19

2B.6 p-Functions
Sometimes it is inconvenient to use the concentration units in Table 2.4. For example, during a reaction a reactant’s concentration may change by many orders of magnitude. If we are interested in viewing the progress of the reaction graphically, we might wish to plot the reactant’s concentration as a function of time or as a function of the volume of a reagent being added to the reaction. Such is the case in Figure 2.1, where the molar concentration of H+ is plotted (y-axis on left side of figure) as a function of the volume of NaOH added to a solution of HCl. The initial [H+] is 0.10 M, and its concentration after adding 75 mL of NaOH is 5.0 × 10–13 M. We can easily follow changes in the [H+] over the first 14 additions of NaOH. For the last ten additions of NaOH, however, changes in the [H+] are too small to be seen. When working with concentrations that span many orders of magnitude, it is often more convenient to express the concentration as a p-function. The p-function of a number X is written as pX and is defined as pX = –log(X) Thus, the pH of a solution that is 0.10 M H+ is pH = –log[H+] = –log(0.10) = 1.00 and the pH of 5.0 × 10–13 M H+ is pH = –log[H+] = –log(5.0 × 10–13) = 12.30 Figure 2.1 shows how plotting pH in place of [H+] provides more detail about how the concentration of H+ changes following the addition of NaOH. EXAMPLE 2.4 What is pNa for a solution of 1.76 × 10–3 M Na3PO4? SOLUTION Since each mole of Na3PO4 contains three moles of Na+, the concentration of Na+ is [Na + ] = and pNa is pNa = –log[Na+] = –log(5.28 × 10–3) = 2.277 3 mol Na + × 1.76 × 10 –3 M = 5.28 × 10 –3 M mol Na3 PO4

p-function A function of the form pX, where pX = -log(X).

20

Modern Analytical Chemistry
0.12 [H+] pH 14 12 10 0.08 [H+] (M) 8 0.06 6 0.04 4 0.02 2 0 0 20 40 60 Volume NaOH (mL) 80 pH

0.10

Figure 2.1
Graph of [H+] versus volume of NaOH and pH versus volume of NaOH for the reaction of 0.10 M HCl with 0.10 M NaOH.

0.00

EXAMPLE 2.5 What is the [H+] in a solution that has a pH of 5.16? SOLUTION The concentration of H+ is pH = –log[H+] = 5.16 log[H+] = –5.16 [H+] = antilog(–5.16) = 10–5.16 = 6.9 × 10–6 M

2C Stoichiometric Calculations
A balanced chemical reaction indicates the quantitative relationships between the moles of reactants and products. These stoichiometric relationships provide the basis for many analytical calculations. Consider, for example, the problem of determining the amount of oxalic acid, H2C2O4, in rhubarb. One method for this analysis uses the following reaction in which we oxidize oxalic acid to CO2. 2Fe3+(aq) + H2C2O4(aq) + 2H2O(l) → 2Fe2+(aq) + 2CO2(g) + 2H3O+(aq) 2.2 The balanced chemical reaction provides the stoichiometric relationship between the moles of Fe3+ used and the moles of oxalic acid in the sample being analyzed— specifically, one mole of oxalic acid reacts with two moles of Fe3+. As shown in Example 2.6, the balanced chemical reaction can be used to determine the amount of oxalic acid in a sample, provided that information about the number of moles of Fe3+ is known.

Chapter 2 Basic Tools of Analytical Chemistry EXAMPLE 2.6 The amount of oxalic acid in a sample of rhubarb was determined by reacting with Fe3+ as outlined in reaction 2.2. In a typical analysis, the oxalic acid in 10.62 g of rhubarb was extracted with a suitable solvent. The complete oxidation of the oxalic acid to CO2 required 36.44 mL of 0.0130 M Fe3+. What is the weight percent of oxalic acid in the sample of rhubarb? SOLUTION We begin by calculating the moles of Fe3+ used in the reaction 0.0130 mol Fe3+ × 0.03644 L = 4.737 × 10 –4 mol Fe3+ L The moles of oxalic acid reacting with the Fe3+, therefore, is 4.737 × 10 –4 mol Fe3+ × 1 mol C2 H 2 O4 = 2.369 × 10 –4 mol C 2 H 2 O4 2 mol Fe3+ 90.03 g C2 H 2 O4 = 2.132 × 10 –2 g oxalic acid mol C 2 H 2 O4

21

Converting moles of oxalic acid to grams of oxalic acid 2.369 × 10 –4 mol C 2 H 2 O4 ×

and converting to weight percent gives the concentration of oxalic acid in the sample of rhubarb as 2.132 × 10 –2 g C 2 H 2 O4 × 100 = 0.201% w/w C 2 H 2 O4 10.62 g rhubarb

In the analysis described in Example 2.6 oxalic acid already was present in the desired form. In many analytical methods the compound to be determined must be converted to another form prior to analysis. For example, one method for the quantitative analysis of tetraethylthiuram disulfide (C10H20N2S4), the active ingredient in the drug Antabuse (disulfiram), requires oxidizing the S to SO2, bubbling the SO2 through H2O2 to produce H2SO4, followed by an acid–base titration of the H2SO4 with NaOH. Although we can write and balance chemical reactions for each of these steps, it often is easier to apply the principle of the conservation of reaction units. A reaction unit is that part of a chemical species involved in a reaction. Consider, for example, the general unbalanced chemical reaction A + B → Products Conservation of reaction units requires that the number of reaction units associated with the reactant A equal the number of reaction units associated with the reactant B. Translating the previous statement into mathematical form gives Number of reaction units per A × moles A = number of reaction units per B × moles B 2.3

If we know the moles of A and the number of reaction units associated with A and B, then we can calculate the moles of B. Note that a conservation of reaction units, as defined by equation 2.3, can only be applied between two species. There are five important principles involving a conservation of reaction units: mass, charge, protons, electron pairs, and electrons.

22

Modern Analytical Chemistry

2C.1 Conservation of Mass
The easiest principle to appreciate is conservation of mass. Except for nuclear reactions, an element’s total mass at the end of a reaction must be the same as that present at the beginning of the reaction; thus, an element serves as the most fundamental reaction unit. Consider, for example, the combustion of butane to produce CO2 and H2O, for which the unbalanced reaction is C4H10(g) + O2(g) → CO2(g) + H2O(g) All the carbon in CO2 comes from the butane, thus we can select carbon as a reaction unit. Since there are four carbon atoms in butane, and one carbon atom in CO2, we write 4 × moles C4H10 = 1 × moles CO2 Hydrogen also can be selected as a reaction unit since all the hydrogen in butane ends up in the H2O produced during combustion. Thus, we can write 10 × moles C4H10 = 2 × moles H2O Although the mass of oxygen is conserved during the reaction, we cannot apply equation 2.3 because the O2 used during combustion does not end up in a single product. Conservation of mass also can, with care, be applied to groups of atoms. For example, the ammonium ion, NH4+, can be precipitated as Fe(NH4)2(SO4)2 ⋅ 6H2O. Selecting NH4+ as the reaction unit gives 2 × moles Fe(NH4)2(SO4)2 · 6H2O = 1 × moles NH4+

2C.2 Conservation of Charge
The stoichiometry between two reactants in a precipitation reaction is governed by a conservation of charge, requiring that the total cation charge and the total anion charge in the precipitate be equal. The reaction units in a precipitation reaction, therefore, are the absolute values of the charges on the cation and anion that make up the precipitate. Applying equation 2.3 to a precipitate of Ca3(PO4)2 formed from the reaction of Ca2+ and PO43–, we write 2 × moles Ca2+ = 3 × moles PO43–

2C.3 Conservation of Protons
In an acid–base reaction, the reaction unit is the proton. For an acid, the number of reaction units is given by the number of protons that can be donated to the base; and for a base, the number of reaction units is the number of protons that the base can accept from the acid. In the reaction between H3PO4 and NaOH, for example, the weak acid H3PO4 can donate all three of its protons to NaOH, whereas the strong base NaOH can accept one proton. Thus, we write 3 × moles H3PO4 = 1 × moles NaOH Care must be exercised in determining the number of reaction units associated with the acid and base. The number of reaction units for an acid, for instance, depends not on how many acidic protons are present, but on how many

Chapter 2 Basic Tools of Analytical Chemistry of the protons are capable of reacting with the chosen base. In the reaction between H3PO4 and NH3 H3PO4(aq) + 2NH3(aq) a conservation of protons requires that 2 × moles H3PO4 = moles of NH3

23

t HPO4–(aq) + 2NH4+(aq)

2C.4 Conservation of Electron Pairs
In a complexation reaction, the reaction unit is an electron pair. For the metal, the number of reaction units is the number of coordination sites available for binding ligands. For the ligand, the number of reaction units is equivalent to the number of electron pairs that can be donated to the metal. One of the most important analytical complexation reactions is that between the ligand ethylenediaminetetracetic acid (EDTA), which can donate 6 electron pairs and 6 coordinate metal ions, such as Cu2+; thus 6 × mole Cu2+ = 6 × moles EDTA

2C.5 Conservation of Electrons
In a redox reaction, the reaction unit is an electron transferred from a reducing agent to an oxidizing agent. The number of reaction units for a reducing agent is equal to the number of electrons released during its oxidation. For an oxidizing agent, the number of reaction units is given by the number of electrons needed to cause its reduction. In the reaction between Fe3+ and oxalic acid (reaction 2.2), for example, Fe3+ undergoes a 1-electron reduction. Each carbon atom in oxalic acid is initially present in a +3 oxidation state, whereas the carbon atom in CO2 is in a +4 oxidation state. Thus, we can write 1 × moles Fe3+ = 2 × moles of H2C2O4 Note that the moles of oxalic acid are multiplied by 2 since there are two carbon atoms, each of which undergoes a 1-electron oxidation.

2C.6 Using Conservation Principles in Stoichiometry Problems
As shown in the following examples, the application of conservation principles simplifies stoichiometric calculations. EXAMPLE 2.7 Rework Example 2.6 using conservation principles. SOLUTION Conservation of electrons for this redox reaction requires that moles Fe3+ = 2 × moles H2C2O4 which can be transformed by writing moles as the product of molarity and volume or as grams per formula weight. MFe 3 + × VFe 3 + = 2 × g H 2 C 2 O4 FW H 2 C 2 O4

24

Modern Analytical Chemistry Solving for g H2C2O4 gives MFe 3 + × VFe 3 + × FW H 2 C 2 O4 (0.0130 M)(0.03644 L)(90.03 g/mole) = 2 2 = 2.132 × 10 –2 g H2 C 2 O4 and the weight percent oxalic acid is 2.132 × 10 –2 g C 2 H 2 O4 × 100 = 0.201% w/w C 2 H 2 O4 10.62 g rhubarb

EXAMPLE 2.8 One quantitative analytical method for tetraethylthiuram disulfide, C10H20N2S4 (Antabuse), requires oxidizing the sulfur to SO2, and bubbling the resulting SO2 through H2O2 to produce H2SO4. The H2SO4 is then reacted with NaOH according to the reaction H2SO4(aq) + 2NaOH(aq) → Na2SO4(aq) + 2H2O(l) Using appropriate conservation principles, derive an equation relating the moles of C 10 H 20 N 2 S 4 to the moles of NaOH. What is the weight percent C10H20N2S4 in a sample of Antabuse if the H2SO4 produced from a 0.4613-g portion reacts with 34.85 mL of 0.02500 M NaOH? SOLUTION The unbalanced reactions converting C10H20N2S4 to H2SO4 are C10H20N2S4 → SO2 SO2 → H2SO4 Using a conservation of mass we have 4 × moles C10H20N2S4 = moles SO2 = moles H2SO4 A conservation of protons for the reaction of H2SO4 with NaOH gives 2 × moles H2SO4 = moles of NaOH Combining the two conservation equations gives the following stoichiometric equation between C10H20N2S4 and NaOH 8 × moles C10H20N2S4 = moles NaOH Now we are ready to finish the problem. Making appropriate substitutions for moles of C10H20N2S4 and moles of NaOH gives 8 × g C10 H 20 N 2 S4 = MNaOH × VNaOH FW C10 H 20 N 2 S4 Solving for g C10H20N2S4 gives g C10 H 20 N 2S 4 = 1 × M NaOH × VNaOH × FW C10 H 20 N 2S 4 8

1 (0.02500 M)(0.03485 L)(296.54 g/mol) = 0.032295 g C10 H 20 N 2 S4 8

Chapter 2 Basic Tools of Analytical Chemistry The weight percent C10H20N2S4 in the sample, therefore, is 0.32295 g C10 H 20 N 2 S4 × 100 = 7.001% w/w C10 H 20 N 2 S4 0.4613 g sample

25

2D Basic Equipment and Instrumentation
Measurements are made using appropriate equipment or instruments. The array of equipment and instrumentation used in analytical chemistry is impressive, ranging from the simple and inexpensive, to the complex and costly. With two exceptions, we will postpone the discussion of equipment and instrumentation to those chapters where they are used. The instrumentation used to measure mass and much of the equipment used to measure volume are important to all analytical techniques and are therefore discussed in this section.

2D.1 Instrumentation for Measuring Mass
An object’s mass is measured using a balance. The most common type of balance is an electronic balance in which the balance pan is placed over an electromagnet (Figure 2.2). The sample to be weighed is placed on the sample pan, displacing the pan downward by a force equal to the product of the sample’s mass and the acceleration due to gravity. The balance detects this downward movement and generates a counterbalancing force using an electromagnet. The current needed to produce this force is proportional to the object’s mass. A typical electronic balance has a capacity of 100–200 g and can measure mass to the nearest ±0.01 to ±1 mg. Another type of balance is the single-pan, unequal arm balance (Figure 2.3). In this mechanical balance the balance pan and a set of removable standard weights on one side of a beam are balanced against a fixed counterweight on the beam’s other side. The beam itself is balanced on a fulcrum consisting of a sharp knife edge. Adding a sample to the balance pan tilts the beam away from its balance point. Selected standard weights are then removed until the beam is brought back into balance. The combined mass of the removed weights equals the sample’s mass. The capacities and measurement limits of these balances are comparable to an electronic balance. balance An apparatus used to measure mass.

Detector

Light source Balance pan N

S

S

Figure 2.2
Control circuitry
(a) Photo of a typical electronic balance. (b) Schematic diagram of electronic balance; adding a sample moves the balance pan down, allowing more light to reach the detector. The control circuitry directs the electromagnetic servomotor to generate an opposing force, raising the sample up until the original intensity of light at the detector is restored.
Photo courtesy of Fisher Scientific.

Electromagnetic servomotor (a) (b)

26

Modern Analytical Chemistry
Fulcrum Balance beam Fulcrum Counterweight Removable weights

Figure 2.3
Schematic diagram of single-arm mechanical balance.

Balance pan

The mass of a sample is determined by difference. If the material being weighed is not moisture-sensitive, a clean and dry container is placed on the balance. The mass of this container is called the tare. Most balances allow the tare to be automatically adjusted to read a mass of zero. The sample is then transferred to the container, the new mass is measured and the sample’s mass determined by subtracting the tare. Samples that absorb moisture from the air are weighed differently. The sample is placed in a covered weighing bottle and their combined mass is determined. A portion of the sample is removed, and the weighing bottle and remaining sample are reweighed. The difference between the two masses gives the mass of the transferred sample. Several important precautions help to minimize errors in measuring an object’s mass. Balances should be placed on heavy surfaces to minimize the effect of vibrations in the surrounding environment and should be maintained in a level position. Analytical balances are sensitive enough that they can measure the mass of a fingerprint. For this reason, materials placed on a balance should normally be handled using tongs or laboratory tissues. Volatile liquid samples should be weighed in a covered container to avoid the loss of sample by evaporation. Air currents can significantly affect a sample’s mass. To avoid air currents, the balance’s glass doors should be closed, or the balance’s wind shield should be in place. A sample that is cooler or warmer than the surrounding air will create convective air currents that adversely affect the measurement of its mass. Finally, samples dried in an oven should be stored in a desiccator to prevent them from reabsorbing moisture from the atmosphere.

2D.2 Equipment for Measuring Volume
Analytical chemists use a variety of glassware to measure volume, several examples of which are shown in Figure 2.4. The type of glassware used depends on how exact the volume needs to be. Beakers, dropping pipets, and graduated cylinders are used to measure volumes approximately, typically with errors of several percent. Pipets and volumetric flasks provide a more accurate means for measuring volume. When filled to its calibration mark, a volumetric flask is designed to contain a specified volume of solution at a stated temperature, usually 20 °C. The actual vol-

volumetric flask Glassware designed to contain a specific volume of solution when filled to its calibration mark.

Chapter 2 Basic Tools of Analytical Chemistry

27

(a)

Figure 2.4
Common examples of glassware used to measure volume: (a) beaker; (b) graduated cylinder; (c) volumetric flask; (d) pipet; (e) dropping pipet.
Photos courtesy of Fisher Scientific.

(b)

(c)

ume contained by the volumetric flask is usually within 0.03–0.2% of the stated value. Volumetric flasks containing less than 100 mL generally measure volumes to the hundredth of a milliliter, whereas larger volumetric flasks measure volumes to the tenth of a milliliter. For example, a 10-mL volumetric flask contains 10.00 mL, but a 250-mL volumetric flask holds 250.0 mL (this is important when keeping track of significant figures). Because a volumetric flask contains a solution, it is useful in preparing solutions with exact concentrations. The reagent is transferred to the volumetric flask, and enough solvent is added to dissolve the reagent. After the reagent is dissolved, additional solvent is added in several portions, mixing the solution after each addition. The final adjustment of volume to the flask’s calibration mark is made using a dropping pipet. To complete the mixing process, the volumetric flask should be inverted at least ten times. A pipet is used to deliver a specified volume of solution. Several different styles of pipets are available (Figure 2.5). Transfer pipets provide the most accurate means for delivering a known volume of solution; their volume error is similar to that from an equivalent volumetric flask. A 250-mL transfer pipet, for instance, will deliver 250.0 mL. To fill a transfer pipet, suction from a rubber bulb is used to pull the liquid up past the calibration mark (never use your mouth to suck a solution into a pipet). After replacing the bulb with your finger, the liquid’s level is adjusted to the calibration mark, and the outside of the pipet is wiped dry. The pipet’s contents are allowed to drain into the receiving container with the tip of the pipet touching the container walls. A small portion of the liquid remains in the pipet’s tip and should not be blown out. Measuring pipets are used to deliver variable volumes, but with less accuracy than transfer pipets. With some measuring

(d)

(e)

pipet Glassware designed to deliver a specific volume of solution when filled to its calibration mark.

28

Modern Analytical Chemistry

(c)

(d)

Figure 2.5
Common types of pipets and syringes: (a) transfer pipet; (b) measuring pipet; (c) digital pipet; (d) syringe.
Photos courtesy of Fisher Scientific.

(a)

(b)

Meniscus Calibration mark

Figure 2.6
Proper means of reading the meniscus on a volumetric flask or pipet.

pipets, delivery of the calibrated volume requires that any solution remaining in the tip be blown out. Digital pipets and syringes can be used to deliver volumes as small as a microliter. Three important precautions are needed when working with pipets and volumetric flasks. First, the volume delivered by a pipet or contained by a volumetric flask assumes that the glassware is clean. Dirt and grease on the inner glass surface prevents liquids from draining evenly, leaving droplets of the liquid on the container’s walls. For a pipet this means that the delivered volume is less than the calibrated volume, whereas drops of liquid above the calibration mark mean that a volumetric flask contains more than its calibrated volume. Commercially available cleaning solutions can be used to clean pipets and volumetric flasks.

Chapter 2 Basic Tools of Analytical Chemistry Second, when filling a pipet or volumetric flask, set the liquid’s level exactly at the calibration mark. The liquid’s top surface is curved into a meniscus, the bottom of which should be exactly even with the glassware’s calibration mark (Figure 2.6). The meniscus should be adjusted with the calibration mark at eye level to avoid parallax errors. If your eye level is above the calibration mark the pipet or volumetric flask will be overfilled. The pipet or volumetric flask will be underfilled if your eye level is below the calibration mark. Finally, before using a pipet or volumetric flask you should rinse it with several small portions of the solution whose volume is being measured. This ensures that any residual liquid remaining in the pipet or volumetric flask is removed.

29

meniscus The curved surface of a liquid contained in a tube.

2D.3 Equipment for Drying Samples
Many materials need to be dried prior to their analysis to remove residual moisture. Depending on the material, heating to a temperature of 110–140 °C is usually sufficient. Other materials need to be heated to much higher temperatures to initiate thermal decomposition. Both processes can be accomplished using a laboratory oven capable of providing the required temperature. Commercial laboratory ovens (Figure 2.7) are used when the maximum desired temperature is 160–325 °C (depending on the model). Some ovens include the ability to circulate heated air, allowing for a more efficient removal of moisture and shorter drying times. Other ovens provide a tight seal for the door, allowing the oven to be evacuated. In some situations a conventional laboratory oven can be replaced with a microwave oven. Higher temperatures, up to 1700° C, can be achieved using a muffle furnace (Figure 2.8). After drying or decomposing a sample, it should be cooled to room temperature in a desiccator to avoid the readsorption of moisture. A desiccator (Figure 2.9) is a closed container that isolates the sample from the atmosphere. A drying agent, called a desiccant, is placed in the bottom of the container. Typical desiccants include calcium chloride and silica gel. A perforated plate sits above the desiccant, providing a shelf for storing samples. Some desiccators are equipped with stopcocks that allow them to be evacuated.

Figure 2.7
Conventional laboratory oven used for drying materials.

Figure 2.8
Example of a muffle furnace used for heating samples to maximum temperatures of 1100–1700 °C.
Courtesy of Fisher Scientific.

desiccator A closed container containing a desiccant; used to store samples in a moisture-free environment. desiccant A drying agent.

(a)

(b)

Figure 2.9
(a) Desiccator. (b) Desiccator with stopcock for evacuating the desiccator.
Photos courtesy of Fisher Scientific.

30

Modern Analytical Chemistry

2E Preparing Solutions
Preparing a solution of known concentration is perhaps the most common activity in any analytical lab. The method for measuring out the solute and solvent depend on the desired concentration units, and how exact the solution’s concentration needs to be known. Pipets and volumetric flasks are used when a solution’s concentration must be exact; graduated cylinders, beakers, and reagent bottles suffice when concentrations need only be approximate. Two methods for preparing solutions are described in this section.

2E.1 Preparing Stock Solutions stock solution A solution of known concentration from which other solutions are prepared.

A stock solution is prepared by weighing out an appropriate portion of a pure solid or by measuring out an appropriate volume of a pure liquid and diluting to a known volume. Exactly how this is done depends on the required concentration units. For example, to prepare a solution with a desired molarity you would weigh out an appropriate mass of the reagent, dissolve it in a portion of solvent, and bring to the desired volume. To prepare a solution where the solute’s concentration is given as a volume percent, you would measure out an appropriate volume of solute and add sufficient solvent to obtain the desired total volume. EXAMPLE 2.9 Describe how you would prepare the following three solutions: (a) 500 mL of approximately 0.20 M NaOH using solid NaOH; (b) 1 L of 150.0 ppm Cu2+ using Cu metal; and (c) 2 L of 4% v/v acetic acid using concentrated glacial acetic acid. SOLUTION (a) Since the concentration only needs to be known to two significant figures, the mass of NaOH and volume of solution do not need to be measured exactly. The desired mass of NaOH is 0.20 mol 40.0 g × × 0.50 L = 4.0 g L mol To prepare the solution we place 4.0 g of NaOH, weighed to the nearest tenth of a gram, in a bottle or beaker and add approximately 500 mL of water. (b) Since the concentration of Cu2+ needs to be exact, the mass of Cu metal and the final solution volume must be measured exactly. The desired mass of Cu metal is 150.0 mg × 1.000 L = 150.0 mg = 0.1500 g L To prepare the solution we measure out exactly 0.1500 g of Cu into a small beaker. To dissolve the Cu we add a small portion of concentrated HNO3 and gently heat until it completely dissolves. The resulting solution is poured into a 1-L volumetric flask. The beaker is rinsed repeatedly with small portions of water, which are added to the volumetric flask. This process, which is called a quantitative transfer, ensures that the Cu2+ is completely transferred to the volumetric flask. Finally, additional water is added to the volumetric flask’s calibration mark.

quantitative transfer The process of moving a sample from one container to another in a manner that ensures all material is transferred.

Chapter 2 Basic Tools of Analytical Chemistry (c) The concentration of this solution is only approximate, so volumes do not need to be measured exactly. The necessary volume of glacial acetic acid is 4 mL CH3COOH × 2000 mL = 80 mL CH3COOH 100 mL To prepare the solution we use a graduated cylinder to transfer 80 mL of glacial acetic acid to a container that holds approximately 2 L, and we then add sufficient water to bring the solution to the desired volume.

31

2E.2 Preparing Solutions by Dilution
Solutions with small concentrations are often prepared by diluting a more concentrated stock solution. A known volume of the stock solution is transferred to a new container and brought to a new volume. Since the total amount of solute is the same before and after dilution, we know that Co × Vo = Cd × Vd 2.4

where Co is the concentration of the stock solution, Vo is the volume of the stock solution being diluted, Cd is the concentration of the dilute solution, and Vd is the volume of the dilute solution. Again, the type of glassware used to measure Vo and Vd depends on how exact the solution’s concentration must be known. EXAMPLE 2.10 A laboratory procedure calls for 250 mL of an approximately 0.10 M solution of NH3. Describe how you would prepare this solution using a stock solution of concentrated NH3 (14.8 M). SOLUTION Substituting known volumes in equation 2.4 14.8 M × Vo = 0.10 M × 0.25 L and solving for Vo gives 1.69 × 10–3 L, or 1.7 mL. Since we are trying to make a solution that is approximately 0.10 M NH3, we can measure the appropriate amount of concentrated NH3 using a graduated cylinder, transfer the NH3 to a beaker, and add sufficient water to bring the total solution volume to approximately 250 mL.

dilution The process of preparing a less concentrated solution from a more concentrated solution.

As shown in the following example, equation 2.4 also can be used to calculate a solution’s original concentration using its known concentration after dilution. EXAMPLE 2.11 A sample of an ore was analyzed for Cu2+ as follows. A 1.25-g sample of the ore was dissolved in acid and diluted to volume in a 250-mL volumetric flask. A 20-mL portion of the resulting solution was transferred by pipet to a 50-mL volumetric flask and diluted to volume. An analysis showed that the concentration of Cu2+ in the final solution was 4.62 ppm. What is the weight percent of Cu in the original ore?

32

Modern Analytical Chemistry SOLUTION Substituting known volumes (with significant figures appropriate for pipets and volumetric flasks) into equation 2.4 (ppm Cu2+)o × 20.00 mL = 4.62 ppm × 50.00 mL and solving for (ppm Cu2+)o gives the original solution concentration as 11.55 ppm. To calculate the grams of Cu2+ we multiply this concentration by the total volume 11.55 µg Cu2+ 1g × 250.0 mL × 6 = 2.888 × 10 –3 g Cu2+ mL 10 µg The weight percent Cu is then given by 2.888 × 10 –3 g Cu2+ × 100 = 0.231% w/w Cu 1.25 g sample

2F The Laboratory Notebook
Finally, we cannot end a chapter on the basic tools of analytical chemistry without mentioning the laboratory notebook. Your laboratory notebook is your most important tool when working in the lab, providing a complete record of all your work. If kept properly, you should be able to look back at your laboratory notebook several years from now and reconstruct the experiments on which you worked. Your instructor will probably provide you with detailed instructions on how he or she wants you to maintain your notebook. Of course, you should expect to bring your notebook to the lab. Everything you do, measure, or observe while working in the lab should be recorded in your notebook as it takes place. Preparing data tables to organize your data will help ensure that you record the data you need and that you can find the data when it is time to calculate and analyze your results. Writing a narrative to accompany your data will help you remember what you did, why you did it, and why you thought it was significant. Reserve space for your calculations, for analyzing your data, and for interpreting your results. Take your notebook with you when you do research in the library. Maintaining a laboratory notebook may seem like a great deal of effort, but if you do it well you have a permanent record of your work. Scientists working in academic, industrial, and governmental research labs rely on their notebooks to provide a written record of their work. Questions about research carried out at some time in the past can be answered by finding the appropriate pages in the laboratory notebook. A laboratory notebook is also a legal document that helps establish patent rights and proof of discovery.

2G

KEY TERMS dilution (p. 31) equivalent (p. 17) equivalent weight (p. 17) formality (p. 15) formula weight (p. 17) meniscus (p. 29) molality (p. 18) molarity (p. 15)

balance (p. 25) concentration (p. 15) desiccant (p. 29) desiccator (p. 29)

Chapter 2 Basic Tools of Analytical Chemistry normality (p. 16) parts per billion (p. 18) parts per million (p. 18) p-function (p. 19) pipet (p. 27) quantitative transfer (p. 30) scientific notation (p. 12) significant figures (p. 13) SI units (p. 12) stock solution (p. 30) volume percent (p. 18) volumetric flask (p. 26) weight percent (p. 18) weight-to-volume percent (p. 18)

33

2H SUMMARY
There are a few basic numerical and experimental tools with which you must be familiar. Fundamental measurements in analytical chemistry, such as mass and volume, use base SI units, such as the kilogram (kg) and the liter (L). Other units, such as power, are defined in terms of these base units. When reporting measurements, we must be careful to include only those digits that are significant and to maintain the uncertainty implied by these significant figures when transforming measurements into results. The relative amount of a constituent in a sample is expressed as its concentration. There are many ways to express concentration, the most common of which are molarity, weight percent, volume percent, weight-to-volume percent, parts per million, and parts per billion. Concentrations also can be expressed using p-functions. Stoichiometric relationships and calculations are important in many quantitative analyses. The stoichiometry between the reactants and products of a chemical reaction is given by the coefficients of a balanced chemical reaction. When it is inconvenient to balance reactions, conservation principles can be used to establish the stoichiometric relationships. Balances, volumetric flasks, pipets, and ovens are standard pieces of laboratory instrumentation and equipment that are routinely used in almost all analytical work. You should be familiar with the proper use of this equipment. You also should be familiar with how to prepare a stock solution of known concentration, and how to prepare a dilute solution from a stock solution.

2I PROBLEMS
1. Indicate how many significant figures are in each of the following numbers. a. 903 b. 0.903 c. 1.0903 d. 0.0903 e. 0.09030 f. 9.03 × 102 2. Round each of the following to three significant figures. a. 0.89377 b. 0.89328 c. 0.89350 d. 0.8997 e. 0.08907 3. Round each of the following to the stated number of significant figures. a. The atomic weight of carbon to four significant figures b. The atomic weight of oxygen to three significant figures c. Avogadro’s number to four significant figures d. Faraday’s constant to three significant figures 4. Report results for the following calculations to the correct number of significant figures. a. 4.591 + 0.2309 + 67.1 = b. 313 – 273.15 = c. 712 × 8.6 = d. 1.43/0.026 = e. (8.314 × 298)/96485 = f. log(6.53 × 10–5) = g. 10–7.14 = h. (6.51 × 10–5) (8.14 × 10–9) = 5. A 12.1374-g sample of an ore containing Ni and Co was carried through Fresenius’ analytical scheme shown in Figure 1.1. At point A the combined mass of Ni and Co was found to be 0.2306 g, and at point B the mass of Co was found to be 0.0813 g. Report the weight percent Ni in the ore to the correct number of significant figures. 6. Hillebrand and Lundell’s analytical scheme (see Figure 1.2) for the analysis of Ni in ores involves precipitating Ni2+ using dimethylgloxime. The formula for the precipitate is Ni(C4H7N2O2)2. Calculate the precipitate’s formula weight to the correct number of significant figures. 7. An analyst wishes to add 256 mg of Cl– to a reaction mixture. How many milliliters of 0.217 M BaCl2 should be added? 8. A solution of 0.10 M SO42– is available. What is the normality of this solution when used in the following reactions? a. Pb2+(aq) + SO42–(aq) PbSO4(s) b. HCl(aq) + SO42–(aq) HSO4–(aq) + Cl–(aq) 2– + 4H O+(aq) + 2e– c. SO4 H2SO3(aq) + 5H2O(l) 3

t t

t

9. The concentration of lead in an industrial waste stream is 0.28 ppm. What is its molar concentration? 10. Commercially available concentrated hydrochloric acid is 37.0% w/w HCl. Its density is 1.18 g/mL. Using this information calculate (a) the molarity of concentrated HCl, and (b) the mass and volume (in milliliters) of solution containing 0.315 mol of HCl. 11. The density of concentrated ammonia, which is 28.0% w/w NH3, is 0.899 g/mL. What volume of this reagent should be diluted to 1.0 × 103 mL to make a solution that is 0.036 M in NH3?

34

Modern Analytical Chemistry
18. A series of dilute NaCl solutions is prepared, starting with an initial stock solution of 0.100 M NaCl. Solution A is prepared by pipeting 10 mL of the stock solution into a 250-mL volumetric flask and diluting to volume. Solution B is prepared by pipeting 25 mL of solution A into a 100-mL volumetric flask and diluting to volume. Solution C is prepared by pipeting 20 mL of solution B into a 500-mL volumetric flask and diluting to volume. What is the molar concentration of NaCl in solutions A, B, and C? 19. Calculate the molar concentration of NaCl, to the correct number of significant figures, if 1.917 g of NaCl is placed in a beaker and dissolved in 50 mL of water measured with a graduated cylinder. This solution is quantitatively transferred to a 250-mL volumetric flask and diluted to volume. Calculate the concentration of this second solution to the correct number of significant figures. 20. What is the molar concentration of NO3– in a solution prepared by mixing 50.0 mL of 0.050 M KNO3 with 40.0 mL of 0.075 M NaNO3? What is pNO3 for the mixture? 21. What is the molar concentration of Cl– in a solution prepared by mixing 25.0 mL of 0.025 M NaCl with 35.0 mL of 0.050 M BaCl2? What is pCl for the mixture? 22. To determine the concentration of ethanol in cognac a 5.00-mL sample of cognac is diluted to 0.500 L. Analysis of the diluted cognac gives an ethanol concentration of 0.0844 M. What is the molar concentration of ethanol in the undiluted cognac?

12. A 250.0-mL aqueous solution contains 45.1 µg of a pesticide. Express the pesticide’s concentration in weight percent, parts per million, and parts per billion. 13. A city’s water supply is fluoridated by adding NaF. The desired concentration of F– is 1.6 ppm. How many milligrams of NaF should be added per gallon of treated water if the water supply already is 0.2 ppm in F–? 14. What is the pH of a solution for which the concentration of H+ is 6.92 × 10–6 M? What is the [H+] in a solution whose pH is 8.923? 15. Using conservation principles, write stoichiometric relationships for the following a. The precipitation of Mg2+ as Mg2P2O7 b. The acid–base reaction between CaCO3 and HCl in which H2CO3 is formed c. The reaction between AgCl and NH3 to form Ag(NH3)2+ d. The redox reaction between Cr2O72– and Fe2+ to form Cr3+ and Fe3+ 16. Calculate the molarity of a potassium dichromate solution prepared by placing 9.67 g of K2Cr2O7 in a 100-mL volumetric flask, dissolving, and diluting to the calibration mark. 17. For each of the following, explain how you would prepare 1.0 L of a solution that is 0.10 M in K+. Repeat for concentrations of 1.0 × 102 ppm K+ and 1.0% w/v K+. a. KCl b. K2SO4 c. K3Fe(CN)6

2J SUGGESTED READINGS
Two useful articles providing additional information on topics covered in this chapter are MacCarthy, P. “A Novel Classification of Concentration Units,” J. Chem. Educ. 1983, 60, 187–189. Schwartz, L. M. “Propagation of Significant Figures,” J. Chem. Educ. 1985, 62, 693–697. A useful resource for information on maintaining a useful laboratory notebook is Kanare, H. M. Writing the Laboratory Notebook, American Chemical Society: Washington, DC; 1985.

2K REFERENCES
1. American Public Health Association. Standard Methods for the Analysis of Waters and Wastewaters, 19th ed., Washington, DC. 1995.

3 Chapter
The Language of Analytical Chemistry

A

nalytical chemists converse using terminology that conveys specific meaning to other analytical chemists. To discuss and learn analytical chemistry you must first understand its language. You are probably already familiar with some analytical terms, such as “accuracy” and “precision,” but you may not have placed them in their appropriate analytical context. Other terms, such as “analyte” and “matrix,” may be less familiar. This chapter introduces many important terms routinely used by analytical chemists. Becoming comfortable with these terms will make the material in the chapters that follow easier to read and understand.

35

36

Modern Analytical Chemistry

3A Analysis, Determination, and Measurement analysis A process that provides chemical or physical information about the constituents in the sample or the sample itself. analytes The constituents of interest in a sample. matrix All other constituents in a sample except for the analytes. determination An analysis of a sample to find the identity, concentration, or properties of the analyte. measurement An experimental determination of an analyte’s chemical or physical properties.

The first important distinction we will make is among the terms “analysis,” “determination,” and “measurement.” An analysis provides chemical or physical information about a sample. The components of interest in the sample are called analytes, and the remainder of the sample is the matrix. In an analysis we determine the identity, concentration, or properties of the analytes. To make this determination we measure one or more of the analyte’s chemical or physical properties. An example helps clarify the differences among an analysis, a determination, and a measurement. In 1974, the federal government enacted the Safe Drinking Water Act to ensure the safety of public drinking water supplies. To comply with this act municipalities regularly monitor their drinking water supply for potentially harmful substances. One such substance is coliform bacteria. Municipal water departments collect and analyze samples from their water supply. To determine the concentration of coliform bacteria, a portion of water is passed through a membrane filter. The filter is placed in a dish containing a nutrient broth and incubated. At the end of the incubation period the number of coliform bacterial colonies in the dish is measured by counting (Figure 3.1). Thus, municipal water departments analyze samples of water to determine the concentration of coliform bacteria by measuring the number of bacterial colonies that form during a specified period of incubation.

3B Techniques, Methods, Procedures, and Protocols
Suppose you are asked to develop a way to determine the concentration of lead in drinking water. How would you approach this problem? To answer this question it helps to distinguish among four levels of analytical methodology: techniques, methods, procedures, and protocols.1 A technique is any chemical or physical principle that can be used to study an analyte. Many techniques have been used to determine lead levels.2 For example, in graphite furnace atomic absorption spectroscopy lead is atomized, and the ability of the free atoms to absorb light is measured; thus, both a chemical principle (atomization) and a physical principle (absorption of light) are used in this technique. Chapters 8–13 of this text cover techniques commonly used to analyze samples. A method is the application of a technique for the determination of a specific analyte in a specific matrix. As shown in Figure 3.2, the graphite furnace atomic absorption spectroscopic method for determining lead levels in water is different from that for the determination of lead in soil or blood. Choosing a method for determining lead in water depends on how the information is to be used and the established design criteria (Figure 3.3). For some analytical problems the best method might use graphite furnace atomic absorption spectroscopy, whereas other problems might be more easily solved by using another technique, such as anodic stripping voltammetry or potentiometry with a lead ion-selective electrode. A procedure is a set of written directions detailing how to apply a method to a particular sample, including information on proper sampling, handling of interferents, and validating results. A method does not necessarily lead to a single procedure, as different analysts or agencies will adapt the method to their specific needs. As shown in Figure 3.2, the American Public Health Agency and the American Society for Testing Materials publish separate procedures for the determination of lead levels in water.

technique A chemical or physical principle that can be used to analyze a sample.

method A means for analyzing a sample for a specific analyte in a specific matrix.

procedure Written directions outlining how to analyze a sample.

Chapter 3 The Language of Analytical Chemistry
Graphite furnace atomic absorption spectroscopy

37

Techniques

Methods

Pb in Water

Pb in Soil

Pb in Blood

Procedures

APHA

ASTM

Protocols

EPA

Figure 3.1
Membrane filter showing colonies of coliform bacteria. The number of colonies are counted and reported as colonies/100 mL of sample.
PourRite™ is a trademark of Hach Company/photo courtesy of Hach Company.

Figure 3.2
Chart showing hierarchical relationship among a technique, methods using that technique, and procedures and protocols for one method. (Abbreviations: APHA = American Public Health Association, ASTM = American Society for Testing Materials, EPA = Environmental Protection Agency)

Finally, a protocol is a set of stringent written guidelines detailing the procedure that must be followed if the agency specifying the protocol is to accept the results of the analysis. Protocols are commonly encountered when analytical chemistry is used to support or define public policy. For purposes of determining lead levels in water under the Safe Drinking Water Act, labs follow a protocol specified by the Environmental Protection Agency. There is an obvious order to these four facets of analytical methodology. Ideally, a protocol uses a previously validated procedure. Before developing and validating a procedure, a method of analysis must be selected. This requires, in turn, an initial screening of available techniques to determine those that have the potential for monitoring the analyte. We begin by considering a useful way to classify analytical techniques.

1. Identify the problem Determine type of information needed (qualitative, quantitative, or characterization) Identify context of the problem

2. Design the experimental procedure Establish design criteria (accuracy, precision, scale of operation, sensitivity, selectivity, cost, speed) Identify interferents Select method Establish validation criteria Establish sampling strategy

3C Classifying Analytical Techniques
Analyzing a sample generates a chemical or physical signal whose magnitude is proportional to the amount of analyte in the sample. The signal may be anything we can measure; common examples are mass, volume, and absorbance. For our purposes it is convenient to divide analytical techniques into two general classes based on whether this signal is proportional to an absolute amount of analyte or a relative amount of analyte. Consider two graduated cylinders, each containing 0.01 M Cu(NO3)2 (Figure 3.4). Cylinder 1 contains 10 mL, or 0.0001 mol, of Cu2+; cylinder 2 contains 20 mL, or 0.0002 mol, of Cu2+. If a technique responds to the absolute amount of analyte in the sample, then the signal due to the analyte, SA, can be expressed as SA = knA 3.1

Figure 3.3
Subsection of the analytical approach to problem solving (see Figure 1.3), of relevance to the selection of a method and the design of an analytical procedure.

where nA is the moles or grams of analyte in the sample, and k is a proportionality constant. Since cylinder 2 contains twice as many moles of Cu2+ as cylinder 1, analyzing the contents of cylinder 2 gives a signal that is twice that of cylinder 1.

protocol A set of written guidelines for analyzing a sample specified by an agency. signal An experimental measurement that is proportional to the amount of analyte (S).

38

Modern Analytical Chemistry A second class of analytical techniques are those that respond to the relative amount of analyte; thus SA = kCA 3.2

total analysis techniques A technique in which the signal is proportional to the absolute amount of analyte; also called “classical” techniques.

concentration techniques A technique in which the signal is proportional to the analyte’s concentration; also called “instrumental” techniques.

where CA is the concentration of analyte in the sample. Since the solutions in both cylinders have the same concentration of Cu2+, their analysis yields identical signals. Techniques responding to the absolute amount of analyte are called total analysis techniques. Historically, most early analytical methods used total analysis techniques, hence they are often referred to as “classical” techniques. Mass, volume, and charge are the most common signals for total analysis techniques, and the corresponding techniques are gravimetry (Chapter 8), titrimetry (Chapter 9), and coulometry (Chapter 11). With a few exceptions, the signal in a total analysis technique results from one or more chemical reactions involving the analyte. These reactions may involve any combination of precipitation, acid–base, complexation, or redox chemistry. The stoichiometry of each reaction, however, must be known to solve equation 3.1 for the moles of analyte. Techniques, such as spectroscopy (Chapter 10), potentiometry (Chapter 11), and voltammetry (Chapter 11), in which the signal is proportional to the relative amount of analyte in a sample are called concentration techniques. Since most concentration techniques rely on measuring an optical or electrical signal, they also are known as “instrumental” techniques. For a concentration technique, the relationship between the signal and the analyte is a theoretical function that depends on experimental conditions and the instrumentation used to measure the signal. For this reason the value of k in equation 3.2 must be determined experimentally.

3D Selecting an Analytical Method
A method is the application of a technique to a specific analyte in a specific matrix. Methods for determining the concentration of lead in drinking water can be developed using any of the techniques mentioned in the previous section. Insoluble lead salts such as PbSO4 and PbCrO4 can form the basis for a gravimetric method. Lead forms several soluble complexes that can be used in a complexation titrimetric method or, if the complexes are highly absorbing, in a spectrophotometric method. Lead in the gaseous free-atom state can be measured by an atomic absorption spectroscopic method. Finally, the availability of multiple oxidation states (Pb, Pb2+, Pb4+) makes coulometric, potentiometric, and voltammetric methods feasible. The requirements of the analysis determine the best method. In choosing a method, consideration is given to some or all the following design criteria: accuracy, precision, sensitivity, selectivity, robustness, ruggedness, scale of operation, analysis time, availability of equipment, and cost. Each of these criteria is considered in more detail in the following sections.

(a)

(b)

Figure 3.4
Graduated cylinders containing 0.01 M Cu(NO3)2. (a) Cylinder 1 contains 10 mL, or 0.0001 mol, of Cu2+. (b) Cylinder 2 contains 20 mL, or 0.0002 mol, of Cu2+.
© David Harvey/Marilyn Culler, photographer.

3D.1 Accuracy
Accuracy is a measure of how closely the result of an experiment agrees with the expected result. The difference between the obtained result and the expected result is usually divided by the expected result and reported as a percent relative error % Error = obtained result – expected result × 100 expected result

accuracy A measure of the agreement between an experimental result and its expected value.

Chapter 3 The Language of Analytical Chemistry Analytical methods may be divided into three groups based on the magnitude of their relative errors.3 When an experimental result is within 1% of the correct result, the analytical method is highly accurate. Methods resulting in relative errors between 1% and 5% are moderately accurate, but methods of low accuracy produce relative errors greater than 5%. The magnitude of a method’s relative error depends on how accurately the signal is measured, how accurately the value of k in equations 3.1 or 3.2 is known, and the ease of handling the sample without loss or contamination. In general, total analysis methods produce results of high accuracy, and concentration methods range from high to low accuracy. A more detailed discussion of accuracy is presented in Chapter 4.

39

5.8 (a)

5.9

6.0 ppm K+

6.1

6.2

5.8 (b)

5.9

6.0 ppm K+

6.1

6.2

Figure 3.5
Two determinations of the concentration of K+ in serum, showing the effect of precision. The data in (a) are less scattered and, therefore, more precise than the data in (b).

3D.2 Precision
When a sample is analyzed several times, the individual results are rarely the same. Instead, the results are randomly scattered. Precision is a measure of this variability. The closer the agreement between individual analyses, the more precise the results. For example, in determining the concentration of K+ in serum, the results shown in Figure 3.5(a) are more precise than those in Figure 3.5(b). It is important to realize that precision does not imply accuracy. That the data in Figure 3.5(a) are more precise does not mean that the first set of results is more accurate. In fact, both sets of results may be very inaccurate. As with accuracy, precision depends on those factors affecting the relationship between the signal and the analyte (equations 3.1 and 3.2). Of particular importance are the uncertainty in measuring the signal and the ease of handling samples reproducibly. In most cases the signal for a total analysis method can be measured with a higher precision than the corresponding signal for a concentration method. Precision is covered in more detail in Chapter 4.

precision An indication of the reproducibility of a measurement or result.

3D.3 Sensitivity
The ability to demonstrate that two samples have different amounts of analyte is an essential part of many analyses. A method’s sensitivity is a measure of its ability to establish that such differences are significant. Sensitivity is often confused with a method’s detection limit.4 The detection limit is the smallest amount of analyte that can be determined with confidence. The detection limit, therefore, is a statistical parameter and is discussed in Chapter 4. Sensitivity is the change in signal per unit change in the amount of analyte and is equivalent to the proportionality constant, k, in equations 3.1 and 3.2. If ∆SA is the smallest increment in signal that can be measured, then the smallest difference in the amount of analyte that can be detected is ∆nA = ∆C A = ∆S A k ∆S A k
(total analysis method) sensitivity A measure of a method’s ability to distinguish between two samples; reported as the change in signal per unit change in the amount of analyte (k). detection limit A statistical statement about the smallest amount of analyte that can be determined with confidence.

(concentration method)

Suppose that for a particular total analysis method the signal is a measurement of mass using a balance whose smallest increment is ±0.0001 g. If the method’s

40

Modern Analytical Chemistry sensitivity is 0.200, then the method can conceivably detect a difference of as little as ±0.0001 g ∆nA = = ±0.0005 g 0.200 in the absolute amount of analyte in two samples. For methods with the same ∆SA, the method with the greatest sensitivity is best able to discriminate among smaller amounts of analyte.

3D.4 Selectivity
An analytical method is selective if its signal is a function of only the amount of analyte present in the sample. In the presence of an interferent, equations 3.1 and 3.2 can be expanded to include a term corresponding to the interferent’s contribution to the signal, SI, Ssamp = SA + SI = kAnA + kInI Ssamp = SA + SI = kACA + kICI
(total analysis method) (concentration method)

3.3 3.4

selectivity A measure of a method’s freedom from interferences as defined by the method’s selectivity coefficient. selectivity coefficient A measure of a method’s sensitivity for an interferent relative to that for the analyte (KA,I).

where Ssamp is the total signal due to constituents in the sample; kA and kI are the sensitivities for the analyte and the interferent, respectively; and nI and CI are the moles (or grams) and concentration of the interferent in the sample. The selectivity of the method for the interferent relative to the analyte is defined by a selectivity coefficient, KA,I K A,I = kI kA 3.5

which may be positive or negative depending on whether the interferent’s effect on the signal is opposite that of the analyte.* A selectivity coefficient greater than +1 or less than –1 indicates that the method is more selective for the interferent than for the analyte. Solving equation 3.5 for kI kI = KA,I × kA substituting into equations 3.3 and 3.4, and simplifying gives Ssamp = kA(nA + KA,I × nI) Ssamp = kA(CA + KA,I × CI)
(total analysis method) (concentration method)

3.6

3.7 3.8

The selectivity coefficient is easy to calculate if kA and kI can be independently determined. It is also possible to calculate KA,I by measuring Ssamp in the presence and absence of known amounts of analyte and interferent. EXAMPLE 3.1 A method for the analysis of Ca2+ in water suffers from an interference in the presence of Zn2+. When the concentration of Ca2+ is 100 times greater than that of Zn2+, an analysis for Ca2+ gives a relative error of +0.5%. What is the selectivity coefficient for this method?

*Although kA and kI are usually positive, they also may be negative. For example, some analytical methods work by measuring the concentration of a species that reacts with the analyte. As the analyte’s concentration increases, the concentration of the species producing the signal decreases, and the signal becomes smaller. If the signal in the absence of analyte is assigned a value of zero, then the subsequent signals are negative.

Chapter 3 The Language of Analytical Chemistry SOLUTION Since only relative concentrations are reported, we can arbitrarily assign absolute concentrations. To make the calculations easy, let CCa = 100 (arbitrary units) and CZn = 1. A relative error of +0.5% means that the signal in the presence of Zn2+ is 0.5% greater than the signal in the absence of zinc. Again, we can assign values to make the calculation easier. If the signal in the absence of zinc is 100 (arbitrary units), then the signal in the presence of zinc is 100.5. The value of kCa is determined using equation 3.2 kCa = SCa 100 = =1 CCa 100

41

In the presence of zinc the signal is Ssamp = 100.5 = kCaCCa + kZnCZn = (1)(100) + kZn(1) Solving for kZn gives a value of 0.5. The selectivity coefficient, therefore, is KCa / Zn = 0.5 kZn = = 0.5 1 kCa

Knowing the selectivity coefficient provides a useful way to evaluate an interferent’s potential effect on an analysis. An interferent will not pose a problem as long as the term KA,I × nI in equation 3.7 is significantly smaller than nA, or KA,I × CI in equation 3.8 is significantly smaller than CA. EXAMPLE 3.2 Barnett and colleagues 5 developed a new method for determining the concentration of codeine during its extraction from poppy plants. As part of their study they determined the method’s response to codeine relative to that for several potential interferents. For example, the authors found that the method’s signal for 6-methoxycodeine was 6 (arbitrary units) when that for an equimolar solution of codeine was 40. (a) What is the value for the selectivity coefficient KA,I when 6-methoxycodeine is the interferent and codeine is the analyte? (b) If the concentration of codeine is to be determined with an accuracy of ±0.50%, what is the maximum relative concentration of 6-methoxycodeine (i.e., [6-methoxycodeine]/[codeine]) that can be present? SOLUTION (a) The signals due to the analyte, SA, and the interferent, SI, are SA = kACA SI = kICI

Solving these two expressions for kA and kI and substituting into equation 3.6 gives S /C K A,I = I I S A /CA

42

Modern Analytical Chemistry Since equimolar concentrations of analyte and interferent were used (CA = CI), we have K A,I = SI 6 = = 0.15 SA 40

(b) To achieve an accuracy of better than ±0.50% the term KA,I × CI in equation 3.8 must be less than 0.50% of CA; thus 0.0050 × CA ≥ KA,I × CI Solving this inequality for the ratio CI/CA and substituting the value for KA,I determined in part (a) gives CI 0.0050 0.0050 ≤ = = 0.033 CA K A,I 0.15 Therefore, the concentration of 6-methoxycodeine cannot exceed 3.3% of codeine’s concentration.

Not surprisingly, methods whose signals depend on chemical reactivity are often less selective and, therefore, more susceptible to interferences. Problems with selectivity become even greater when the analyte is present at a very low concentration.6

3D.5 Robustness and Ruggedness
For a method to be useful it must provide reliable results. Unfortunately, methods are subject to a variety of chemical and physical interferences that contribute uncertainty to the analysis. When a method is relatively free from chemical interferences, it can be applied to the determination of analytes in a wide variety of sample matrices. Such methods are considered robust. Random variations in experimental conditions also introduce uncertainty. If a method’s sensitivity is highly dependent on experimental conditions, such as temperature, acidity, or reaction time, then slight changes in those conditions may lead to significantly different results. A rugged method is relatively insensitive to changes in experimental conditions.

robust A method that can be applied to analytes in a wide variety of matrices is considered robust. rugged A method that is insensitive to changes in experimental conditions is considered rugged.

3D.6 Scale of Operation
Another way to narrow the choice of methods is to consider the scale on which the analysis must be conducted. Three limitations of particular importance are the amount of sample available for the analysis, the concentration of analyte in the sample, and the absolute amount of analyte needed to obtain a measurable signal. The first and second limitations define the scale of operations shown in Figure 3.6; the last limitation positions a method within the scale of operations.7 The scale of operations in Figure 3.6 shows the analyte’s concentration in weight percent on the y-axis and the sample’s size on the x-axis. For convenience, we divide analytes into major (>1% w/w), minor (0.01% w/w – 1% w/w), trace (10–7% w/w – 0.01% w/w) and ultratrace (0.1 g), meso (10 mg – 100 mg), micro (0.1 mg – 10 mg) and ultramicro ( µ – HA: X < µ
(c) Values

(a)

Values

(b)

Values

Figure 4.10
Examples of (a) two-tailed, (b) and (c) onetailed, significance tests. The shaded areas in each curve represent the values for which the null hypothesis is rejected.

two-tailed significance test Significance test in which the null hypothesis is rejected for values at either end of the normal distribution. one-tailed significance test Significance test in which the null hypothesis is rejected for values at only one end of the normal distribution.

for which the null hypothesis is rejected if µ falls within the shaded areas shown in Figure 4.10(b) and Figure 4.10(c), respectively. In each case the shaded area represents 5% of the area under the probability distribution curve. These are examples of one-tailed significance tests. For a fixed confidence level, a two-tailed test is always the more conservative test – because it requires a larger difference between X and µ to reject the null hypothesis. Most significance tests are applied when there is no a priori expectation about the relative magnitudes of the parameters being compared. A two-tailed significance test, therefore, is usually the appropriate choice. One-tailed significance tests are reserved for situations when we have reason to expect one parameter to be larger or smaller than the other. For example, a one-tailed significance test would be appropriate for our earlier example regarding a medication’s effect on blood glucose levels since we believe that the medication will lower the concentration of glucose.

4E.4 Errors in Significance Testing
Since significance tests are based on probabilities, their interpretation is naturally subject to error. As we have already seen, significance tests are carried out at a significance level, α, that defines the probability of rejecting a null hypothesis that is true. For example, when a significance test is conducted at α = 0.05, there is a 5% probability that the null hypothesis will be incorrectly rejected. This is known as a type 1 error, and its risk is always equivalent to α. Type 1 errors in two-tailed and one-tailed significance tests are represented by the shaded areas under the probability distribution curves in Figure 4.10. The second type of error occurs when the null hypothesis is retained even though it is false and should be rejected. This is known as a type 2 error, and its probability of occurrence is β. Unfortunately, in most cases β cannot be easily calculated or estimated.

type 1 error The risk of falsely rejecting the null hypothesis (α).

type 2 error The risk of falsely retaining the null hypothesis (β).

Chapter 4 Evaluating Analytical Data The probability of a type 1 error is inversely related to the probability of a type 2 error. Minimizing a type 1 error by decreasing α, for example, increases the likelihood of a type 2 error. The value of α chosen for a particular significance test, therefore, represents a compromise between these two types of error. Most of the examples in this text use a 95% confidence level, or α = 0.05, since this is the most frequently used confidence level for the majority of analytical work. It is not unusual, however, for more stringent (e.g. α = 0.01) or for more lenient (e.g. α = 0.10) confidence levels to be used. texps n

85

4F Statistical Methods for Normal Distributions
The most commonly encountered probability distribution is the normal, or Gaussian, distribution. A normal distribution is characterized by a true mean, µ, and vari– ance, σ2, which are estimated using X and s2. Since the area between any two limits of a normal distribution is well defined, the construction and evaluation of significance tests are straightforward.

X–

X+

texps n

(a)

X–

texps n

X+

texps n

– 4F.1 Comparing X to µ
One approach for validating a new analytical method is to analyze a standard sample containing a known amount of analyte, µ. The method’s accuracy is judged – by determining the average amount of analyte in several samples, X, and using – a significance test to compare it with µ. The null hypothesis is that X and µ are the same and that any difference between the two values can be explained by in– determinate errors affecting the determination of X . The alternative hypothesis is – that the difference between X and µ is too large to be explained by indeterminate error. The equation for the test (experimental) statistic, texp, is derived from the confidence interval for µ t exp s µ = X± 4.14 n Rearranging equation 4.14 t exp = µ−X × n s 4.15
(b)

t (α,ν)s X– n X+

t (α,ν)s n

X–

texps n

X+

texps n

t (α,ν)s X–
(c)

t (α,ν)s X+ n

n

Figure 4.11
Relationship between confidence intervals and results of a significance test. (a) The shaded area under the normal distribution curves shows the apparent confidence intervals for the sample based on texp. The solid bars in (b) and (c) show the actual confidence intervals that can be explained by indeterminate error using the critical value of (α,ν). In part (b) the null hypothesis is rejected and the alternative hypothesis is accepted. In part (c) the null hypothesis is retained.

gives the value of texp when µ is at either the right or left edge of the sample’s apparent confidence interval (Figure 4.11a). The value of texp is compared with a critical value, t(α ,ν), which is determined by the chosen significance level, α , the degrees of freedom for the sample, ν, and whether the significance test is onetailed or two-tailed. Values for t(α ,ν) are found in Appendix 1B. The critical value t(α ,ν) defines the confidence interval that can be explained by indeterminate errors. If texp is greater than t(α ,ν), then the confidence interval for the data is wider than that expected from indeterminate errors (Figure 4.11b). In this case, the null hypothesis is rejected and the alternative hypothesis is accepted. If texp is less than or equal to t(α ,ν), then the confidence interval for the data could be attributed to indeterminate error, and the null hypothesis is retained at the stated significance level (Figure 4.11c). – A typical application of this significance test, which is known as a t-test of X to µ, is outlined in the following example.

t-test Statistical test for comparing two mean values to see if their difference is too large to be explained by indeterminate error.

86

Modern Analytical Chemistry EXAMPLE 4.16 Before determining the amount of Na2CO3 in an unknown sample, a student decides to check her procedure by analyzing a sample known to contain 98.76% w/w Na2CO3. Five replicate determinations of the %w/w Na2CO3 in the standard were made with the following results 98.71% 98.59% 98.62% 98.44% 98.58%

Is the mean for these five trials significantly different from the accepted value at the 95% confidence level (α = 0.05)? SOLUTION The mean and standard deviation for the five trials are – X = 98.59 s = 0.0973 – Since there is no reason to believe that X must be either larger or smaller than µ, the use of a two-tailed significance test is appropriate. The null and alternative hypotheses are – – H0: X = µ HA: X ≠ µ The test statistic is t exp = µ−X × n s = 98.76 − 98.59 × 5 0.0973 = 3.91

The critical value for t(0.05,4), as found in Appendix 1B, is 2.78. Since texp is greater than t(0.05, 4), we must reject the null hypothesis and accept the alternative hypothesis. At the 95% confidence level the difference between – X and µ is significant and cannot be explained by indeterminate sources of error. There is evidence, therefore, that the results are affected by a determinate source of error.

If evidence for a determinate error is found, as in Example 4.16, its source should be identified and corrected before analyzing additional samples. Failing to reject the null hypothesis, however, does not imply that the method is accurate, but only indicates that there is insufficient evidence to prove the method inaccurate at the stated confidence level. – The utility of the t-test for X and µ is improved by optimizing the conditions – used in determining X. Examining equation 4.15 shows that increasing the number of replicate determinations, n, or improving the precision of the analysis enhances the utility of this significance test. A t-test can only give useful results, however, if the standard deviation for the analysis is reasonable. If the standard deviation is substantially larger than the expected standard deviation, σ, the con– fidence interval around X will be so large that a significant difference between – X and µ may be difficult to prove. On the other hand, if the standard deviation is – significantly smaller than expected, the confidence interval around X will be too – small, and a significant difference between X and µ may be found when none exists. A significance test that can be used to evaluate the standard deviation is the subject of the next section.

Chapter 4 Evaluating Analytical Data

87

4F.2 Comparing s2 to σ2
When a particular type of sample is analyzed on a regular basis, it may be possible to determine the expected, or true variance, σ2, for the analysis. This often is the case in clinical labs where hundreds of blood samples are analyzed each day. Replicate analyses of any single sample, however, results in a sample variance, s2. A statistical comparison of s2 to σ2 provides useful information about whether the analysis is in a state of “statistical control.” The null hypothesis is that s2 and σ2 are identical, and the alternative hypothesis is that they are not identical. The test statistic for evaluating the null hypothesis is called an F-test, and is given as either s2 σ2 Fexp = 2 or Fexp = 2 σ s 4.16
(s2 > σ2) (σ2 > s2)

F-test Statistical test for comparing two variances to see if their difference is too large to be explained by indeterminate error.

depending on whether s2 is larger or smaller than σ2. Note that Fexp is defined such that its value is always greater than or equal to 1. If the null hypothesis is true, then Fexp should equal 1. Due to indeterminate errors, however, the value for Fexp usually is greater than 1. A critical value, F(α, νnum, νden), gives the largest value of F that can be explained by indeterminate error. It is chosen for a specified significance level, α, and the degrees of freedom for the variances in the numerator, νnum, and denominator, νden. The degrees of freedom for s2 is n – 1, where n is the number of replicates used in determining the sample’s variance. Critical values of F for α = 0.05 are listed in Appendix 1C for both one-tailed and two-tailed significance tests. EXAMPLE 4.17 A manufacturer’s process for analyzing aspirin tablets has a known variance of 25. A sample of ten aspirin tablets is selected and analyzed for the amount of aspirin, yielding the following results 254 249 252 252 249 249 250 247 251 252

Determine whether there is any evidence that the measurement process is not under statistical control at α = 0.05. SOLUTION The variance for the sample of ten tablets is 4.3. A two-tailed significance test is used since the measurement process is considered out of statistical control if the sample’s variance is either too good or too poor. The null hypothesis and alternative hypotheses are H0: The test statistic is Fexp = s2 = σ2 HA: s2 ≠ σ2

σ2 25 = = 5.8 2 4.3 s

The critical value for F(0.05, ∞, 9) from Appendix 1C is 3.33. Since F is greater than F(0.05,∞, 9), we reject the null hypothesis and accept the alternative hypothesis that the analysis is not under statistical control. One explanation for the unreasonably small variance could be that the aspirin tablets were not selected randomly.

88

Modern Analytical Chemistry

4F.3 Comparing Two Sample Variances
The F-test can be extended to the comparison of variances for two samples, A and B, by rewriting equation 4.16 as Fexp =
2 sA 2 sB

2 2 where A and B are defined such that sA is greater than or equal to sB. An example of this application of the F-test is shown in the following example.

EXAMPLE 4.18 Tables 4.1 and 4.8 show results for two separate experiments to determine the mass of a circulating U.S. penny. Determine whether there is a difference in the precisions of these analyses at α = 0.05. SOLUTION Letting A represent the results in Table 4.1 and B represent the results in Table 2 2 4.8, we find that the variances are sA = 0.00259 and sB = 0.00138. A two-tailed significance test is used since there is no reason to suspect that the results for one analysis will be more precise than that of the other. The null and alternative hypotheses are H0: and the test statistic is Fexp =
2 sA 0.00259 = = 1.88 2 0.00138 sB 2 2 sA = sB

HA:

2 2 sA ≠ sB

The critical value for F(0.05, 6, 4) is 9.197. Since Fexp is less than F(0.05, 6, 4), the null hypothesis is retained. There is no evidence at the chosen significance level to suggest that the difference in precisions is significant.

4F.4 Comparing Two Sample Means
The result of an analysis is influenced by three factors: the method, the sample, and the analyst. The influence of these factors can be studied by conducting a pair of experiments in which only one factor is changed. For example, two methods can be compared by having the same analyst apply both methods to the same sample and examining the resulting means. In a similar fashion, it is possible to compare two analysts or two samples. Significance testing for comparing two mean values is divided into two categories depending on the source of the data. Data are said to be unpaired when each mean is derived from the analysis of several samples drawn from the same source. Paired data are encountered when analyzing a series of samples drawn from different sources. – – Unpaired Data Consider two samples, A and B, for which mean values, XA and XB, and standard deviations, sA and sB, have been measured. Confidence intervals for µA and µB can be written for both samples

unpaired data Two sets of data consisting of results obtained using several samples drawn from a single source. paired data Two sets of data consisting of results obtained using several samples drawn from different sources.

Chapter 4 Evaluating Analytical Data µA = XA ± ts A nA tsB nB 4.18 4.17

89

µB = XB ±

where nA and nB are the number of replicate trials conducted on samples A and B. A – – comparison of the mean values is based on the null hypothesis that XA and XB are identical, and an alternative hypothesis that the means are significantly different. A test statistic is derived by letting µA equal µB, and combining equations 4.17 and 4.18 to give ts ts X A ± A = XB ± B nA nB – – Solving for  XA – XB and using a propagation of uncertainty, gives  X A − XB = t ×
2 sA s2 + B n A nB

Finally, solving for t, which we replace with texp, leaves us with t exp = X A − XB
2 (s 2 / nA ) + (s B / nB ) A

4.19

The value of texp is compared with a critical value, t(α, ν), as determined by the chosen significance level, α, the degrees of freedom for the sample, ν, and whether the significance test is one-tailed or two-tailed. It is unclear, however, how many degrees of freedom are associated with t(α, ν) 2 2 since there are two sets of independent measurements. If the variances sA and sB esti2, then the two standard deviations can be factored out of equation mate the same σ 4.19 and replaced by a pooled standard deviation, spool, which provides a better estimate for the precision of the analysis. Thus, equation 4.19 becomes t exp = X A − XB s pool (1/ nA ) + (1/ nB ) 4.20

with the pooled standard deviation given as spool =
2 2 (n A − 1)s A + (nB − 1)sB n A + nB − 2

4.21

As indicated by the denominator of equation 4.21, the degrees of freedom for the pooled standard deviation is nA + nB – 2. If sA and sB are significantly different, however, then texp must be calculated using equation 4.19. In this case, the degrees of freedom is calculated using the following imposing equation. ν=
2 2 [(s A /nA ) + (s B /nB )]2 –2 2 [(s 2 /nA )2 /(nA + 1)] + [(s B /nB )2 /(nB + 1)] A

4.22

Since the degrees of freedom must be an integer, the value of ν obtained using equation 4.22 is rounded to the nearest integer. Regardless of whether equation 4.19 or 4.20 is used to calculate texp, the null hypothesis is rejected if texp is greater than t(α, ν), and retained if texp is less than or equal to t(α, ν).

90

Modern Analytical Chemistry EXAMPLE 4.19 Tables 4.1 and 4.8 show results for two separate experiments to determine the mass of a circulating U.S. penny. Determine whether there is a difference in the means of these analyses at α = 0.05. SOLUTION To begin with, we must determine whether the variances for the two analyses are significantly different. This is done using an F-test as outlined in Example 4.18. Since no significant difference was found, a pooled standard deviation with 10 degrees of freedom is calculated s pool = =
2 2 (nA − 1)s A + (nB − 1)s B nA + nB − 2

(7 − 1)(0.00259) + (5 − 1)(0.00138) 7+5−2

= 0.0459 where the subscript A indicates the data in Table 4.1, and the subscript B indicates the data in Table 4.8. The comparison of the means for the two analyses is based on the null hypothesis – – H0: XA = XB and a two-tailed alternative hypothesis HA: – – XA ≠ XB

Since the standard deviations can be pooled, the test statistic is calculated using equation 4.20 t exp = X A − XB s pool (1/ nA + 1/ nB ) = 3.117 − 3.081 0.0459 (1/ 7 + 1/ 5) = 1.34

The critical value for t(0.05, 10), from Appendix 1B, is 2.23. Since texp is less than t(0.05, 10) the null hypothesis is retained, and there is no evidence that the two sets of pennies are significantly different at the chosen significance level.

EXAMPLE 4.20 The %w/w Na2CO3 in soda ash can be determined by an acid–base titration. The results obtained by two analysts are shown here. Determine whether the difference in their mean values is significant at α = 0.05.
Analyst A
86.82 87.04 86.93 87.01 86.20 87.00

Analyst B
81.01 86.15 81.73 83.19 80.27 83.94

Chapter 4 Evaluating Analytical Data SOLUTION We begin by summarizing the mean and standard deviation for the data reported by each analyst. These values are – XA = 86.83% s = 0.32 –A XB = 82.71% sB = 2.16 A two-tailed F-test of the following null and alternative hypotheses H0:
2 2 sA = sB 2 2 HA: sA ≠ sB

91

is used to determine whether a pooled standard deviation can be calculated. The test statistic is Fexp =
2 sB (2.16)2 = = 45.6 2 sA (0.32)2

Since Fexp is larger than the critical value of 7.15 for F(0.05, 5, 5), the null hypothesis is rejected and the alternative hypothesis that the variances are significantly different is accepted. As a result, a pooled standard deviation cannot be calculated. The mean values obtained by the two analysts are compared using a twotailed t-test. The null and alternative hypotheses are – – – – H0: XA = XB HA: XA ≠ XB Since a pooled standard deviation could not be calculated, the test statistic, texp, is calculated using equation 4.19 t exp = X A − XB
2 (s 2 / nA ) + (s B / nB ) A

=

86.83 − 82.71 [(0.32)2 /6] + [(2.16)2 /6]

= 4.62

and the degrees of freedom are calculated using equation 4.22 ν= {(0.322 /6)2 /(6 [(0.322 /6) + (2.162 /6)]2 − 2 = 5.3 ≈ 5 + 1)} + {(2.162 /6)2 /(6 + 1)}

The critical value for t(0.05, 5) is 2.57. Since the calculated value of texp is greater than t(0.05, 5) we reject the null hypothesis and accept the alternative hypothesis that the mean values for %w/w Na2 CO 3 reported by the two analysts are significantly different at the chosen significance level.

Paired Data In some situations the variation within the data sets being compared is more significant than the difference between the means of the two data sets. This is commonly encountered in clinical and environmental studies, where the data being compared usually consist of a set of samples drawn from several populations. For example, a study designed to investigate two procedures for monitoring the concentration of glucose in blood might involve blood samples drawn from ten patients. If the variation in the blood glucose levels among the patients is significantly larger than the anticipated variation between the methods, then an analysis in which the data are treated as unpaired will fail to find a significant difference between the

92

Modern Analytical Chemistry methods. In general, paired data sets are used whenever the variation being investigated is smaller than other potential sources of variation. In a study involving paired data the difference, di, between the paired values for – each sample is calculated. The average difference, d, and standard deviation of the – differences, sd, are then calculated. The null hypothesis is that d is 0, and that there is no difference in the results for the two data sets. The alternative hypothesis is that – the results for the two sets of data are significantly different, and, therefore, d is not equal to 0. – The test statistic, texp, is derived from a confidence interval around d 0=d ± tsd n where n is the number of paired samples. Replacing t with texp and rearranging gives t exp = d n sd

paired t-test Statistical test for comparing paired data to determine if their difference is too large to be explained by indeterminate error.

The value of texp is then compared with a critical value, t(α, ν), which is determined by the chosen significance level, α, the degrees of freedom for the sample, ν, and whether the significance test is one-tailed or two-tailed. For paired data, the degrees of freedom is n – 1. If texp is greater than t(α, ν), then the null hypothesis is rejected and the alternative hypothesis is accepted. If texp is less than or equal to t(α, ν), then the null hypothesis is retained, and a significant difference has not been demonstrated at the stated significance level. This is known as the paired t-test. EXAMPLE 4.21 Marecek and colleagues developed a new electrochemical method for the rapid quantitative analysis of the antibiotic monensin in the fermentation vats used during its production.9 The standard method for the analysis, which is based on a test for microbiological activity, is both difficult and time-consuming. As part of the study, samples taken at different times from a fermentation production vat were analyzed for the concentration of monensin using both the electrochemical and microbiological procedures. The results, in parts per thousand (ppt),* are reported in the following table.
Sample
1 2 3 4 5 6 7 8 9 10 11

Microbiological
129.5 89.6 76.6 52.2 110.8 50.4 72.4 141.4 75.0 34.1 60.3

Electrochemical
132.3 91.0 73.6 58.2 104.2 49.9 82.1 154.1 73.4 38.1 60.1

Determine whether there is a significant difference between the methods at α = 0.05.
*1 ppt is equivalent to 0.1%.

Chapter 4 Evaluating Analytical Data SOLUTION This is an example of a paired data set since the acquisition of samples over an extended period introduces a substantial time-dependent change in the concentration of monensin. The comparison of the two methods must be done with the paired t-test, using the following null and two-tailed alternative hypotheses – – H0: d = 0 HA: d ≠ 0 Defining the difference between the methods as d = Xelect – Xmicro we can calculate the difference for each sample Sample d 1 2.8 2 1.4 3 –3.0 4 6.0 5 –6.6 6 –0.5 7 9.7 8 12.7 9 –1.6 10 4.0 11 –0.2

93

The mean and standard deviation for the differences are 2.25 and 5.63, respectively. The test statistic is t exp = d n sd = 2.25 11 5.63 = 1.33

which is smaller than the critical value of 2.23 for t(0.05, 10). Thus, the null hypothesis is retained, and there is no evidence that the two methods yield different results at the stated significance level.

A paired t-test can only be applied when the individual differences, di, belong to the same population. This will only be true if the determinate and indeterminate errors affecting the results are independent of the concentration of analyte in the samples. If this is not the case, a single sample with a larger error could result in a value of di that is substantially larger than that for the remaining samples. Including – this sample in the calculation of d and sd leads to a biased estimate of the true mean and standard deviation. For samples that span a limited range of analyte concentrations, such as that in Example 4.21, this is rarely a problem. When paired data span a wide range of concentrations, however, the magnitude of the determinate and indeterminate sources of error may not be independent of the analyte’s concentration. In such cases the paired t-test may give misleading results since the paired data – with the largest absolute determinate and indeterminate errors will dominate d. In this situation a comparison is best made using a linear regression, details of which are discussed in the next chapter.

4F.5 Outliers
On occasion, a data set appears to be skewed by the presence of one or more data points that are not consistent with the remaining data points. Such values are called outliers. The most commonly used significance test for identifying outliers is Dixon’s Q-test. The null hypothesis is that the apparent outlier is taken from the same population as the remaining data. The alternative hypothesis is that the outlier comes from a different population, and, therefore, should be excluded from consideration. The Q-test compares the difference between the suspected outlier and its nearest numerical neighbor to the range of the entire data set. Data are ranked from smallest to largest so that the suspected outlier is either the first or the last data outlier Data point whose value is much larger or smaller than the remaining data. Dixon’s Q-test Statistical test for deciding if an outlier can be removed from a set of data.

94

Modern Analytical Chemistry point. The test statistic, Qexp, is calculated using equation 4.23 if the suspected outlier is the smallest value (X1) Qexp = X 2 − X1 X n − X1 X n − Xn −1 X n − X1 4.23

or using equation 4.24 if the suspected outlier is the largest value (Xn) Qexp = 4.24

where n is the number of members in the data set, including the suspected outlier. It is important to note that equations 4.23 and 4.24 are valid only for the detection of a single outlier. Other forms of Dixon’s Q-test allow its extension to the detection of multiple outliers.10 The value of Qexp is compared with a critical value, Q(α, n), at a significance level of α. The Q-test is usually applied as the more conservative twotailed test, even though the outlier is the smallest or largest value in the data set. Values for Q(α, n) can be found in Appendix 1D. If Qexp is greater than Q(α, n), then the null hypothesis is rejected and the outlier may be rejected. When Qexp is less than or equal to Q(α, n) the suspected outlier must be retained. EXAMPLE 4.22 The following masses, in grams, were recorded in an experiment to determine the average mass of a U.S. penny. 3.067 3.049 3.039 2.514 3.048 3.079 3.094 3.109 3.102 Determine if the value of 2.514 g is an outlier at α = 0.05. SOLUTION To begin with, place the masses in order from smallest to largest 2.514 3.039 3.048 3.049 3.067 3.079 3.094 3.102 3.109

and calculate Qexp Qexp = 3.039 − 2.514 X 2 − X1 = = 0.882 3.109 − 2.514 X 9 − X1

The critical value for Q(0.05, 9) is 0.493. Since Qexp > Q(0.05, 9) the value is assumed to be an outlier, and can be rejected.

The Q-test should be applied with caution since there is a probability, equal to α, that an outlier identified by the Q-test actually is not an outlier. In addition, the Q-test should be avoided when rejecting an outlier leads to a precision that is unreasonably better than the expected precision determined by a propagation of uncertainty. Given these two concerns it is not surprising that some statisticians caution against the removal of outliers.11 On the other hand, testing for outliers can provide useful information if we try to understand the source of the suspected outlier. For example, the outlier identified in Example 4.22 represents a significant change in the mass of a penny (an approximately 17% decrease in mass), due to a change in the composition of the U.S. penny. In 1982, the composition of a U.S. penny was changed from a brass alloy consisting of 95% w/w Cu and 5% w/w Zn, to a zinc core covered with copper.12 The pennies in Example 4.22 were therefore drawn from different populations.

Chapter 4 Evaluating Analytical Data

95

4G Detection Limits
The focus of this chapter has been the evaluation of analytical data, including the use of statistics. In this final section we consider how statistics may be used to characterize a method’s ability to detect trace amounts of an analyte. A method’s detection limit is the smallest amount or concentration of analyte that can be detected with statistical confidence. The International Union of Pure and Applied Chemistry (IUPAC) defines the detection limit as the smallest concentration or absolute amount of analyte that has a signal significantly larger than the signal arising from a reagent blank. Mathematically, the analyte’s signal at the detection limit, (SA)DL, is (SA)DL = Sreag + zσreag 4.25 where Sreag is the signal for a reagent blank, σreag is the known standard deviation for the reagent blank’s signal, and z is a factor accounting for the desired confidence level. The concentration, (C A ) DL , or absolute amount of analyte, (nA)DL, at the detection limit can be determined from the signal at the detection limit. (C A )DL (S ) = A DL k (S ) = A DL k

detection limit The smallest concentration or absolute amount of analyte that can be reliably detected.

Probability distribution for blank

(a)

Sreag

(SA)DL

Probability distribution for blank

(n A )DL

Probability distribution for sample

The value for z depends on the desired significance level for reporting the detection limit. Typically, z is set to 3, which, from Appendix 1A, corresponds to a significance level of α = 0.00135. Consequently, only 0.135% of measurements made on the blank will yield signals that fall outside this range (Figure 4.12a). When σreag is unknown, the term zσreag may be replaced with tsreag, where t is the appropriate value from a t-table for a one-tailed analysis.13 In analyzing a sample to determine whether an analyte is present, the signal for the sample is compared with the signal for the blank. The null hypothesis is that the sample does not contain any analyte, in which case (SA)DL and Sreag are identical. The alternative hypothesis is that the analyte is present, and (SA)DL is greater than Sreag. If (SA)DL exceeds Sreag by zσ(or ts), then the null hypothesis is rejected and there is evidence for the analyte’s presence in the sample. The probability that the null hypothesis will be falsely rejected, a type 1 error, is the same as the significance level. Selecting z to be 3 minimizes the probability of a type 1 error to 0.135%. Significance tests, however, also are subject to type 2 errors in which the null hypothesis is falsely retained. Consider, for example, the situation shown in Figure 4.12b, where SA is exactly equal to (SA)DL. In this case the probability of a type 2 error is 50% since half of the signals arising from the sample’s population fall below the detection limit. Thus, there is only a 50:50 probability that an analyte at the IUPAC detection limit will be detected. As defined, the IUPAC definition for the detection limit only indicates the smallest signal for which we can say, at a significance level of α, that an analyte is present in the sample. Failing to detect the analyte, however, does not imply that it is not present. An alternative expression for the detection limit, which minimizes both type 1 and type 2 errors, is the limit of identification, (SA)LOI, which is defined as 14 (SA)LOI = Sreag + zσreag + zσsamp

(b)

Sreag

(SA)DL Probability distribution for sample

Probability distribution for blank

(c)

Sreag

(SA)LOI

Figure 4.12
Normal distribution curves showing the definition of detection limit and limit of identification (LOI). The probability of a type 1 error is indicated by the dark shading, and the probability of a type 2 error is indicated by light shading.

limit of identification The smallest concentration or absolute amount of analyte such that the probability of type 1 and type 2 errors are equal (LOI).

96

Modern Analytical Chemistry As shown in Figure 4.12c, the limit of identification is selected such that there is an equal probability of type 1 and type 2 errors. The American Chemical Society’s Committee on Environmental Analytical Chemistry recommends the limit of quantitation, (SA)LOQ, which is defined as15 (SA)LOQ = Sreag + 10σreag Other approaches for defining the detection limit have also been developed.16 The detection limit is often represented, particularly when used in debates over public policy issues, as a distinct line separating analytes that can be detected from those that cannot be detected.17 This use of a detection limit is incorrect. Defining the detection limit in terms of statistical confidence levels implies that there may be a gray area where the analyte is sometimes detected and sometimes not detected. This is shown in Figure 4.13 where the upper and lower confidence limits are defined by the acceptable probabilities for type 1 and type 2 errors. Analytes producing signals greater than that defined by the upper confidence limit are always detected, and analytes giving signals smaller than the lower confidence limit are never detected. Signals falling between the upper and lower confidence limits, however, are ambiguous because they could belong to populations representing either the reagent blank or the analyte. Figure 4.12c represents the smallest value of SA for which no such ambiguity exists.

limit of quantitation The smallest concentration or absolute amount of analyte that can be reliably determined (LOQ).

Sreag

SA

Never detected

???

Always detected

Lower confidence interval

Upper confidence interval

Figure 4.13
Establishment of areas where the signal is never detected, always detected, and where results are ambiguous. The upper and lower confidence limits are defined by the probability of a type 1 error (dark shading), and the probability of a type 2 error (light shading).

4H KEY TERMS alternative hypothesis (p. 83) binomial distribution (p. 72) central limit theorem (p. 79) confidence interval (p. 75) constant determinate error (p. 60) degrees of freedom (p. 80) detection limit (p. 95) determinate error (p. 58) Dixon’s Q-test (p. 93) error (p. 64) F-test (p. 87) heterogeneous (p. 58) histogram (p. 77) homogeneous (p. 72) indeterminate error (p. 62) limit of identification (p. 95) limit of quantitation (p. 96) mean (p. 54) measurement error (p. 58) median (p. 55) method error (p. 58) normal distribution (p. 73) null hypothesis (p. 83) one-tailed significance test (p. 84) outlier (p. 93) paired data (p. 88) paired t-test (p. 92) personal error (p. 60) population (p. 71) probability distribution (p. 71) proportional determinate error (p. 61) range (p. 56) repeatability (p. 62) reproducibility (p. 62) sample (p. 71) sampling error (p. 58) significance test (p. 83) standard deviation (p. 56) standard reference material (p. 61) tolerance (p. 58) t-test (p. 85) two-tailed significance test (p. 84) type 1 error (p. 84) type 2 error (p. 84) uncertainty (p. 64) unpaired data (p. 88) variance (p. 57)

4I SUMMARY
The data we collect are characterized by their central tendency (where the values are clustered), and their spread (the variation of individual values around the central value). Central tendency is reported by stating the mean or median. The range, standard deviation, or variance may be used to report the data’s spread. Data also are characterized by their errors, which include determinate errors affecting the data’s accuracy, and indeterminate errors affecting the data’s precision. A propagation of uncertainty allows us to estimate the affect of these determinate and indeterminate errors on results determined from our data. The distribution of the results of an analysis around a central value is often described by a probability distribution, two examples

Chapter 4 Evaluating Analytical Data of which are the binomial distribution and the normal distribution. Knowing the type of distribution allows us to determine the probability of obtaining results within a specified range. For a normal distribution this range is best expressed as a confidence interval. A statistical analysis allows us to determine whether our results are significantly different from known values, or from values obtained by other analysts, by other methods of analysis, or for other samples. A t-test is used to compare mean values, and an F-test to compare precisions. Comparisons between two sets of data require an initial evaluation of whether the data

97

is paired or unpaired. For unpaired data it is also necessary to decide if the standard deviations can be pooled. A decision about whether to retain an outlying value can be made using Dixon’s Q-test. Finally, we have seen that the detection limit is a statistical statement about the smallest amount of analyte that can be detected with confidence. A detection limit is not exact because its value depends on how willing we are to falsely report the analyte’s presence or absence in a sample. When reporting a detection limit, you should clearly indicate how you arrived at its value.

4J

Suggested EXPERIMENTS
The following experiments may be used to introduce the statistical analysis of data in the analytical chemistry laboratory. Each experiment is annotated with a brief description of the data collected and the type of statistical analysis used in evaluating the data. Cunningham, C. C.; Brown, G. R.; St Pierre, L. E. “Evaluation of Experimental Data,” J. Chem. Educ. 1981, 58, 509–511. In this experiment students determine the density of glass marbles and the radius of the bore of a glass capillary tube. Density is determined by measuring a marble’s mass and volume, the latter by measuring a marble’s diameter and assuming a spherical shape. Results are compared with those expected for a normal distribution. The radius of a glass capillary tube is determined using Poiseuille’s equation by measuring the volume flow rate of water as a function of the hydrostatic head. In both experiments the experimentally obtained standard deviation is compared with that estimated by a propagation of uncertainty. Gordus, A. A. “Statistical Evaluation of Class Data for Two Buret Readings,” J. Chem. Educ. 1987, 64, 376–377. The volumes of water in two burets are read, and the difference between the volumes are calculated. Students analyze the data by drawing histograms for each of the three volumes, comparing results with those predicted for a normal distribution. Harvey, D. T. “Statistical Evaluation of Acid/Base Indicators,” J. Chem. Educ. 1991, 68, 329–331. In this experiment students standardize a solution of HCl by titration using several different indicators to signal the titration’s end point. A statistical analysis of the data using ttests and F-tests allows students to compare results obtained using the same indicator, with results obtained using different indicators. The results of this experiment can be used later when discussing the selection of appropriate indicators. O’Reilley, J. E. “The Length of a Pestle,” J. Chem. Educ. 1986, 63, 894–896. In this experiment students measure the length of a pestle using a wooden meter stick, a stainless-steel ruler, and a vernier caliper. The data collected in this experiment provide an opportunity to discuss significant figures and sources of error. Statistical analysis includes the Q-test, t-test, and F-test. Paselk, R. A. “An Experiment for Introducing Statistics to Students of Analytical and Clinical Chemistry,” J. Chem. Educ. 1985, 62, 536. Students use a commercial diluter to prepare five sets of dilutions of a stock dye solution (each set contains ten replicates) using two different diluters. Results are compared using t-tests and F-tests. Richardson, T. H. “Reproducible Bad Data for Instruction in Statistical Methods,” J. Chem. Educ. 1991, 68, 310–311. This experiment uses the change in the mass of a U.S. penny to create data sets with outliers. Students are given a sample of ten pennies, nine of which are from one population. The Q-test is used to verify that the outlier can be rejected. Class data from each of the two populations of pennies are pooled and compared with results predicted for a normal distribution. Sheeran, D. “Copper Content in Synthetic Copper Carbonate: A Statistical Comparison of Experimental and Expected Results,” J. Chem. Educ. 1998, 75, 453–456. In this experiment students synthesize basic copper(II) carbonate and determine the %w/w Cu by reducing the copper to Cu. A statistical analysis of the results shows that the synthesis does not produce CuCO3, the compound that many predict to be the product (although it does not exist). Results are shown to be consistent with a hemihydrate of malachite, Cu2(OH)2(CO3) • 1/2H2O, or azurite, Cu3(OH)2(CO3)2. —Continued

Experiments

Experiments

98

Modern Analytical Chemistry

Experiments

Continued from page 97 Spencer, R. D. “The Dependence of Strength in Plastics upon Polymer Chain Length and Chain Orientation,” J. Chem. Educ. 1984, 61, 555–563. The stretching properties of polymers are investigated by examining the effect of polymer orientation, polymer chain length, stretching rate, and temperature. Homogeneity of polymer films and consistency between lots of polymer films also are investigated. Statistical analysis of data includes Qtests and t-tests. Thomasson, K.; Lofthus-Merschman, S.; Humbert, M.; et al. “Applying Statistics in the Undergraduate Chemistry Laboratory: Experiments with Food Dyes,” J. Chem. Educ. 1998, 75, 231–233. The absorbance of solutions of food dyes is used to explore the treatment of outliers and the application of the t-test for comparing means. Vitha, M. F.; Carr, P. W. “A Laboratory Exercise in Statistical Analysis of Data,” J. Chem. Educ. 1997, 74, 998–1000. Students determine the average weight of vitamin E pills using several different methods (one at a time, in sets of ten pills, and in sets of 100 pills). The data collected by the class are pooled together, plotted as histograms, and compared with results predicted by a normal distribution. The histograms and standard deviations for the pooled data also show the effect of sample size on the standard error of the mean.

4K PROBLEMS
1. The following masses were recorded for 12 different U.S. quarters (all given in grams):
5.683 5.620 5.551 5.549 5.536 5.552 5.548 5.539 5.554 5.552 5.684 5.632

Report the mean, median, range, standard deviation, and variance for these data. 2. Shown in the following rows are results for the determination of acetaminophen (in milligrams) in ten separate tablets of Excedrin Extra Strength Pain Reliever.18
224.3 261.7 240.4 229.4 246.3 255.5 239.4 235.5 253.1 249.7

(a) For each dosage, calculate the mean and standard deviation for the milligrams of morphine hydrochloride per – tablet. (b) Assuming that X and s2 are good approximations for µ and σ2, and that the population is normally distributed, what percentage of tablets at each dosage level are expected to contain more than the nominal amount of morphine hydrochloride per tablet? 4. Daskalakis and co-workers recently evaluated several procedures for digesting the tissues of oysters and mussels prior to analyzing the samples for silver.20 One of the methods used to evaluate the procedure is a spike recovery in which a known amount of silver is added to the tissue sample and the percent of the added silver found on analysis is reported. Ideally, spike recoveries should fall within the range 100 ± 15%. The results for one method are
106% 108% 92% 99% 104% 101% 93% 93%

(a) Report the mean, median, range, standard deviation, and – variance for these data. (b) Assuming that X and s2 are good 2, and that the population is approximations for µ and σ normally distributed, what percentage of tablets are expected to contain more than the standard amount of 250 mg acetaminophen per tablet? 3. Salem and Galan have developed a new method for determining the amount of morphine hydrochloride in tablets.19 Results, in milligrams, for several tablets containing different nominal dosages follow
100-mg tablets
99.17 94.31 95.92 94.55 93.83

Assuming that the spike recoveries are normally distributed, what is the probability that any single spike recovery will be within the accepted range? 5. The formula weight (FW) of a gas can be determined using the following form of the ideal gas law FW = gRT PV

60-mg tablets
54.21 55.62 57.40 57.51 52.59

30-mg tablets
28.51 26.25 25.92 28.62 24.93

10-mg tablets
19.06 8.83 9.08

where g is the mass in grams, R is the gas constant, T is the temperature in kelvins, P is the pressure in atmospheres, and V is the volume in liters. In a typical analysis the following data are obtained (with estimated uncertainties in parentheses)

Chapter 4 Evaluating Analytical Data g R T P V = = = = = 0.118 (±0.002) 0.082056 (±0.000001) 298.2 (±0.1) 0.724 (±0.005) 0.250 (±0.005) 13. Obtain a sample of a metal from your instructor, and determine its density by one or both of the following methods:

99

(a) What is the compound’s formula weight and its estimated uncertainty? (b) To which variable(s) should you direct your attention if you wish to improve the uncertainty in the compound’s molecular weight? 6. A standard solution of Mn2+ was prepared by dissolving 0.250 g of Mn in 10 mL of concentrated HNO3 (measured with a graduated cylinder). The resulting solution was quantitatively transferred to a 100-mL volumetric flask and diluted to volume with distilled water. A 10-mL aliquot of the solution was pipeted into a 500-mL volumetric flask and diluted to volume. (a) Express the concentration of Mn in parts per million, and estimate uncertainty by a propagation of uncertainty calculation. (b) Would the uncertainty in the solution’s concentration be improved by using a pipet to measure the HNO3, instead of a graduated cylinder? 7. Hydroscopic materials often are measured by the technique of weighing by difference. In this technique the material is placed in a sealed container and weighed. A portion of the material is removed, and the container and the remaining material are reweighed. The difference between the two masses gives the amount of material that was sampled. A solution of a hydroscopic material with a gram formula weight of 121.34 (±0.01) was prepared in the following manner. A sample of the compound and its container has a mass of 23.5811 g. A portion of the compound was transferred to a 100-mL volumetric flask and diluted to volume. The mass of the compound and container after the transfer is 22.1559 g. Calculate the molarity of the solution, and estimate its uncertainty by a propagation of uncertainty calculation. 8. Show by a propagation of uncertainty calculation that the standard error of the mean for n determinations is given as — s / √ n. 9. What is the smallest mass that can be measured on an analytical balance with a tolerance of ±0.1 mg, such that the relative error is less than 0.1%? 10. Which of the following is the best way to dispense 100.0 mL of a reagent: (a) use a 50-mL pipet twice; (b) use a 25-mL pipet four times; or (c) use a 10-mL pipet ten times? 11. A solution can be diluted by a factor of 200 using readily available pipets (1-mL to 100-mL) and volumetric flasks (10-mL to 1000-mL) in either one, two, or three steps. Limiting yourself to glassware listed in Table 4.2, determine the proper combination of glassware to accomplish each dilution, and rank them in order of their most probable uncertainties. 12. Explain why changing all values in a data set by a constant – amount will change X but will have no effect on s.

Method A: Obtain the sample’s mass with a balance. Calculate the sample’s volume using appropriate linear dimensions. Method B: Obtain the sample’s mass with a balance. Calculate the sample’s volume by measuring the amount of water that it displaces. This can be done by adding water to a graduated cylinder, reading the volume, adding the object, and reading the new volume. The difference in volumes is equal to the object’s volume. Determine the density at least five times. (a) Report the mean, the standard deviation, and the 95% confidence interval for your results. (b) Find the accepted value for the density of your metal, and determine the absolute and relative error for your experimentally determined density. (c) Use the propagation of uncertainty to determine the uncertainty for your chosen method. Are the results of this calculation consistent with your experimental results? If not, suggest some possible reasons for this disagreement. 14. How many carbon atoms must a molecule have if the mean number of 13C atoms per molecule is 1.00? What percent of such molecules will have no atoms of 13C? 15. In Example 4.10 we determined the probability that a molecule of cholesterol, C27H44O, had no atoms of 13C. (a) Calculate the probability that a molecule of cholesterol, has one atom of 13C. (b) What is the probability that a molecule of cholesterol will have two or more atoms of 13C? 16. Berglund and Wichart investigated the quantitative determination of Cr in high-alloy steels by a potentiometric titration of Cr6+.21 Before titrating the steel was dissolved in acid and the chromium oxidized to Cr6+ by peroxydisulfate. Following are their results (%w/w Cr) for the analysis of a single reference steel.
16.968 16.887 16.922 16.977 16.840 16.857 16.883 16.728

Calculate the mean, the standard deviation, and the 95% confidence interval about the mean. What does this confidence interval mean? 17. Ketkar and co-workers developed a new analytical method for measuring trace levels of atmospheric gases.22 The analysis of a sample containing 40.0 parts per thousand (ppt) 2-chloroethylsulfide yielded the following results
43.3 34.8 31.9 37.8 34.4 31.9 42.1 33.6 35.3

(a)Determine whether there is a significant difference between the experimental mean and the expected value at α = 0.05. (b) As part of this study a reagent blank was analyzed 12 times, giving a mean of 0.16 ppt and a standard deviation of 1.20 ppt. What are the IUPAC detection limit, the limit of identification, and limit of quantitation for this method assuming α = 0.05?

100

Modern Analytical Chemistry
Atmospheric Origin: Chemical Origin: 2.31017 2.31024 2.30143 2.29869 2.30986 2.31010 2.29890 2.29940 2.31010 2.31028 2.29816 2.29849 2.31001 2.30182 2.29889

18. To test a spectrophotometer for its accuracy, a solution of 60.06 ppm K2Cr2O7 in 5.0 mM H2SO4 is prepared and analyzed. This solution has a known absorbance of 0.640 at 350.0 nm in a 1.0-cm cell when using 5.0 mM H2SO4 as a reagent blank. Several aliquots of the solution are analyzed with the following results
0.639 0.638 0.640 0.639 0.640 0.639 0.638

Explain why these data led Rayleigh to look for and discover Ar. 23. Gács and Ferraroli reported a new method for monitoring the concentration of SO2 in air.25 They compared their method with the standard method by sampling and analyzing urban air from a single location. Air samples were collected by drawing air through a collection solution for 6 min. Following is a summary of their results with SO2 concentrations reported in microliters per cubic meter.
Standard method: 21.62 New method: 21.54 22.20 20.51 24.27 22.31 23.54 21.30 24.25 24.62 23.09 21.02 25.72 21.54

Determine whether there is a significant difference between the experimental mean and the expected value at α = 0.01. 19. Monna and co-workers studied the use of radioactive isotopes as a means of dating sediments collected from the bottom of lakes and estuaries.23 To verify this method they analyzed a 208Po standard known to have an activity of 77.5 decays/min, obtaining the following results
77.09 78.03 75.37 74.96 72.42 77.54 76.84 76.09 77.84 81.12 76.69 75.75

Determine whether there is a significant difference between the mean and the expected value at α = 0.05. 20. A 2.6540-g sample of an iron ore known to contain 53.51% w/w Fe is dissolved in a small portion of concentrated HCl and diluted to volume in a 250-mL volumetric flask. A spectrophotometric method is used to determine the concentration of Fe in this solution, yielding results of 5840, 5770, 5650, and 5660 ppm. Determine whether there is a significant difference between the experimental mean and the expected value at α = 0.05. 21. Horvat and colleagues investigated the application of atomic absorption spectroscopy to the analysis of Hg in coal fly ash.24 Of particular interest was the development of an appropriate procedure for digesting the samples in order to release the Hg for analysis. As part of their study they tested several reagents for digesting samples. Results obtained with HNO3 and with a 1 + 3 mixture of HNO3 and HCl are shown here. All concentrations are given as nanograms of Hg per gram of sample.
HNO3: 1 + 3 HNO3–HCl: 161 159 165 145 160 140 167 147 166 143 156

Using an appropriate statistical test, determine whether there is any significant difference between the standard and new methods at α = 0.05. 24. The accuracy of a spectrophotometer can be checked by measuring absorbances for a series of standard dichromate solutions that can be obtained in sealed cuvettes from the National Institute of Standards and Technology. Absorbances are measured at 257 nm and compared with the accepted values. The results obtained when testing a newly purchased spectrophotometer are shown here. Determine if the tested spectrophotometer is accurate at α = 0.05.
Standard: Measured absorbance: Accepted absorbance: 1 2 3 0.8674 0.8677 4 1.1623 1.1608 5 1.4559 1.4565

0.2872 0.5773 0.2871 0.5760

Determine whether there is a significant difference between these methods at α = 0.05. 22. Lord Rayleigh, John William Strutt (1842–1919) was one of the most well-known scientists of the late nineteenth and early twentieth centuries, publishing over 440 papers and receiving the Nobel Prize in chemistry in 1904 for the discovery of argon. An important turning point in the discovery of Ar was Rayleigh’s experimental measurements of the density of N2. Rayleigh approached this experiment in two ways: first by taking atmospheric air and removing any O2 and H2 that was present; and second, by chemically producing N2 by decomposing nitrogen-containing compounds (NO, N2O, and NH4NO3) and again removing any O2 and H2. His results for the density of N2, published in Proc. Roy. Soc. 1894, LV, 340 (publication 210), follow (all values are for grams of gas at equivalent volume, pressure, and temperature).

25. Maskarinec and associates investigated the stability of volatile organics in environmental water samples.26 Of particular interest was establishing proper conditions for maintaining the sample’s integrity between its collection and analysis. Two preservatives were investigated (ascorbic acid and sodium bisulfate), and maximum holding times were determined for a number of volatile organics and water matrices. Results (in days) for surface waters follow.
Ascorbic acid methylene chloride carbon disulfide trichloroethane benzene 1,1,2-trichloroethane 1,1,2,2-tetrachloroethane tetrachloroethene toluene chlorobenzene 77 23 52 62 57 33 41 32 36

Sodium bisulfate
62 54 51 42 53 85 63 94 86

Chapter 4 Evaluating Analytical Data
Determine whether there is a significant difference in the effectiveness of the two preservatives at α = 0.10. 26. Using X-ray diffraction, Karstang and Kvalhein reported a new method for determining the weight percent of kalonite in complex clay minerals.27 To test the method, nine samples containing known amounts of kalonite were prepared and analyzed. The results (as %w/w kalonite) are shown.
Actual: 5.0 10.0 Found: 6.8 11.7 20.0 40.0 19.8 40.5 50.0 53.6 60.0 61.7 80.0 78.9 90.0 95.0 91.7 94.7

101

29. The following data were collected during a study of the concentration of Zn in samples drawn from several locations in Lake Erie (all concentrations in parts per million).
[Zn2+] at the air–water interface
0.430 0.266 0.567 0.531 0.707 0.716

Location
1 2 3 4 5 6

[Zn2+] at the sediment–water interface
0.415 0.238 0.390 0.410 0.605 0.609

Evaluate the accuracy of the method at α = 0.05. 27. Mizutani and colleagues reported the development of a new method for the analysis of l-malate.28 As part of their study they analyzed a series of beverages using both their method and a standard spectrophotometric procedure based on a clinical kit purchased from Boerhinger Scientific. A summary follows of their results (in parts per million).
Sample
Apple juice 1 Apple juice 2 Apple juice 3 Apple juice 4 Grape juice 1 Grape juice 2 Mixed fruit juice 1 Mixed fruit juice 2 White wine 1 White wine 2 White wine 3 White wine 4

Determine whether there is a significant difference between the concentration of Zn2+ at the air–water interface and the sediment–water interface at α = 0.05. 30. Ten laboratories were asked to determine the concentration of an analyte A in three standard test samples. Following are the results, in parts per million.30
Laboratory
1 2 3 4 5 6 7 8 9 10

Electrode
34.0 22.6 29.7 24.9 17.8 14.8 8.6 31.4 10.8 17.3 15.7 18.4

Spectrophotometric
33.4 28.4 29.5 24.8 18.3 15.4 8.5 31.9 11.5 17.6 15.4 18.3

Sample 1
22.6 23.0 21.5 21.9 21.3 22.1 23.1 21.7 22.2 21.7

Sample 2
13.6 14.2 13.9 13.9 13.5 13.5 13.9 13.5 12.9 13.8

Sample 3
16.0 15.9 16.3 16.9 16.7 17.4 17.5 16.8 17.2 16.7

Determine whether there is a significant difference between the methods at α = 0.05. 28. Alexiev and associates describe an improved photometric method for the determination of Fe3+ based on its catalytic effect on the oxidation of sulphanilic acid by KIO4.29 As part of their study the concentration of Fe3+ in human serum samples was determined by the proposed method and the standard method. Following are the results, with concentrations in micromoles/L.
Sample
1 2 3 4 5 6 7 8

Determine if there are any potential outliers in Sample 1, Sample 2, or Sample 3 at a significance level of α = 0.05. 31. When copper metal and powdered sulfur are placed in a crucible and ignited, the product is a sulfide with an empirical formula of CuxS. The value of x can be determined by weighing the Cu and S before ignition, and finding the mass of CuxS when the reaction is complete. Following are the Cu/S ratios from 62 such experiments.
1.764 1.897 1.920 1.939 1.957 1.968 1.993 1.838 1.899 1.922 1.940 1.957 1.969 1.993 1.865 1.900 1.927 1.941 1.957 1.973 1.995 1.866 1.906 1.931 1.941 1.959 1.975 1.995 1.872 1.908 1.935 1.942 1.962 1.976 1.995 1.877 1.910 1.936 1.943 1.963 1.977 2.017 1.890 1.911 1.936 1.948 1.963 1.981 2.029 1.891 1.916 1.937 1.953 1.963 1.981 2.042 1.891 1.919 1.939 1.955 1.966 1.988

Proposed Method
8.25 9.75 9.75 9.75 10.75 11.25 13.88 14.25

Standard Method
8.06 8.84 8.36 8.73 13.13 13.65 13.85 13.43

Determine whether there is a significant difference between the two methods at α = 0.05.

(a) Calculate the mean and standard deviation for these data. (b) Construct a histogram for this data set. From a visual inspection of your histogram, do the data appear to be normally distributed? (c) In a normally distributed population, 68.26% of all members lie within the range µ ± 1σ. What percentage of the data lies within the range – X ± 1s? Does this support your answer to the previous

102

Modern Analytical Chemistry
– question? (d) Assuming that X and σ2 are good 2, what percentage of all approximations for µ and σ experimentally determined Cu/S ratios will be greater than 2? How does this compare with the experimental data? Does this support your conclusion about whether the data are normally distributed? (e) It has been reported that this method for preparing copper sulfide results in a nonstoichiometric compound with a Cu/S ratio of less than 2. Determine if the mean value for these data is significantly less than 2 at a significance level of α = 0.01.

4L SUGGESTED READINGS
A more comprehensive discussion of the analysis of data, covering all topics considered in this chapter as well as additional material, can be found in any textbook on statistics or data analysis; following are several such texts. Anderson, R. L. Practical Statistics for Analytical Chemists. Van Nostrand Reinhold: New York, 1987. Graham, R. C. Data Analysis for the Chemical Sciences. VCH Publishers: New York, 1993. Mark, H.; Workman, J. Statistics in Spectroscopy. Academic Press: Boston, 1991. Mason, R. L.; Gunst, R. F.; Hess, J. L. Statistical Design and Analysis of Experiments. Wiley: New York, 1989. Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry, 3rd ed. Ellis Horwood PTR Prentice-Hall: New York, 1993. Sharaf, M. H.; Illman, D. L.; Kowalski, B. R. Chemometrics. WileyInterscience: New York, 1986. The difference between precision and accuracy and a discussion of indeterminate and determinate sources of error is covered in the following paper. Treptow, R. S. “Precision and Accuracy in Measurements,” J. Chem. Educ. 1998, 75, 992–995. The detection of outliers, particularly when working with a small number of samples, is discussed in the following papers. Efstathiou, C. “Stochastic Calculation of Critical Q-Test Values for the Detection of Outliers in Measurements,” J. Chem. Educ. 1992, 69, 773–736. Kelly, P. C. “Outlier Detection in Collaborative Studies,” Anal. Chem. 1990, 73, 58–64. Mitschele, J. “Small Sample Statistics,” J. Chem. Educ. 1991, 68, 470–473. The following papers provide additional information on error and uncertainty, including the propagation of uncertainty. Andraos, J. “On the Propagation of Statistical Errors for a Function of Several Variables,” J. Chem. Educ. 1996, 73, 150–154. Donato, H.; Metz, C. “A Direct Method for the Propagation of Error Using a Personal Computer Spreadsheet Program,” J. Chem. Educ. 1988, 65, 867–868. Gordon, R.; Pickering, M.; Bisson, D. “Uncertainty Analysis by the ‘Worst Case’ Method,” J. Chem. Educ. 1984, 61, 780–781. Guare, C. J. “Error, Precision and Uncertainty,” J. Chem. Educ. 1991, 68, 649–652. Guedens, W. J.; Yperman, J.; Mullens, J.; et al. “Statistical Analysis of Errors: A Practical Approach for an Undergraduate Chemistry Lab,” Part 1. The Concept, J. Chem. Educ. 1993, 70, 776–779; Part 2. Some Worked Examples, J. Chem. Educ. 1993, 70, 838–841. Heydorn, K. “Detecting Errors in Micro and Trace Analysis by Using Statistics,” Anal. Chim. Acta 1993, 283, 494–499. Taylor, B. N.; Kuyatt, C. E. “Guidelines for Evaluating and Expressing the Uncertainty of NIST Measurement Results,” NIST Technical Note 1297, 1994. A further discussion of detection limits is found in Currie, L. A., ed. Detection in Analytical Chemistry: Importance, Theory and Practice. American Chemical Society: Washington, DC, 1988.

4M REFERENCES
1. Goedhart, M. J.; Verdonk, A. H. J. Chem. Educ. 1991, 68, 1005–1009. 2. Rousseeuw, P. J. J. Chemom. 1991, 5, 1–20. 3. Ellison, S.; Wegscheider, W.; Williams, A. Anal. Chem. 1997, 69, 607A–613A. 4. Shoemaker, D. P.; Garland, C. W.; Nibler, J. W. Experiments in Physical Chemistry, 5th ed. McGraw-Hill: New York, 1989, pp. 55–63. 5. Lam, R. B.; Isenhour, T. L. Anal. Chem. 1980, 52, 1158–1161. 6. Mark, H.; Workman, J. Spectroscopy, 1988, 3(1), 44–48. 7. Winn, R. L. Statistics for Scientists and Engineers, Prentice-Hall: Englewood Cliffs, NJ, 1964; pp. 165–174. 8. Mark, H.; Workman, J. Spectroscopy, 1989, 4(3), 56–58. 9. Marecek, V.; Janchenova, H.; Brezina, M.; et al. Anal. Chim. Acta 1991, 244, 15–19. 10. Rorabacher, D. B. Anal. Chem. 1991, 63, 139–146. 11. Deming, W. E. Statistical Adjustment of Data. Wiley: New York, 1943 (republished by Dover: New York, 1961); p. 171. 12. Richardson, T. H. J. Chem. Educ. 1991, 68, 310–311. 13. Kirchner, C. J. “Estimation of Detection Limits for Environmental Analytical Procedures,” In Currie, L. A., ed. Detection in Analytical Chemistry: Importance, Theory and Practice. American Chemical Society: Washington, DC, 1988. 14. Long, G. L.; Winefordner, J. D. Anal. Chem. 1983, 55, 712A–724A.

Chapter 4 Evaluating Analytical Data
15. “Guidelines for Data Acquisition and Data Quality Control Evaluation in Environmental Chemistry,” Anal. Chem. 1980, 52, 2242–2249. 16. (a) Ferrus, R.; Egea, M. R. Anal. Chim. Acta 1994, 287, 119–145; (b) Glaser, J. A.; Foerst, D. L.; McKee, G. D.; et al. Environ. Sci. Technol. 1981, 15, 1426–1435; (c) Boumans, P. W. J. M. Anal. Chem. 1994, 66, 459A–467A; (d) Kimbrough, D. E.; Wakakuwa, J. Environ. Sci. Technol. 1994, 28, 338–345; (e) Currie, L. A. Anal. Chem. 1968, 40, 586–593. 17. (a) Rogers, L. B. J. Chem. Educ. 1986, 63, 3–6; (b) Mark, H.; Workman, J. Spectroscopy, 1989, 4(1), 52–55. 18. Simonian, M. H.; Dinh, S.; Fray, L. A. Spectroscopy 1993, 8(6), 37–47. 19. Salem, I. I.; Galan, A. C. Anal. Chim. Acta 1993, 283, 334–337. 20. Daskalakis, K. D.; O’Connor, T. P.; Crecelius, E. A. Environ. Sci. Technol. 1997, 31, 2303–2306. 21. Berglund, B.; Wichardt, C. Anal. Chim. Acta 1990, 236, 399–410. 22. Ketkar, S. N.; Dulak, J. G.; Dheandhanou, S.; et al. Anal. Chim. Acta. 1991, 245, 267–270.

103

23. Monna, F.; Mathieu, D.; Marques Jr., et al. Anal. Chim. Acta 1996, 330, 107–116. 24. Horvat, M.; Lupsina, V.; Pihlar, B. Anal. Chim. Acta. 1991, 243, 71–79. 25. Gács, I.; Ferraroli, R. Anal. Chim. Acta 1992, 269, 177–185. 26. Maskarinec, M. P.; Johnson, L. H.; Holladay, S. K.; et al. Environ. Sci. Technol. 1990, 24, 1665–1670. 27. Karstang, T. V.; Kvalhein, O. M. Anal. Chem. 1991, 63, 767–772. 28. Mizutani, F.; Yabuki, S.; Asai, M. Anal. Chim. Acta. 1991, 245, 145–150. 29. Alexiev, A.; Rubino, S.; Deyanova, M.; et al. Anal. Chem. Acta 1994, 295, 211–219. 30. These data are adapted from Steiner, E. H. “Planning and Analysis of Results of Collaborative Tests” published in Statistical Manual of the Association of Official Analytical Chemists, Association of Official Analytical Chemists: Washington, DC, 1975.

5 Chapter
Calibrations, Standardizations, and Blank Corrections

I

n Chapter 3 we introduced a relationship between the measured signal, Smeas, and the absolute amount of analyte
Smeas = knA + Sreag 5.1

or the relative amount of analyte in a sample
Smeas = kCA + Sreag 5.2

where nA is the moles of analyte, CA is the analyte’s concentration, k is the method’s sensitivity, and Sreag is the contribution to Smeas from constant errors introduced by the reagents used in the analysis. To obtain an accurate value for nA or CA it is necessary to avoid determinate errors affecting Smeas, k, and Sreag. This is accomplished by a combination of calibrations, standardizations, and reagent blanks.

104

Chapter 5 Calibrations, Standardizations, and Blank Corrections

105

5A Calibrating Signals
Signals are measured using equipment or instruments that must be properly calibrated if Smeas is to be free of determinate errors. Calibration is accomplished against a standard, adjusting Smeas until it agrees with the standard’s known signal. Several common examples of calibration are discussed here. When the signal is a measurement of mass, Smeas is determined with an analytical balance. Before a balance can be used, it must be calibrated against a reference weight meeting standards established by either the National Institute for Standards and Technology or the American Society for Testing and Materials. With an electronic balance the sample’s mass is determined by the current required to generate an upward electromagnetic force counteracting the sample’s downward gravitational force. The balance’s calibration procedure invokes an internally programmed calibration routine specifying the reference weight to be used. The reference weight is placed on the balance’s weighing pan, and the relationship between the displacement of the weighing pan and the counteracting current is automatically adjusted. Calibrating a balance, however, does not eliminate all sources of determinate error. Due to the buoyancy of air, an object’s weight in air is always lighter than its weight in vacuum. If there is a difference between the density of the object being weighed and the density of the weights used to calibrate the balance, then a correction to the object’s weight must be made.1 An object’s true weight in vacuo, Wv, is related to its weight in air, Wa, by the equation  Wv = Wa × 1 +     1 1  –   × 0.0012  Do Dw   

where Do is the object’s density, Dw is the density of the calibration weight, and 0.0012 is the density of air under normal laboratory conditions (all densities are in units of g/cm3). Clearly the greater the difference between Do and Dw the more serious the error in the object’s measured weight. The buoyancy correction for a solid is small, and frequently ignored. It may be significant, however, for liquids and gases of low density. This is particularly important when calibrating glassware. For example, a volumetric pipet is calibrated by carefully filling the pipet with water to its calibration mark, dispensing the water into a tared beaker and determining the mass of water transferred. After correcting for the buoyancy of air, the density of water is used to calculate the volume of water dispensed by the pipet. EXAMPLE 5.1 A 10-mL volumetric pipet was calibrated following the procedure just outlined, using a balance calibrated with brass weights having a density of 8.40 g/cm3. At 25 °C the pipet was found to dispense 9.9736 g of water. What is the actual volume dispensed by the pipet? SOLUTION At 25 °C the density of water is 0.99705 g/cm3 . The water’s true weight, therefore, is    1 1  Wv = 9.9736 g × 1 +  –  × 0.0012 = 9.9842 g  0.99705 8.40     

106

Modern Analytical Chemistry and the actual volume of water dispensed by the pipet is 9.9842 g = 10.014 cm 3 = 10.014 mL 0.99705 g/cm 3 If the buoyancy correction is ignored, the pipet’s volume is reported as 9.9736 g = 10.003 cm 3 = 10.003 mL 0.99705 g/cm 3 introducing a negative determinate error of –0.11%.

Balances and volumetric glassware are examples of laboratory equipment. Laboratory instrumentation also must be calibrated using a standard providing a known response. For example, a spectrophotometer’s accuracy can be evaluated by measuring the absorbance of a carefully prepared solution of 60.06 ppm K2Cr2O7 in 0.0050 M H2SO4, using 0.0050 M H2SO4 as a reagent blank.2 The spectrophotometer is considered calibrated if the resulting absorbance at a wavelength of 350.0 nm is 0.640 ± 0.010 absorbance units. Be sure to read and carefully follow the calibration instructions provided with any instrument you use.

5B Standardizing Methods
The American Chemical Society’s Committee on Environmental Improvement defines standardization as the process of determining the relationship between the measured signal and the amount of analyte.3 A method is considered standardized when the value of k in equation 5.1 or 5.2 is known. In principle, it should be possible to derive the value of k for any method by considering the chemical and physical processes responsible for the signal. Unfortunately, such calculations are often of limited utility due either to an insufficiently developed theoretical model of the physical processes or to nonideal chemical behavior. In such situations the value of k must be determined experimentally by analyzing one or more standard solutions containing known amounts of analyte. In this section we consider several approaches for determining the value of k. For simplicity we will assume that Sreag has been accounted for by a proper reagent blank, allowing us to replace Smeas in equations 5.1 and 5.2 with the signal for the species being measured.

5B.1 Reagents Used as Standards
The accuracy of a standardization depends on the quality of the reagents and glassware used to prepare standards. For example, in an acid–base titration, the amount of analyte is related to the absolute amount of titrant used in the analysis by the stoichiometry of the chemical reaction between the analyte and the titrant. The amount of titrant used is the product of the signal (which is the volume of titrant) and the titrant’s concentration. Thus, the accuracy of a titrimetric analysis can be no better than the accuracy to which the titrant’s concentration is known. primary reagent A reagent of known purity that can be used to make a solution of known concentration.

Primary Reagents Reagents used as standards are divided into primary reagents and secondary reagents. A primary reagent can be used to prepare a standard containing an accurately known amount of analyte. For example, an accurately weighed sample of 0.1250 g K2Cr2O7 contains exactly 4.249 × 10–4 mol of K2Cr2O7. If this

Chapter 5 Calibrations, Standardizations, and Blank Corrections same sample is placed in a 250-mL volumetric flask and diluted to volume, the concentration of the resulting solution is exactly 1.700 × 10–3 M. A primary reagent must have a known stoichiometry, a known purity (or assay), and be stable during long-term storage both in solid and solution form. Because of the difficulty in establishing the degree of hydration, even after drying, hydrated materials usually are not considered primary reagents. Reagents not meeting these criteria are called secondary reagents. The purity of a secondary reagent in solid form or the concentration of a standard prepared from a secondary reagent must be determined relative to a primary reagent. Lists of acceptable primary reagents are available.4 Appendix 2 contains a selected listing of primary standards. Other Reagents Preparing a standard often requires additional substances that are not primary or secondary reagents. When a standard is prepared in solution, for example, a suitable solvent and solution matrix must be used. Each of these solvents and reagents is a potential source of additional analyte that, if unaccounted for, leads to a determinate error. If available, reagent grade chemicals conforming to standards set by the American Chemical Society should be used.5 The packaging label included with a reagent grade chemical (Figure 5.1) lists either the maximum allowed limit for specific impurities or provides the actual assayed values for the impurities as reported by the manufacturer. The purity of a reagent grade chemical can be improved by purification or by conducting a more accurate assay. As discussed later in the chapter, contributions to Smeas from impurities in the sample matrix can be compensated for by including an appropriate blank determination in the analytical procedure.

107

secondary reagent A reagent whose purity must be established relative to a primary reagent.

reagent grade Reagents conforming to standards set by the American Chemical Society.

Figure 5.1
Examples of typical packaging labels from reagent grade chemicals. Label (a) provides the actual lot assay for the reagent as determined by the manufacturer. Note that potassium has been flagged with an asterisk (*) because its assay exceeds the maximum limit established by the American Chemical Society (ACS). Label (b) does not provide assayed values, but indicates that the reagent meets the specifications of the ACS for the listed impurities. An assay for the reagent also is provided.

(a)

(b)

© David Harvey/Marilyn Culler, photographer.

108

Modern Analytical Chemistry Preparing Standard Solutions Solutions of primary standards generally are prepared in class A volumetric glassware to minimize determinate errors. Even so, the relative error in preparing a primary standard is typically ±0.1%. The relative error can be improved if the glassware is first calibrated as described in Example 5.1. It also is possible to prepare standards gravimetrically by taking a known mass of standard, dissolving it in a solvent, and weighing the resulting solution. Relative errors of ±0.01% can typically be achieved in this fashion. It is often necessary to prepare a series of standard solutions, each with a different concentration of analyte. Such solutions may be prepared in two ways. If the range of concentrations is limited to only one or two orders of magnitude, the solutions are best prepared by transferring a known mass or volume of the pure standard to a volumetric flask and diluting to volume. When working with larger concentration ranges, particularly those extending over more than three orders of magnitude, standards are best prepared by a serial dilution from a single stock solution. In a serial dilution a volume of a concentrated stock solution, which is the first standard, is diluted to prepare a second standard. A portion of the second standard is then diluted to prepare a third standard, and the process is repeated until all necessary standards have been prepared. Serial dilutions must be prepared with extra care because a determinate error in the preparation of any single standard is passed on to all succeeding standards.

5B.2 Single-Point versus Multiple-Point Standardizations* single-point standardization Any standardization using a single standard containing a known amount of analyte.

The simplest way to determine the value of k in equation 5.2 is by a singlepoint standardization. A single standard containing a known concentration of analyte, CS, is prepared and its signal, Sstand, is measured. The value of k is calculated as k = Sstand CS 5.3

Assumed relationship

Actual relationship

Sstand Cs

Concentration reported

CA

Actual concentration

Figure 5.2
Example showing how an improper use of a single-point standardization can lead to a determinate error in the reported concentration of analyte.

A single-point standardization is the least desirable way to standardize a method. When using a single standard, all experimental errors, both determinate and indeterminate, are carried over into the calculated value for k. Any uncertainty in the value of k increases the uncertainty in the analyte’s concentration. In addition, equation 5.3 establishes the standardization relationship for only a single concentration of analyte. Extending equation 5.3 to samples containing concentrations of analyte different from that in the standard assumes that the value of k is constant, an assumption that is often not true.6 Figure 5.2 shows how assuming a constant value of k may lead to a determinate error. Despite these limitations, single-point standardizations are routinely used in many laboratories when the analyte’s range of expected concentrations is limited. Under these conditions it is often safe to assume that k is constant (although this assumption should be verified experimentally). This is the case, for example, in clinical laboratories where many automated analyzers use only a single standard. The preferred approach to standardizing a method is to prepare a series of standards, each containing the analyte at a different concentration. Standards are chosen such that they bracket the expected range for the
*The following discussion of standardizations assumes that the amount of analyte is expressed as a concentration. It also applies, however, when the absolute amount of analyte is given in grams or moles.

Signal

Chapter 5 Calibrations, Standardizations, and Blank Corrections analyte’s concentration. Thus, a multiple-point standardization should use at least three standards, although more are preferable. A plot of Sstand versus CS is known as a calibration curve. The exact standardization, or calibration relationship, is determined by an appropriate curve-fitting algorithm.* Several approaches to standardization are discussed in the following sections.

109

multiple-point standardization Any standardization using two or more standards containing known amounts of analyte.

5B.3 External Standards
The most commonly employed standardization method uses one or more external standards containing known concentrations of analyte. These standards are identified as external standards because they are prepared and analyzed separately from the samples. A quantitative determination using a single external standard was described at the beginning of this section, with k given by equation 5.3. Once standardized, the concentration of analyte, CA, is given as CA = EXAMPLE 5.2 A spectrophotometric method for the quantitative determination of Pb2+ levels in blood yields an Sstand of 0.474 for a standard whose concentration of lead is 1.75 ppb. How many parts per billion of Pb2+ occur in a sample of blood if Ssamp is 0.361? SOLUTION Equation 5.3 allows us to calculate the value of k for this method using the data for the standard k = 0.474 Sstand = = 0.2709 ppb –1 1.75 ppb CS Pb2+ in the sample of blood can be
CA
(a) external standard A standard solution containing a known amount of analyte, prepared separately from samples containing the analyte.

Ssamp k

5.4

Once k is known, the concentration of calculated using equation 5.4 CA =

A multiple-point external standardization is accomplished by constructing a calibration curve, two examples of which are shown in Figure 5.3. Since this is the most frequently employed method of standardization, the resulting relationship often is called a normal calibration curve. When the calibration curve is a linear (Figure 5.3a), the slope of the line gives the value of k. This is the most desirable situation since the method’s sensitivity remains constant throughout the standard’s concentration range. When the calibration curve is nonlinear, the method’s sensitivity is a function of the analyte’s concentration. In Figure 5.3b, for example, the value of k is greatest when the analyte’s concentration is small and decreases continuously as the amount of analyte is increased. The value of k at any point along the calibration curve is given by the slope at that point. In
*Linear regression, also known as the method of least squares, is covered in Section 5C.

Sstand

Ssamp 0.361 = = 1.33 ppb k 0.2709 ppb –1

Sstand

CA
(b)

Figure 5.3
Examples of (a) straight-line and (b) curved normal calibration curves.

normal calibration curve A calibration curve prepared using several external standards.

110

Modern Analytical Chemistry either case, the calibration curve provides a means for relating Ssamp to the analyte’s concentration. EXAMPLE 5.3

Colorplate 1 shows an example of a set of external standards and their corresponding normal calibration curve.

A second spectrophotometric method for the quantitative determination of Pb2+ levels in blood gives a linear normal calibration curve for which Sstand = (0.296 ppb–1) × CS + 0.003 What is the Pb2+ level (in ppb) in a sample of blood if Ssamp is 0.397? SOLUTION To determine the concentration of Pb2+ in the sample of blood, we replace Sstand in the calibration equation with Ssamp and solve for CA CA = Ssamp – 0.003 0.296 ppb –1 = 0.397 – 0.003 = 1.33 ppb 0.296 ppb –1

It is worth noting that the calibration equation in this problem includes an extra term that is not in equation 5.3. Ideally, we expect the calibration curve to give a signal of zero when CS is zero. This is the purpose of using a reagent blank to correct the measured signal. The extra term of +0.003 in our calibration equation results from uncertainty in measuring the signal for the reagent blank and the standards.

An external standardization allows a related series of samples to be analyzed using a single calibration curve. This is an important advantage in laboratories where many samples are to be analyzed or when the need for a rapid Calibration curve obtained in standard’s matrix throughput of samples is critical. Not surprisingly, many of the most commonly encountered quantitative analytical methods are based on an external Calibration curve obtained standardization. in sample’s matrix There is a serious limitation, however, to an external standardization. The relationship between Sstand and CS in equation 5.3 is determined when the analyte is present in the external standard’s matrix. In using an external standardization, we assume that any difference between the matrix of the standards and the sample’s matrix has no effect on the value of k. A proportional determinate error is introduced when differences between the two matrices cannot be ignored. This is shown in Figure 5.4, where the reReported Actual lationship between the signal and the amount of analyte is shown for both Amount of analyte the sample’s matrix and the standard’s matrix. In this example, using a Figure 5.4 normal calibration curve results in a negative determinate error. When Effect of the sample’s matrix on a normal matrix problems are expected, an effort is made to match the matrix of the calibration curve. standards to that of the sample. This is known as matrix matching. When the sample’s matrix is unknown, the matrix effect must be shown to be negligimatrix matching ble, or an alternative method of standardization must be used. Both approaches Adjusting the matrix of an external are discussed in the following sections. standard so that it is the same as the matrix of the samples to be analyzed. method of standard additions A standardization in which aliquots of a standard solution are added to the sample.

Signal

5B.4 Standard Additions
The complication of matching the matrix of the standards to that of the sample can be avoided by conducting the standardization in the sample. This is known as the method of standard additions. The simplest version of a standard addi-

Chapter 5 Calibrations, Standardizations, and Blank Corrections
Add Vo of CA Add Vo of CA Add VS of CS

111

Dilute to Vf

Dilute to Vf

Figure 5.5
Total concentration of analyte Total concentration of analyte
Illustration showing the method of standard additions in which separate aliquots of sample are diluted to the same final volume. One aliquot of sample is spiked with a known volume of a standard solution of analyte before diluting to the final volume.

CA

Vo Vf

CA

Vo VS + CS Vf Vf

tion is shown in Figure 5.5. A volume, Vo, of sample is diluted to a final volume, Vf, and the signal, Ssamp is measured. A second identical aliquot of sample is spiked with a volume, Vs, of a standard solution for which the analyte’s concentration, CS, is known. The spiked sample is diluted to the same final volume and its signal, Sspike, is recorded. The following two equations relate Ssamp and Sspike to the concentration of analyte, CA, in the original sample Ssamp = kCA Vo Vf 5.5

aliquot A portion of a solution.

 V V  Sspike = k  C A o + C S s  Vf Vf  

5.6

where the ratios Vo/Vf and Vs/Vf account for the dilution. As long as Vs is small relative to Vo, the effect of adding the standard to the sample’s matrix is insignificant, and the matrices of the sample and the spiked sample may be considered identical. Under these conditions the value of k is the same in equations 5.5 and 5.6. Solving both equations for k and equating gives Ssamp Sspike = C A (Vo /Vf ) C A (Vo /Vf ) + CS (Vs /Vf )

5.7 Equation 5.7 can be solved for the concentration of analyte in the original sample.

112

Modern Analytical Chemistry EXAMPLE 5.4 A third spectrophotometric method for the quantitative determination of the concentration of Pb2+ in blood yields an Ssamp of 0.193 for a 1.00-mL sample of blood that has been diluted to 5.00 mL. A second 1.00-mL sample is spiked with 1.00 µL of a 1560-ppb Pb2+ standard and diluted to 5.00 mL, yielding an Sspike of 0.419. Determine the concentration of Pb2+ in the original sample of blood. SOLUTION The concentration of Pb2+ in the original sample of blood can be determined by making appropriate substitutions into equation 5.7 and solving for CA. Note that all volumes must be in the same units, thus Vs is converted from 1.00 µL to 1.00 × 10–3 mL. 0.193 0.419 =  1.00 mL   1.00 × 10 –3 mL   1.00 mL  CA  CA    + 1560 ppb  5.00 mL  5.00 mL   5.00 mL    0.193 0.419 = 0.200C A 0.200C A + 0.312 ppb 0.0386C A + 0.0602 ppb = 0.0838C A 0.0452C A = 0.0602 ppb C A = 1.33 ppb Thus, the concentration of Pb2+ in the original sample of blood is 1.33 ppb.

It also is possible to make a standard addition directly to the sample after measuring Ssamp (Figure 5.6). In this case, the final volume after the standard addition is Vo + Vs and equations 5.5–5.7 become Ssamp = kCA  Vo Vs  Sspike = k  C A + CS  Vo + Vs Vo + Vs   5.8

Add VS of CS

Vo

Vo

Figure 5.6
Illustration showing an alternative form of the method of standard additions. In this case a sample containing the analyte is spiked with a known volume of a standard solution of analyte without further diluting either the sample or the spiked sample.

Total concentration of analyte

Total concentration of analyte

CA

CA

Vo VS + CS Vo + VS Vo + VS

Chapter 5 Calibrations, Standardizations, and Blank Corrections Ssamp Sspike = CA C A[Vo /(Vo + Vs )] + C S[Vs /(Vo + Vs )] EXAMPLE 5.5 A fourth spectrophotometric method for the quantitative determination of the concentration of Pb2+ in blood yields an Ssamp of 0.712 for a 5.00-mL sample of blood. After spiking the blood sample with 5.00 µL of a 1560-ppb Pb 2+ standard, an Sspike of 1.546 is measured. Determine the concentration of Pb2+ in the original sample of blood. SOLUTION The concentration of Pb2+ in the original sample of blood can be determined by making appropriate substitutions into equation 5.9 and solving for CA.
0.712 CA = 1.546 CA 

113

5.9

   5.00 × 10 –3 mL   + 1560 ppb –3 –3  (5.00 mL + 5.00 × 10 mL)   (5.00 mL + 5.00 × 10 mL)  
5.00 mL 0.712 CA = 1.546 0.9990C A + 1.558 ppb

0.7113C A + 1.109 ppb = 1.546C A C A = 1.33 ppb

Thus, the concentration of

Pb2+

in the original sample of blood is 1.33 ppb.

The single-point standard additions outlined in Examples 5.4 and 5.5 are easily adapted to a multiple-point standard addition by preparing a series of spiked samples containing increasing amounts of the standard. A calibration curve is prepared by plotting Sspike versus an appropriate measure of the amount of added standard. Figure 5.7 shows two examples of a standard addition calibration curve based on equation 5.6. In Figure 5.7(a) Sspike is plotted versus the volume of the standard solution spikes, Vs. When k is constant, the calibration curve is linear, and it is easy to show that the x-intercept’s absolute value is CAVo/CS.

Colorplate 2 shows an example of a set of standard additions and their corresponding standard additions calibration curve.

EXAMPLE 5.6 Starting with equation 5.6, show that the equations for the slope, y-intercept, and x-intercept in Figure 5.7(a) are correct. SOLUTION We begin by rewriting equation 5.6 as Sspike = kCAVo kCS + × Vs Vf Vf

which is in the form of the linear equation Y = y-intercept + slope × X

114

Modern Analytical Chemistry

Sspiked

Slope = –CAVo CS

kCS Vf

x-intercept =

y-intercept =

kCAVo Vf

VS
(a)

Sspiked

Slope = k –CAVo Vf

Figure 5.7
Examples of calibration curves for the method of standard additions. In (a) the signal is plotted versus the volume of the added standard, and in (b) the signal is plotted versus the concentration of the added standard after dilution.

x-intercept =

y-intercept =

kCAVo Vf

CS
(b)

(V ) V
S f

where Y is Sspike and X is Vs. The slope of the line, therefore, is kCS/Vf, and the y-intercept is kCAVo/Vf. The x-intercept is the value of X when Y is 0, or 0 = kCAVo kCS + × (x -intercept) Vf Vf (kCAVo /Vf ) C V = – A o (kCS /Vf ) CS

x -intercept = –

Thus, the absolute value of the x-intercept is CAVo/CS.

Since both Vo and CS are known, the x-intercept can be used to calculate the analyte’s concentration. EXAMPLE

5.7

A fifth spectrophotometric method for the quantitative determination of the concentration of Pb2+ in blood uses a multiple-point standard addition based on equation 5.6. The original blood sample has a volume of 1.00 mL, and the standard used for spiking the sample has a concentration of 1560 ppb Pb2+. All samples were diluted to 5.00 mL before measuring the signal. A calibration curve of Sspike versus Vs is described by

Chapter 5 Calibrations, Standardizations, and Blank Corrections Sspike = 0.266 + 312 mL–1 × Vs Determine the concentration of Pb2+ in the original sample of blood. SOLUTION To find the x-intercept we let Sspike equal 0 0 = 0.266 + 312 mL–1 × (x-intercept) and solve for the x-intercept’s absolute value, giving a value of 8.526 × 10–4 mL. Thus x -intercept = 8.526 × 10 –4 mL = C AVo C × (1.00 mL) = A CS 1560 ppb

115

and the concentration of Pb2+ in the blood sample, CA, is 1.33 ppb.

Figure 5.7(b) shows the relevant relationships when Sspike is plotted versus the concentrations of the spiked standards after dilution. Standard addition calibration curves based on equation 5.8 are also possible. Since a standard additions calibration curve is constructed in the sample, it cannot be extended to the analysis of another sample. Each sample, therefore, requires its own standard additions calibration curve. This is a serious drawback to the routine application of the method of standard additions, particularly in laboratories that must handle many samples or that require a quick turnaround time. For example, suppose you need to analyze ten samples using a three-point calibration curve. For a normal calibration curve using external standards, only 13 solutions need to be analyzed (3 standards and 10 samples). Using the method of standard additions, however, requires the analysis of 30 solutions, since each of the 10 samples must be analyzed three times (once before spiking and two times after adding successive spikes). The method of standard additions can be used to check the validity of an external standardization when matrix matching is not feasible. To do this, a normal calibration curve of Sstand versus CS is constructed, and the value of k is determined from its slope. A standard additions calibration curve is then constructed using equation 5.6, plotting the data as shown in Figure 5.7(b). The slope of this standard additions calibration curve gives an independent determination of k. If the two values of k are identical, then any difference between the sample’s matrix and that of the external standards can be ignored. When the values of k are different, a proportional determinate error is introduced if the normal calibration curve is used.

5B.5 Internal Standards
The successful application of an external standardization or the method of standard additions, depends on the analyst’s ability to handle samples and standards reproducibly. When a procedure cannot be controlled to the extent that all samples and standards are treated equally, the accuracy and precision of the standardization may suffer. For example, if an analyte is present in a volatile solvent, its concentration will increase if some solvent is lost to evaporation. Suppose that you have a sample and a standard with identical concentrations of analyte and identical signals. If both experience the same loss of solvent their concentrations of analyte and signals will continue to be identical. In effect, we can ignore changes in concentration due to evaporation provided that the samples and standards experience an equivalent loss of solvent. If an identical standard and sample experience different losses of solvent,

116

Modern Analytical Chemistry however, their concentrations and signals will no longer be equal. In this case, an external standardization or standard addition results in a determinate error. A standardization is still possible if the analyte’s signal is referenced to a signal generated by another species that has been added at a fixed concentration to all samples and standards. The added species, which must be different from the analyte, is called an internal standard. Since the analyte and internal standard in any sample or standard receive the same treatment, the ratio of their signals will be unaffected by any lack of reproducibility in the procedure. If a solution contains an analyte of concentration CA, and an internal standard of concentration, CIS, then the signals due to the analyte, SA, and the internal standard, SIS, are SA = kACA SIS = kISCIS where kA and kIS are the sensitivities for the analyte and internal standard, respectively. Taking the ratio of the two signals gives SA k C C = A × A = K× A SIS kIS CIS CIS 5.10

internal standard A standard, whose identity is different from the analyte’s, that is added to all samples and standards containing the analyte.

Because equation 5.10 is defined in terms of a ratio, K, of the analyte’s sensitivity and the internal standard’s sensitivity, it is not necessary to independently determine values for either kA or kIS. In a single-point internal standardization, a single standard is prepared, and K is determined by solving equation 5.10  C  S  K =  IS   A   C A   SIS  stand Once the method is standardized, the analyte’s concentration is given by  C  S  C A =  IS   A   K   SIS  samp EXAMPLE 5.8 A sixth spectrophotometric method for the quantitative determination of Pb2+ levels in blood uses Cu2+ as an internal standard. A standard containing 1.75 ppb Pb2+ and 2.25 ppb Cu2+ yields a ratio of SA/SIS of 2.37. A sample of blood is spiked with the same concentration of Cu 2+, giving a signal ratio of 1.80. Determine the concentration of Pb2+ in the sample of blood. SOLUTION Equation 5.11 allows us to calculate the value of K using the data for the standard  C  S  2.25 K =  IS   A  = × 2.37 = 3.05 1.75  C A   SIS  stand The concentration of Pb2+, therefore, is  C  S  2.25 C A =  IS   A  = × 1.80 = 1.33 ppb Pb 2+ K   SIS  samp 3.05  5.11

Chapter 5 Calibrations, Standardizations, and Blank Corrections A single-point internal standardization has the same limitations as a singlepoint normal calibration. To construct an internal standard calibration curve, it is necessary to prepare several standards containing different concentrations of analyte. These standards are usually prepared such that the internal standard’s concentration is constant. Under these conditions a calibration curve of (SA/SIS)stand versus CA is linear with a slope of K/CIS. EXAMPLE 5.9 A seventh spectrophotometric method for the quantitative determination of Pb2+ levels in blood gives a linear internal standards calibration curve for which  SA  = (2.11 ppb –1 ) × C A – 0.006    SIS  stand What is the concentration (in ppb) of Pb2+ in a sample of blood if (SA/SIS)samp is 2.80? SOLUTION To determine the concentration of Pb2+ in the sample of blood, we replace (SA/SIS)stand in the calibration equation with (SA/SIS)samp and solve for CA CA = (S A / SIS )samp + 0.006 2.11 ppb –1 = 2.80 + 0.006 = 1.33 ppb 2.11 ppb –1

117

The concentration of Pb2+ in the sample of blood is 1.33 ppb.

When the internal standard’s concentration cannot be held constant the data must be plotted as (SA/SIS)stand versus CA/CIS, giving a linear calibration curve with a slope of K.

5C Linear Regression and Calibration Curves
In a single-point external standardization, we first determine the value of k by measuring the signal for a single standard containing a known concentration of analyte. This value of k and the signal for the sample are then used to calculate the concentration of analyte in the sample (see Example 5.2). With only a single determination of k, a quantitative analysis using a single-point external standardization is straightforward. This is also true for a single-point standard addition (see Examples 5.4 and 5.5) and a single-point internal standardization (see Example 5.8). A multiple-point standardization presents a more difficult problem. Consider the data in Table 5.1 for a multiple-point external standardization. What is the best estimate of the relationship between Table 5.1 Data for Hypothetical MultipleSmeas and CS? It is tempting to treat this data as five separate Point External Standardization single-point standardizations, determining k for each stanCS Smeas dard and reporting the mean value. Despite its simplicity, this is not an appropriate way to treat a multiple-point 0.000 0.00 standardization. 0.100 12.36 In a single-point standardization, we assume that 0.200 24.83 the reagent blank (the first row in Table 5.1) corrects for 0.300 35.91 all constant sources of determinate error. If this is not 0.400 48.79 the case, then the value of k determined by a single0.500 60.42 point standardization will have a determinate error.

118

Modern Analytical Chemistry

Table 5.2
CA
1.00 2.00 3.00 4.00 5.00

Effect of a Constant Determinate Error on the Value of k Calculated Using a Single-Point Standardization k (true)
1.00 1.00 1.00 1.00 1.00 1.00

Smeas (true)
1.00 2.00 3.00 4.00 5.00 mean k(true) =

Smeas (with constant error)
1.50 2.50 3.50 4.50 5.50 mean k (apparent) =

k (apparent)
1.50 1.25 1.17 1.13 1.10 1.23

80

60

40

20

0 0.0

0.1

0.2

0.3 CA

0.4

0.5

0.6

Figure 5.8
Normal calibration plot of hypothetical data from Table 5.1.

Table 5.2 demonstrates how an uncorrected constant error affects our determination of k. The first three columns show the concentration of analyte, the true measured signal (no constant error) and the true value of k for five standards. As expected, the value of k is the same for each standard. In the fourth column a constant determinate error of +0.50 has been added to the measured signals. The corresponding values of k are shown in the last column. Note that a different value of k is obtained for each standard and that all values are greater than the true value. As we noted in Section 5B.2, this is a significant limitation to any single-point standardization. How do we find the best estimate for the relationship between the measured signal and the concentration of analyte in a multiple-point standardization? Figure 5.8 shows the data in Table 5.1 plotted as a normal calibration curve. Although the data appear to fall along a straight line, the actual calibration curve is not intuitively obvious. The process of mathematically determining the best equation for the calibration curve is called regression.

Smeas

5C.1 Linear Regression of Straight-Line Calibration Curves
A calibration curve shows us the relationship between the measured signal and the analyte’s concentration in a series of standards. The most useful calibration curve is a straight line since the method’s sensitivity is the same for all concentrations of analyte. The equation for a linear calibration curve is y = β0 + β1x linear regression A mathematical technique for fitting an equation, such as that for a straight line, to experimental data. residual error The difference between an experimental value and the value predicted by a regression equation.

5.12

where y is the signal and x is the amount of analyte. The constants β0 and β1 are the true y-intercept and the true slope, respectively. The goal of linear regression is to determine the best estimates for the slope, b1, and y-intercept, b0. This is accomplished by minimizing the residual error between the experimental valˆ ues, yi, and those values, yi, predicted by equation 5.12 (Figure 5.9). For obvious reasons, a regression analysis is also called a least-squares treatment. Several approaches to the linear regression of equation 5.12 are discussed in the following sections.

Chapter 5 Calibrations, Standardizations, and Blank Corrections

119

5C.2 Unweighted Linear Regression with Errors in y
The most commonly used form of linear regression is based on three assumptions: (1) that any difference between the experimental data and the calculated regression line is due to indeterminate errors affecting the values of y, (2) that these indeterminate errors are normally distributed, and (3) that the indeterminate errors in y do not depend on the value of x. Because we assume that indeterminate errors are the same for all standards, each standard contributes equally in estimating the slope and y-intercept. For this reason the result is considered an unweighted linear regression. The second assumption is generally true because of the central limit theorem outlined in Chapter 4. The validity of the two remaining assumptions is less certain and should be evaluated before accepting the results of a linear regression. In particular, the first assumption is always suspect since there will certainly be some indeterminate errors affecting the values of x. In preparing a calibration curve, however, it is not unusual for the relative standard deviation of the measured signal (y) to be significantly larger than that for the concentration of analyte in the standards (x). In such circumstances, the first assumption is usually reasonable. Finding the Estimated Slope and y-Intercept The derivation of equations for calculating the estimated slope and y-intercept can be found in standard statistical texts7 and is not developed here. The resulting equation for the slope is given as b1 = n ∑ x i yi – ∑ x i ∑ yi n ∑ x i2 – (∑ x i )2 5.13

Regression line

y ˆi

Residual error = yi – ˆ i y

yi
Total residual error =

∑(y – ˆy ) i i

2

Figure 5.9
Residual error in linear regression, where the filled circle shows the experimental value yi, and the open circle shows the predicted ˆ value yi.

and the equation for the y-intercept is b0 = ∑ yi – b1 ∑ x i n 5.14

Although equations 5.13 and 5.14 appear formidable, it is only necessary to evaluate four summation terms. In addition, many calculators, spreadsheets, and other computer software packages are capable of performing a linear regression analysis based on this model. To save time and to avoid tedious calculations, learn how to use one of these tools. For illustrative purposes, the necessary calculations are shown in detail in the following example.

EXAMPLE 5.10 Using the data from Table 5.1, determine the relationship between Smeas and CS by an unweighted linear regression. SOLUTION Equations 5.13 and 5.14 are written in terms of the general variables x and y. As you work through this example, remember that x represents the concentration of analyte in the standards (CS), and that y corresponds to the signal (Smeas). We begin by setting up a table to help in the calculation of the summation terms Σxi, Σyi, Σx i2, and Σxiyi which are needed for the calculation of b0 and b1

120

Modern Analytical Chemistry xi 0.000 0.100 0.200 0.300 0.400 0.500

yi
0.00 12.36 24.83 35.91 48.79 60.42

xi2
0.000 0.010 0.040 0.090 0.160 0.250

xiyi
0.000 1.236 4.966 10.773 19.516 30.210

Adding the values in each column gives Σxi = 1.500 Σyi = 182.31 Σx 2 = 0.550 i Σxiyi = 66.701

Substituting these values into equations 5.12 and 5.13 gives the estimated slope b1 = (6)(66.701) – (1.500)(182.31) = 120.706 (6)(0.550) – (1.500)2

and the estimated y-intercept b0 = 182.31 – (120.706)(1.500) = 0.209 6 Smeas = 120.70 × CS + 0.21 Note that for now we keep enough significant figures to match the number of decimal places to which the signal was measured. The resulting calibration curve is shown in Figure 5.10.
80

The relationship between the signal and the analyte, therefore, is

60

Smeas

40

20

0 0.0

0.1

0.2

0.3 CA

0.4

0.5

0.6

Figure 5.10
Normal calibration curve for the hypothetical data in Table 5.1, showing the regression line.

Uncertainty in the Regression Analysis As shown in Figure 5.10, the regression line need not pass through the data points (this is the consequence of indeterminate errors affecting the signal). The cumulative deviation of the data from the regression line is used to calculate the uncertainty in the regression due to

Chapter 5 Calibrations, Standardizations, and Blank Corrections indeterminate error. This is called the standard deviation about the regression, sr, and is given as sr = ˆ ∑ n=1 (yi – yi )2 i n–2 5.15

121

standard deviation about the regression The uncertainty in a regression analysis due to indeterminate error (sr).

ˆ where yi is the ith experimental value, and yi is the corresponding value predicted by the regression line ˆ yi = b0 + b1xi There is an obvious similarity between equation 5.15 and the standard deviation introduced in Chapter 4, except that the sum of squares term for sr is determined rela– ˆ tive to yi instead of y, and the denominator is n – 2 instead of n – 1; n – 2 indicates that the linear regression analysis has only n – 2 degrees of freedom since two paˆ rameters, the slope and the intercept, are used to calculate the values of yi. A more useful representation of uncertainty is to consider the effect of indeterminate errors on the predicted slope and intercept. The standard deviation of the slope and intercept are given as s b1 = n∑ x i2
2 ns r = – (∑ x i )2 2 sr ∑ (x i – x )2

5.16

s b0 =

2 s r ∑ x i2 = 2 n ∑ x i – (∑ x i )2

2 s r ∑ x i2 n ∑ (x i – x )2

5.17

These standard deviations can be used to establish confidence intervals for the true slope and the true y-intercept β1 = b1 ± tsb1 βo = bo ± tsb0 5.18 5.19

where t is selected for a significance level of α and for n – 2 degrees of freedom. Note that the terms tsb1 and tsb0 do not contain a factor of ( n ) –1 because the confidence interval is based on a single regression line. Again, many calculators, spreadsheets, and computer software packages can handle the calculation of sb0 and sb1 and the corresponding confidence intervals for β0 and β1. Example 5.11 illustrates the calculations. EXAMPLE 5.11 Calculate the 95% confidence intervals for the slope and y-intercept determined in Example 5.10. SOLUTION Again, as you work through this example, remember that x represents the concentration of analyte in the standards (CS), and y corresponds to the signal (Smeas). To begin with, it is necessary to calculate the standard deviation about ˆ the regression. This requires that we first calculate the predicted signals, yi, using the slope and y-intercept determined in Example 5.10. Taking the first standard as an example, the predicted signal is ˆ yi = b0 + b1x = 0.209 + (120.706)(0.100) = 12.280

122

Modern Analytical Chemistry The results for all six solutions are shown in the following table. xi 0.000 0.100 0.200 0.300 0.400 0.500

yi
0.00 12.36 24.83 35.91 48.79 60.42

yi ˆ
0.209 12.280 24.350 36.421 48.491 60.562

(yi – yi)2 ˆ
0.0437 0.0064 0.2304 0.2611 0.0894 0.0202

Adding together the data in the last column gives the numerator of equation ˆ 5.15, Σ(y i – y i ) 2 , as 0.6512. The standard deviation about the regression, therefore, is sr = 0.6512 = 0.4035 6–2

Next we calculate sb1 and sb0 using equations 5.16 and 5.17. Values for the summation terms Σx2 and Σxi are found in Example 5.10. i s b1 = n∑ x i2
2 ns r = – (∑ x i )2

(6)(0.4035)2 = 0.965 (6)(0.550) – (1.500)2 (0.4035)2 (0.550) = 0.292 (6)(0.550) – (1.500)2

s b0 =

2 s r ∑ x i2 = n ∑ x i2 – (∑ x i )2

Finally, the 95% confidence intervals (α = 0.05, 4 degrees of freedom) for the slope and y-intercept are β1 = b1 ± tsb1 = 120.706 ± (2.78)(0.965) = 120.7 ± 2.7 β0 = b0 ± tsb0 = 0.209 ± (2.78)(0.292) = 0.2 ± 0.8 The standard deviation about the regression, sr, suggests that the measured signals are precise to only the first decimal place. For this reason, we report the slope and intercept to only a single decimal place.

To minimize the uncertainty in the predicted slope and y-intercept, calibration curves are best prepared by selecting standards that are evenly spaced over a wide range of concentrations or amounts of analyte. The reason for this can be rationalized by examining equations 5.16 and 5.17. For example, both sb0 and sb1 can be – minimized by increasing the value of the term Σ(xi – x)2, which is present in the denominators of both equations. Thus, increasing the range of concentrations used in preparing standards decreases the uncertainty in the slope and the y-intercept. Furthermore, to minimize the uncertainty in the y-intercept, it also is necessary to decrease the value of the term Σx2 in equation 5.17. This is accomplished by spreading i the calibration standards evenly over their range. Using the Regression Equation Once the regression equation is known, we can use it to determine the concentration of analyte in a sample. When using a normal calibration curve with external standards or an internal standards calibration curve, we – measure an average signal for our sample, YX, and use it to calculate the value of X

Chapter 5 Calibrations, Standardizations, and Blank Corrections X = YX – b0 b1

123

5.20 The standard deviation for the calculated value of X is given by the following equation 1/ 2   1 (YX – y )2  sr  1 sX = 5.21   + + 2 b1  m n b 1 ∑ (x i – x )2    – where m is the number of replicate samples used to establish YX, n is the number of – is the average signal for the standards, and x and – are the calibration standards, y x i individual and mean concentrations of the standards.8 Once sX is known the confidence interval for the analyte’s concentration can be calculated as µX= X ± tsX where µX is the expected value of X in the absence of determinate errors, and the value of t is determined by the desired level of confidence and for n – 2 degrees of freedom. The following example illustrates the use of these equations for an analysis using a normal calibration curve with external standards.

EXAMPLE 5.12 Three replicate determinations are made of the signal for a sample containing an unknown concentration of analyte, yielding values of 29.32, 29.16, and 29.51. Using the regression line from Examples 5.10 and 5.11, determine the analyte’s concentration, CA, and its 95% confidence interval. SOLUTION The equation for a normal calibration curve using external standards is Smeas = b0 + b1 × CA – thus, Y X is the average signal of 29.33, and X is the analyte’s concentra– tion. Substituting the value of YX into equation 5.20 along with the estimated slope and the y-intercept for the regression line gives the analyte’s concentration as CA = X = YX – b0 29.33 – 0.209 = = 0.241 b1 120.706

To calculate the standard deviation for the analyte’s concentration, we must determine the values for – and Σ(xi – – 2. The former is just the average signal y x) for the standards used to construct the calibration curve. From the data in Table 5.1, we easily calculate that – is 30.385. Calculating Σ(xi – – 2 y x) looks formidable, but we can simplify the calculation by recognizing that this sum of squares term is simply the numerator in a standard deviation equation; thus, – Σ(xi – x)2 = s2(n – 1) where s is the standard deviation for the concentration of analyte in the standards used to construct the calibration curve. Using the data in Table 5.1, we find that s is 0.1871 and – Σ(x – x)2 = (0.1871)2(6 – 1) = 0.175 i 124

Modern Analytical Chemistry Substituting known values into equation 5.21 gives sA = sX 0.4035 = 120.706  1 1 (29.33 – 30.385)2   + +   3 6 (120.706)2 (0.175)   
1/ 2

= 0.0024

Finally, the 95% confidence interval for 4 degrees of freedom is µA = CA ± tsA = 0.241 ± (2.78)(0.0024) = 0.241 ± 0.007

In a standard addition the analyte’s concentration is determined by extrapolating the calibration curve to find the x-intercept. In this case the value of X is X = x -intercept =
Residual error

–b0 b1
1/ 2

and the standard deviation in X is
0

sX =

sr b1

 1 (y )2     + 2 n b1 ∑ (x i – x )2    

xi
(a)

Residual error

0

where n is the number of standards used in preparing the standard additions calibration curve (including the sample with no added standard), and – is the average y signal for the n standards. Because the analyte’s concentration is determined by extrapolation, rather than by interpolation, sX for the method of standard additions generally is larger than for a normal calibration curve. A linear regression analysis should not be accepted without evaluating the validity of the model on which the calculations were based. Perhaps the simplest way to evaluate a regression analysis is to calculate and plot the residual error for each value of x. The residual error for a single calibration standard, ri, is given as ˆ ri = yi – yi If the regression model is valid, then the residual errors should be randomly distributed about an average residual error of 0, with no apparent trend toward either smaller or larger residual errors (Figure 5.11a). Trends such as those shown in Figures 5.11b and 5.11c provide evidence that at least one of the assumptions on which the regression model is based are incorrect. For example, the trend toward larger residual errors in Figure 5.11b suggests that the indeterminate errors affecting y are not independent of the value of x. In Figure 5.11c the residual errors are not randomly distributed, suggesting that the data cannot be modeled with a straight-line relationship. Regression methods for these two cases are discussed in the following sections.

xi
(b)

Residual error

0

xi
(c)

5C.3 Weighted Linear Regression with Errors in y
Equations 5.13 for the slope, b1, and 5.14 for the y-intercept, b0, assume that indeterminate errors equally affect each value of y. When this assumption is false, as shown in Figure 5.11b, the variance associated with each value of y must be included when estimating β0 and β1. In this case the predicted slope and intercept are b1 = n ∑ w i x i y i – ∑ w i x i ∑ w i yi n ∑ w i x 2 – (∑ w i x i )2 i 5.22

Figure 5.11
Plot of the residual error in y as a function of x. The distribution of the residuals in (a) indicates that the regression model was appropriate for the data, and the distributions in (b) and (c) indicate that the model does not provide a good fit for the data.

Chapter 5 Calibrations, Standardizations, and Blank Corrections and b0 = ∑ w i yi – b1 ∑ w i x i n 5.23

125

where wi is a weighting factor accounting for the variance in measuring yi. Values of wi are calculated using equation 5.24. wi = ns –2 i ∑ s –2 i 5.24

where si is the standard deviation associated with yi. The use of a weighting factor ensures that the contribution of each pair of xy values to the regression line is proportional to the precision with which yi is measured. EXAMPLE 5.13 The following data were recorded during the preparation of a calibration curve, – where Smeas and s are the mean and standard deviation, respectively, for three replicate measurements of the signal.
CA
0.000 0.100 0.200 0.300 0.400 0.500

– Smeas
0.00 12.36 24.83 35.91 48.79 60.42

s
0.02 0.02 0.07 0.13 0.22 0.33

– Determine the relationship between Smeas and CA using a weighted linear regression model. SOLUTION Once again, as you work through this example, remember that x represents the concentration of analyte in the standards (C S ), and y corresponds to the – average signal (Smeas). We begin by setting up a table to aid in the calculation of the weighting factor. xi 0.000 0.100 0.200 0.300 0.400 0.500

yi
0.00 12.36 24.83 35.91 48.79 60.42

si
0.02 0.02 0.07 0.13 0.22 0.33

–2 si

wi
2.8339 2.8339 0.2313 0.0671 0.0234 0.0104

2500.00 2500.00 204.08 59.17 20.66 9.18

Adding together the values in the forth column gives

∑ s –2 i

= 5293.09

which is used to calculate the weights in the last column. As a check on the calculation, the sum of the weights in the last column should equal the number of calibration standards, n. In this case Σwi = 6.0000

126

Modern Analytical Chemistry After the individual weights have been calculated, a second table is used to aid in calculating the four summation terms in equations 5.22 and 5.23. xi 0.000 0.100 0.200 0.300 0.400 0.500

yi
0.00 12.36 24.83 35.91 48.79 60.42

wi
2.8339 2.8339 0.2313 0.0671 0.0234 0.0104

wixi
0.0000 0.2834 0.0463 0.0201 0.0094 0.0052

wiyi
0.0000 35.0270 5.7432 2.4096 1.1417 0.6284

2 wixi

wixiyi
0.0000 3.5027 1.1486 0.7229 0.4567 0.3142

0.0000 0.0283 0.0093 0.0060 0.0037 0.0026

Adding the values in the last four columns gives

Σwixi = 0.3644 Σwiyi = 44.9499 Σwixi2= 0.0499 Σwixiyi = 6.1451
Substituting these values into the equations 5.22 and 5.23 gives the estimated slope b1 = (6)(6.1451) – (0.3644)(44.9499) = 122.985 (6)(0.0499) – (0.3644)2

and the estimated y-intercept b0 = 44.9499 – (122.985)(0.3644) = 0.0224 6

The relationship between the signal and the concentration of the analyte, therefore, is – Smeas = 122.98 × CA + 0.02 with the calibration curve shown in Figure 5.12.
80

60

Smeas

40

20

0 0.0

0.1

0.2

0.3 CA

0.4

0.5

0.6

Figure 5.12
Weighted normal calibration curve for the data in Example 5.13. The lines through the data points show the standard deviation of the signal for the standards. These lines have been scaled by a factor of 50 so that they can be seen on the same scale as the calibration curve.

Chapter 5 Calibrations, Standardizations, and Blank Corrections Equations for calculating confidence intervals for the slope, the y-intercept, and the concentration of analyte when using a weighted linear regression are not as easily defined as for an unweighted linear regression.9 The confidence interval for the concentration of an analyte, however, will be at its optimum value when the analyte’s signal is near the weighted centroid, – of the calibration y, curve y = 1 n

127

∑ wi y i

5C.4 Weighted Linear Regression with Errors in Both x and y
If we remove the assumption that the indeterminate errors affecting a calibration curve are found only in the signal (y), then indeterminate errors affecting the preparation of standards containing known amounts of analyte (x) must be factored into the regression model. The solution for the resulting regression line is computationally more involved than that for either the unweighted or weighted regression lines, and is not presented in this text. The suggested readings at the end of the chapter list several papers discussing algorithms for this regression method.

5C.5 Curvilinear and Multivariate Regression
Regression models based on a straight line, despite their apparent complexity, use the simplest functional relationship between two variables. In many cases, calibration curves show a pronounced curvature at high concentrations of analyte (see Figure 5.3b). One approach to constructing a calibration curve when curvature exists is to seek a transformation function that will make the data linear. Logarithms, exponentials, reciprocals, square roots, and trigonometric functions have all been used in this capacity. A plot of y versus log x is a typical example. Such transformations are not without complications. Perhaps the most obvious is that data that originally has a uniform variance for the y values will not maintain that uniform variance when the variable is transformed. A more rigorous approach to developing a regression model for a nonlinear calibration curve is to fit a polynomial equation such as y = a + bx + cx2 to the data. Equations for calculating the parameters a, b, and c are derived in the same manner as that described earlier for the straight-line model.10 When a single polynomial equation cannot be fitted to the calibration data, it may be possible to fit separate polynomial equations to short segments of the calibration curve. The result is a single continuous calibration curve known as a spline function. The regression models considered earlier apply only to functions containing a single independent variable. Analytical methods, however, are frequently subject to determinate sources of error due to interferents that contribute to the measured signal. In the presence of a single interferent, equations 5.1 and 5.2 become Smeas = kAnA + kInI + Sreag Smeas = kACA + kICI + Sreag where kI is the interferent’s sensitivity, nI is the moles of interferent, and CI is the interferent’s concentration. Multivariate calibration curves can be prepared using standards that contain known amounts of analyte and interferent.11

128

Modern Analytical Chemistry

5D Blank Corrections
In discussing ways to standardize a method, we assumed that an appropriate reagent blank had been used to correct Smeas for signals originating from sources other than the analyte. At that time we did not ask an important question— “What constitutes an appropriate reagent blank?” Surprisingly, the answer is not intuitively obvious. In one study,12 analytical chemists were asked to evaluate a data set consisting of a normal calibration curve, three samples of different size but drawn from the same source, and an analyte-free sample (Table 5.3). At least four different approaches for correcting the signals were used by the participants: (1) ignore the correction entirely, which clearly is incorrect; (2) use the y-intercept of the calibration curve as a calibration blank, CB; (3) use the analyte-free sample as a reagent blank, RB; and (4) use both the calibration and reagent blanks. Equations for calculating the concentration of analyte using each approach are shown in Table 5.4, along with the resulting concentration for the analyte in each of the three samples. That all four methods give a different result for the concentration of analyte underscores the importance of choosing a proper blank but does not tell us which of the methods is correct. In fact, the variation within each method for the reported concentration of analyte indicates that none of these four methods has adequately corrected for the blank. Since the three samples were drawn from the same source, they must have the same true concentration of analyte. Since all four methods predict concentrations of analyte that are dependent on the size of the sample, we can conclude that none of these blank corrections has accounted for an underlying constant source of determinate error. To correct for all constant method errors, a blank must account for signals due to the reagents and solvent used in the analysis and any bias due to interac-

Table 5.3
Wsa
1.6667 5.0000 8.3333 9.5507 11.6667 18.1600 19.9333

Hypothetical Data Used to Study Procedures for Method Blanks
Sstand
0.2500 0.5000 0.7500 0.8413 1.0000 1.4870 1.6200

Sample Number
1 2 3

Wxb
62.4746 82.7915 103.1085 analyte-freec

Ssamp
0.8000 1.0000 1.2000 0.1000

Calibration equation: Sstand = 0.0750 × Ws + 0.1250
Source: Modified from Cardone, M. J. Anal. Chem. 1986, 58, 433–438. aW = weight of analyte used to prepare standard solution by diluting to a fixed volume, V. s bW = weight of sample used to prepare sample solution by diluting to a fixed volume, V. x cAnalyte-free sample prepared in the same fashion as samples, but without the analyte being present.

Chapter 5 Calibrations, Standardizations, and Blank Corrections tions between the analyte and the sample matrix. Both the calibration blank and the reagent blank correct for signals due to the reagents and solvents. Any difference in their values is due to the number and composition of samples contributing to the determination of the blank. Unfortunately, neither the calibration blank nor the reagent blank can correct for bias due to analyte–matrix interactions because the analyte is missing in the reagent blank, and the sample’s matrix is missing from the calibration blank. The true method blank must include both the matrix and the analyte and, consequently, can only be determined using the sample itself. One approach is to measure the signal for samples of different size and determine the regression line from a plot of signal versus the amount of sample. The resulting y-intercept gives the signal for the condition of no sample and is known as the total Youden blank.13 This is the true blank correction. The regression line for the sample data in Table 5.3 is Ssamp = 0.009844 × Wx + 0.185 giving a true blank correction of 0.185. Using this value to correct the signals gives identical values for the concentration of analyte in all three samples (see Table 5.4, bottom row). The total Youden blank is not encountered frequently in analytical work, because most chemists rely on a calibration blank when using calibration curves and rely on reagent blanks when using a single-point standardization. As long as any constant bias due to analyte–matrix interactions can be ignored, which is often the case, the accuracy of the method will not suffer. It is always a good idea, however, to check for constant sources of error, by analyzing samples of different sizes, before relying on either a calibration or reagent blank.

129

total Youden blank A blank that corrects the signal for analyte–matrix interactions.

Table 5.4

Equations and Resulting Concentrations for Different Approaches to Correcting for the Method Blank
Concentration of Analyte in
Equationa
CA = CA = CA = CA = CA = Ssamp Wa = Wx kW x Ssamp – CB Wa = Wx kW x Ssamp – RB Wa = Wx kW x Ssamp – CB – RB Wa = Wx kW x Ssamp – TYB Wa = Wx kW x

Approach for Correcting Method Blank
Ignore blank corrections Use calibration blank Use reagent blank Use both calibration and reagent blank

Sample 1
0.1707 0.1441 0.1494 0.1227

Sample 2
0.1610 0.1409 0.1449 0.1248

Sample 3
0.1552 0.1390 0.1422 0.1261

Use total Youden blank

0.1313

0.1313

0.1313

aC = concentration of analyte; W = weight of analyte; W = weight of sample; k = slope of calibration curve = 0.075 (see Table 5.3). A a x Abbreviations: CB = calibration blank = 0.125 (see Table 5.3); RB = reagent blank = 0.100 (see Table 5.3); TYB = total Youden blank = 0.185 (see text).

130

Modern Analytical Chemistry

5E KEY TERMS aliquot (p. 111) external standard (p. 109) internal standard (p. 116) linear regression (p. 118) matrix matching (p. 110) method of standard additions multiple-point standardization (p. 109) normal calibration curve (p. 109) primary reagent (p. 106) reagent grade (p. 107) residual error (p. 118) (p. 110) secondary reagent (p. 107) single-point standardization (p. 108) standard deviation about the regression (p. 121) total Youden blank (p. 129)

5F SUMMARY
In a quantitative analysis, we measure a signal and calculate the amount of analyte using one of the following equations. Smeas = knA + Sreag Smeas = kCA + Sreag To obtain accurate results we must eliminate determinate errors affecting the measured signal, Smeas, the method’s sensitivity, k, and any signal due to the reagents, Sreag. To ensure that Smeas is determined accurately, we calibrate the equipment or instrument used to obtain the signal. Balances are calibrated using standard weights. When necessary, we can also correct for the buoyancy of air. Volumetric glassware can be calibrated by measuring the mass of water contained or delivered and using the density of water to calculate the true volume. Most instruments have calibration standards suggested by the manufacturer. An analytical method is standardized by determining its sensitivity. There are several approaches to standardization, including the use of external standards, the method of standard addition, and the use of an internal standard. The most desirable standardization strategy is an external standardization. The method of standard additions, in which known amounts of analyte are added to the sample, is used when the sample’s matrix complicates the analysis. An internal standard, which is a species (not analyte) added to all samples and standards, is used when the procedure does not allow for the reproducible handling of samples and standards. Standardizations using a single standard are common, but also are subject to greater uncertainty. Whenever possible, a multiplepoint standardization is preferred. The results of a multiple-point standardization are graphed as a calibration curve. A linear regression analysis can provide an equation for the standardization. A reagent blank corrects the measured signal for signals due to reagents other than the sample that are used in an analysis. The most common reagent blank is prepared by omitting the sample. When a simple reagent blank does not compensate for all constant sources of determinate error, other types of blanks, such as the total Youden blank, can be used.

5G Suggested EXPERIMENTS

Experiments

The following exercises and experiments help connect the material in this chapter to the analytical laboratory.

Calibration—Volumetric glassware (burets, pipets, and volumetric flasks) can be calibrated in the manner described in Example 5.1. Most instruments have a calibration sample that can be prepared to verify the instrument’s accuracy and precision. For example, as described in this chapter, a solution of 60.06 ppm K2Cr2O7 in 0.0050 M H2SO4 should give an absorbance of 0.640 ± 0.010 at a wavelength of 350.0 nm when using 0.0050 M H2SO4 as a reagent blank. These exercises also provide practice with using volumetric glassware, weighing samples, and preparing solutions.

Standardization—External standards, standard additions, and internal standards are a common feature of many quantitative analyses. Suggested experiments using these standardization methods are found in later chapters. A good project experiment for introducing external standardization, standard additions, and the importance of the sample’s matrix is to explore the effect of pH on the quantitative analysis of an acid–base indicator. Using bromothymol blue as an example, external standards can be prepared in a pH 9 buffer and used to analyze samples buffered to different pHs in the range of 6–10. Results can be compared with those obtained using a standard addition.

Chapter 5 Calibrations, Standardizations, and Blank Corrections

131

5H PROBLEMS
1. In calibrating a 10-mL pipet, a measured volume of water was transferred to a tared flask and weighed, yielding a mass of 9.9814 g. (a) Calculate, with and without correcting for buoyancy, the volume of water delivered by the pipet. Assume that the density of water is 0.99707 g/cm3 and that the density of the weights is 8.40 g/cm3. (b) What are the absolute and relative errors introduced by failing to account for the effect of buoyancy? Is this a significant source of determinate error for the calibration of a pipet? Explain. 2. Repeat the questions in problem 1 for the case when a mass of 0.2500 g is measured for a solid that has a density of 2.50 g/cm3. 3. Is the failure to correct for buoyancy a constant or proportional source of determinate error? 4. What is the minimum density of a substance necessary to keep the buoyancy correction to less than 0.01% when using brass calibration weights with a density of 8.40 g/cm3? 5. Describe how you would use a serial dilution to prepare 100 mL each of a series of standards with concentrations of 1.000 × 10–5, 1.000 × 10–4, 1.000 × 10–3, and 1.000 × 10–2 M from a 0.1000 M stock solution. Calculate the uncertainty for each solution using a propagation of uncertainty, and compare to the uncertainty if each solution was prepared by a single dilution of the stock solution. Tolerances for different types of volumetric glassware and digital pipets are found in Tables 4.2 and 4.4. Assume that the uncertainty in the molarity of the stock solution is ±0.0002. 6. Three replicate determinations of the signal for a standard solution of an analyte at a concentration of 10.0 ppm give values of 0.163, 0.157, and 0.161 (arbitrary units), respectively. The signal for a method blank was found to be 0.002. Calculate the concentration of analyte in a sample that gives a signal of 0.118. 7. A 10.00-g sample containing an analyte was transferred to a 250-mL volumetric flask and diluted to volume. When a 10.00-mL aliquot of the resulting solution was diluted to 25.00 mL it was found to give a signal of 0.235 (arbitrary units). A second 10.00-mL aliquot was spiked with 10.00 mL of a 1.00ppm standard solution of the analyte and diluted to 25.00 mL. The signal for the spiked sample was found to be 0.502. Calculate the weight percent of analyte in the original sample. 8. A 50.00-mL sample containing an analyte gives a signal of 11.5 (arbitrary units). A second 50-mL aliquot of the sample, which is spiked with 1.00-mL of a 10.0-ppm standard solution of the analyte, gives a signal of 23.1. What is the concentration of analyte in the original sample? 9. An appropriate standard additions calibration curve based on equation 5.8 plots Sspike(Vo + Vs) on the y-axis and CsVs on the x-axis. Clearly explain why you cannot plot Sspike on the yaxis and Cs[Vs/(Vo + Vs)] on the x-axis. Derive equations for the slope and y-intercept, and explain how the amount of analyte in a sample can be determined from the calibration curve. 10. A standard sample was prepared containing 10.0 ppm of an analyte and 15.0 ppm of an internal standard. Analysis of the sample gave signals for the analyte and internal standard of 0.155 and 0.233 (arbitrary units), respectively. Sufficient internal standard was added to a sample to make it 15.0 ppm in the internal standard. Analysis of the sample yielded signals for the analyte and internal standard of 0.274 and 0.198, respectively. Report the concentration of analyte in the sample. 11. For each of the pairs of calibration curves in Figure 5.13 on page 132, select the calibration curve with the better set of standards. Briefly explain the reasons for your selections. The scales for the x-axes and y-axes are the same for each pair. 12. The following standardization data were provided for a series of external standards of Cd2+ that had been buffered to a pH of 4.6.14
[Cd2+] (nM) Smeas (nA) 15.4 4.8 30.4 11.4 44.9 18.2 59.0 26.6 72.7 32.3 86.0 37.7

(a) Determine the standardization relationship by a linear regression analysis, and report the confidence intervals for the slope and y-intercept. (b) Construct a plot of the residuals, and comment on their significance. At a pH of 3.7 the following data were recorded
[Cd2+] (nM) Smeas (nA) 15.4 15.0 30.4 42.7 44.9 58.5 59.0 77.0 72.7 101 86.0 118

(c) How much more or less sensitive is this method at the lower pH? (d) A single sample is buffered to a pH of 3.7 and analyzed for cadmium, yielding a signal of 66.3. Report the concentration of Cd2+ in the sample and its 95% confidence interval. 13. To determine the concentration of analyte in a sample, a standard additions was performed. A 5.00-mL portion of the sample was analyzed and then successive 0.10-mL spikes of a 600.0-ppb standard of the analyte

132

Modern Analytical Chemistry

Signal

CA
(a)

Signal

CA

Signal

CA
(b)

Signal

CA

Signal

CA
(c)

Signal

CA

Figure 5.13

Chapter 5 Calibrations, Standardizations, and Blank Corrections were added, analyzing after each spike. The following results were obtained
Volume of Spike (mL)
0.00 0.10 0.20 0.30

133

Signal (arbitrary units)
0.119 0.231 0.339 0.442

15. In Chapter 4 we used a paired t-test to compare two methods that had been used to independently analyze a series of samples of variable composition. An alternative approach is to plot the results for one method versus those for the other. If the two methods yield identical results, then the plot should have a true slope (β1) of 1.00 and a true y-intercept (β0) of 0.0. A t-test can be used to compare the actual slope and yintercept with these ideal values. The appropriate test statistic for the y-intercept is found by rearranging equation 5.18 t exp = β 0 – b0 s b0 = b0 s b0

Construct an appropriate standard additions calibration curve, and use a linear regression analysis to determine the concentration of analyte in the original sample and its 95% confidence interval. 14. Troost and Olavesen investigated the application of an internal standardization to the quantitative analysis of polynuclear aromatic hydrocarbons.15 The following results were obtained for the analysis of the analyte phenanthrene using isotopically labeled phenanthrene as an internal standard SA/SIS
CA/CIS
0.50 1.25 2.00 3.00 4.00

Rearranging equation 5.17 gives the test statistic for the slope t exp = β1 – b1 s b1 = 1.00 – b1 s b1

Reevaluate the data in problem 24 in Chapter 4 using the same significance level as in the original problem.* 16. Franke and co-workers evaluated a standard additions method for a voltammetric determination of Tl.16 A summary of their results is tabulated here. ppm Tl added
0.000 0.387 1.851 5.734 2.53 8.42 29.65 84.8

Replicate 1
0.514 0.993 1.486 2.044 2.342

Replicate 2
0.522 1.024 1.471 2.080 2.550

Instrument Response for Replicates (µA)
2.50 7.96 28.70 85.6 2.70 8.54 29.05 86.0 2.63 8.18 28.30 85.2 2.70 7.70 29.20 84.2 2.80 8.34 29.95 86.4 2.52 7.98 28.95 87.8

(a) Determine the standardization relationship by a linear regression, and report the confidence intervals for the slope and y-intercept. (b) Based on your results, explain why the authors of this paper concluded that the internal standardization was inappropriate.

Determine the standardization relationship using a weighted linear regression.

5I SUGGESTED READINGS
In addition to the texts listed as suggested readings in Chapter 4, the following text provides additional details on regression Draper, N. R.; Smith, H. Applied Regression Analysis, 2nd. ed. Wiley: New York, 1981. Several articles providing more details about linear regression follow. Boqué, R.; Rius, F. X.; Massart, D. L. “Straight Line Calibration: Something More Than Slopes, Intercepts, and Correlation Coefficients,” J. Chem. Educ. 1993, 70, 230–232. Henderson, G. “Lecture Graphic Aids for Least-Squares Analysis,” J. Chem. Educ. 1988, 65, 1001–1003. Renman, L., Jagner, D. “Asymmetric Distribution of Results in Calibration Curve and Standard Addition Evaluations,” Anal. Chim. Acta 1997, 357, 157–166. Two useful papers providing additional details on the method of standard additions are Bader, M. “A Systematic Approach to Standard Addition Methods in Instrumental Analysis,” J. Chem. Educ. 1980, 57, 703–706.

*Although this is a commonly used procedure for comparing two methods, it does violate one of the assumptions of an ordinary linear regression. Since both methods are expected to have indeterminate errors, an unweighted regression with errors in y may produce a biased result, with the slope being underestimated and the y-intercept being overestimated. This limitation can be minimized by placing the more precise method on the x-axis, using ten or more samples to increase the degrees of freedom in the analysis, and by using samples that uniformly cover the range of concentrations. For more information see Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry, 3rd ed. Ellis Horwood PTR Prentice-Hall: New York, 1993. Alternative approaches are discussed in Hartman, C.; Smeyers-Verbeke, J.; Penninckx, W.; Massart, D. L. Anal. Chim. Acta 1997, 338, 19–40 and Zwanziger, H. W.; Sârbu, C. Anal. Chem. 1998, 70, 1277–1280.

134

Modern Analytical Chemistry
Harris, D. C. “Nonlinear Least-Squares Curve Fitting with Microsoft Excel Solver,” J. Chem. Educ. 1998, 75, 119–121. Lieb, S. G. “Simplex Method of Nonlinear Least-Squares—A Logical Complementary Method to Linear Least-Squares Analysis of Data,” J. Chem. Educ. 1997, 74, 1008–1011. Machuca-Herrera, J. G. “Nonlinear Curve Fitting with Spreadsheets,” J. Chem. Educ. 1997, 74, 448–449. Zielinski, T. J.; Allendoerfer, R. D. “Least Squares Fitting of Nonlinear Data in the Undergraduate Laboratory,” J. Chem. Educ. 1997, 74, 1001–1007. More information on multivariate regression can be found in Lang, P. M.; Kalivas, J. H. “A Global Perspective on Multivariate Calibration Methods,” J. Chemometrics 1993, 7, 153–164. Kowalski, B. R.; Seasholtz, M. B. “Recent Developments in Multivariate Calibration,” J. Chemometrics 1991, 5 129–145. An additional discussion on method blanks is found in the following two papers. Cardone, M. J. “Detection and Determination of Error in Analytical Methodology. Part II. Correction for Corrigible Systematic Error in the Course of Real Sample Analysis,” J. Assoc. Off. Anal. Chem. 1983, 66, 1283–1294. Cardone, M. J. “Detection and Determination of Error in Analytical Methodology. Part IIB. Direct Calculational Technique for Making Corrigible Systematic Error Corrections,” J. Assoc. Off. Anal. Chem. 1985, 68, 199–202.

Nimura, Y.; Carr, M. R. “Reduction of the Relative Error in the Standard Additions Method,” Analyst 1990, 115, 1589–1595. The following paper discusses the importance of weighting experimental data when using linear regression Karolczak, M. “To Weight or Not to Weight? An Analyst’s Dilemma,” Curr. Separations 1995, 13, 98–104. Algorithms for performing a linear regression with errors in both x and y are discussed in Irvin, J. A.; Quickenden, T. L. “Linear Least Squares Treatment When There Are Errors in Both x and y,” J. Chem. Educ. 1983, 60, 711–712. Kalantar, A. H. “Kerrich’s Method for y = αx Data When Both y and x Are Uncertain,” J. Chem. Educ. 1991, 68, 368–370. Macdonald, J. R.; Thompson, W. J. “Least-Squares Fitting When Both Variables Contain Errors: Pitfalls and Possibilities,” Am. J. Phys. 1992, 60, 66–73. Ogren, P. J.; Norton, J. R. “Applying a Simple Linear LeastSquares Algorithm to Data with Uncertainties in Both Variables,” J. Chem. Educ. 1992, 69, A130–A131. The following paper discusses some of the problems that may be encountered when using linear regression to model data that have been mathematically transformed into a linear form. Chong, D. P. “On the Use of Least Squares to Fit Data in Linear Form,” J. Chem. Educ. 1994, 71, 489–490. The analysis of nonlinear data is covered in the following papers.

5J REFERENCES
1. Battino, R.; Williamson, A. G. J. Chem. Educ. 1984, 61, 51–52. 2. Ebel, S. Fresenius J. Anal. Chem. 1992, 342, 769. 3. ACS Committee on Environmental Improvement “Guidelines for Data Acquisition and Data Quality Evaluation in Environmental Chemistry,” Anal. Chem. 1980, 52, 2242–2249. 4. Moody, J. R.; Greenburg, P. R.; Pratt, K. W.; et al. Anal. Chem. 1988, 60, 1203A–1218A. 5. Committee on Analytical Reagents, Reagent Chemicals, 8th ed., American Chemical Society: Washington, DC, 1993. 6. Cardone, M. J.; Palmero, P. J.; Sybrandt, L. B. Anal. Chem. 1980, 52, 1187–1191. 7. Draper, N. R.; Smith, H. Applied Regression Analysis, 2nd ed. Wiley: New York, 1981. 8. (a) Miller, J. N. Analyst 1991, 116, 3–14; and (b) Sharaf, M. A.; Illman, D. L.; Kowalski, B. R. Chemometrics, Wiley-Interscience: New York, 1986; pp. 126–127. 9. Bonate, P. J. Anal. Chem. 1993, 65, 1367–1372. 10. (a) Sharaf, M. A.; Illman, D. L.; Kowalski, B. R. Chemometrics, Wiley-Interscience: New York, 1986; (b) Deming, S. N.; Morgan, S. L. Experimental Design: A Chemometric Approach, Elsevier: Amsterdam, 1987. 11. Beebe, K. R.; Kowalski, B. R. Anal. Chem. 1987, 59, 1007A–1017A. 12. Cardone, M. J. Anal. Chem. 1986, 58, 433–438. 13. Cardone, M. J. Anal. Chem. 1986, 58, 438–445. 14. Wojciechowski, M; Balcerzak, J. Anal. Chim. Acta 1991, 249, 433–445. 15. Troost, J. R.; Olavesen, E. Y. Anal. Chem. 1996, 68, 708–711. 16. Franke, J. P.; de Zeeuw, R. A.; Hakkert, R. Anal. Chem. 1978, 50, 1374–1380.

Chapter 6
Equilibrium Chemistry

R

egardless of the problem on which an analytical chemist is working, its solution ultimately requires a knowledge of chemistry and the ability to reason with that knowledge. For example, an analytical chemist developing a method for studying the effect of pollution on spruce trees needs to know, or know where to find, the structural and chemical differences between p-hydroxybenzoic acid and p-hydroxyacetophenone, two common phenols found in the needles of spruce trees (Figure 6.1). Chemical reasoning is a product of experience and is constructed from knowledge acquired in the classroom, the laboratory, and the chemical literature. The material in this text assumes familiarity with topics covered in the courses and laboratory work you have already completed. This chapter provides a review of equilibrium chemistry. Much of the material in this chapter should be familiar to you, but other ideas are natural extensions of familiar topics.

135

136
O

Modern Analytical Chemistry
OH O CH3

6A Reversible Reactions and Chemical Equilibria
In 1798, the chemist Claude Berthollet (1748–1822) accompanied a French military expedition to Egypt. While visiting the Natron Lakes, a series of salt water lakes carved from limestone, Berthollet made an observation that contributed to an important discovery. Upon analyzing water from the Natron Lakes, Berthollet found large quantities of common salt, NaCl, and soda ash, Na2CO3, a result he found surprising. Why would Berthollet find this result surprising and how did it contribute to an important discovery? Answering these questions provides an example of chemical reasoning and introduces the topic of this chapter. Berthollet “knew” that a reaction between Na2CO3 and CaCl2 goes to completion, forming NaCl and a precipitate of CaCO3 as products. Na2CO3 + CaCl2 → 2NaCl + CaCO3 Understanding this, Berthollet expected that large quantities of NaCl and Na2CO3 could not coexist in the presence of CaCO3. Since the reaction goes to completion, adding a large quantity of CaCl2 to a solution of Na2CO3 should produce NaCl and CaCO3, leaving behind no unreacted Na2CO3. In fact, this result is what he observed in the laboratory. The evidence from Natron Lakes, where the coexistence of NaCl and Na2CO3 suggests that the reaction has not gone to completion, ran counter to Berthollet’s expectations. Berthollet’s important insight was recognizing that the chemistry occurring in the Natron Lakes is the reverse of what occurs in the laboratory. CaCO3 + 2NaCl → Na2CO3 + CaCl2

OH (a) (b)

OH

Figure 6.1
Structures of (a) p-hydroxybenzoic acid and (b) p-hydroxyacetophenone.

CaCO3

Using this insight Berthollet reasoned that the reaction is reversible, and that the relative amounts of “reactants” and “products” determine the direction in which the reaction occurs, and the final composition of the reaction mixture. We recognize a reaction’s ability to move in both directions by using a double arrow when writing the reaction.
Ca2+

Grams

Na2CO3 + CaCl2

t 2NaCl + CaCO3

Time

Figure 6.2
Change in mass of undissolved Ca2+ and solid CaCO3 over time during the precipitation of CaCO3.

equilibrium A system is at equilibrium when the concentrations of reactants and products remain constant.

Berthollet’s reasoning that reactions are reversible was an important step in understanding chemical reactivity. When we mix together solutions of Na2CO3 and CaCl2, they react to produce NaCl and CaCO3. If we monitor the mass of dissolved Ca 2+ remaining and the mass of CaCO 3 produced as a function of time, the result will look something like the graph in Figure 6.2. At the start of the reaction the mass of dissolved Ca2+ decreases and the mass of CaCO3 increases. Eventually, however, the reaction reaches a point after which no further changes occur in the amounts of these species. Such a condition is called a state of equilibrium. Although a system at equilibrium appears static on a macroscopic level, it is important to remember that the forward and reverse reactions still occur. A reaction at equilibrium exists in a “steady state,” in which the rate at which any species forms equals the rate at which it is consumed.

6B Thermodynamics and Equilibrium Chemistry
Thermodynamics is the study of thermal, electrical, chemical, and mechanical forms of energy. The study of thermodynamics crosses many disciplines, including physics, engineering, and chemistry. Of the various branches of thermodynamics,

Chapter 6 Equilibrium Chemistry the most important to chemistry is the study of the changes in energy occurring during a chemical reaction. Consider, for example, the general equilibrium reaction shown in equation 6.1, involving the solutes A, B, C, and D, with stoichiometric coefficients a, b, c, and d. aA + bB

137

t cC + dD

6.1

By convention, species to the left of the arrows are called reactants, and those on the right side of the arrows are called products. As Berthollet discovered, writing a reaction in this fashion does not guarantee that the reaction of A and B to produce C and D is favorable. Depending on initial conditions, the reaction may move to the left, to the right, or be in a state of equilibrium. Understanding the factors that determine the final position of a reaction is one of the goals of chemical thermodynamics. Chemical systems spontaneously react in a fashion that lowers their overall free energy. At a constant temperature and pressure, typical of many bench-top chemical reactions, the free energy of a chemical reaction is given by the Gibb’s free energy function ∆G = ∆H – T ∆S 6.2 where T is the temperature in kelvins, and ∆G, ∆H, and ∆S are the differences in the Gibb’s free energy, the enthalpy, and the entropy between the products and reactants. Enthalpy is a measure of the net flow of energy, as heat, during a chemical reaction. Reactions in which heat is produced have a negative ∆H and are called exothermic. Endothermic reactions absorb heat from their surroundings and have a positive ∆H. Entropy is a measure of randomness, or disorder. The entropy of an individual species is always positive and tends to be larger for gases than for solids and for more complex rather than simpler molecules. Reactions that result in a large number of simple, gaseous products usually have a positive ∆S. The sign of ∆G can be used to predict the direction in which a reaction moves to reach its equilibrium position. A reaction is always thermodynamically favored when enthalpy decreases and entropy increases. Substituting the inequalities ∆H < 0 and ∆S > 0 into equation 6.2 shows that ∆G is negative when a reaction is thermodynamically favored. When ∆G is positive, the reaction is unfavorable as written (although the reverse reaction is favorable). Systems at equilibrium have a ∆G of zero. As a system moves from a nonequilibrium to an equilibrium position, ∆G must change from its initial value to zero. At the same time, the species involved in the reaction undergo a change in their concentrations. The Gibb’s free energy, therefore, must be a function of the concentrations of reactants and products. As shown in equation 6.3, the Gibb’s free energy can be divided into two terms. ∆G = ∆G° + RT ln Q 6.3 The first term, ∆G°, is the change in Gibb’s free energy under standard-state conditions; defined as a temperature of 298 K, all gases with partial pressures of 1 atm, all solids and liquids pure, and all solutes present with 1 M concentrations. The second term, which includes the reaction quotient, Q, accounts for nonstandard-state pressures or concentrations. For reaction 6.1 the reaction quotient is Q = [C]c [D]d [A]a [B]b 6.4

Gibb’s free energy A thermodynamic function for systems at constant temperature and pressure that indicates whether or not a reaction is favorable (∆G < 0), unfavorable (∆G > 0), or at equilibrium (∆G = 0). enthalpy A change in enthalpy indicates the heat absorbed or released during a chemical reaction at constant pressure. entropy A measure of disorder.

standard state Condition in which solids and liquids are in pure form, gases have partial pressures of 1 atm, solutes have concentrations of 1 M, and the temperature is 298 K.

where the terms in brackets are the molar concentrations of the solutes. Note that the reaction quotient is defined such that the concentrations of products are placed

138

Modern Analytical Chemistry in the numerator, and the concentrations of reactants are placed in the denominator. In addition, each concentration term is raised to a power equal to its stoichiometric coefficient in the balanced chemical reaction. Partial pressures are substituted for concentrations when the reactant or product is a gas. The concentrations of pure solids and pure liquids do not change during a chemical reaction and are excluded from the reaction quotient. At equilibrium the Gibb’s free energy is zero, and equation 6.3 simplifies to ∆G° = –RT ln K

equilibrium constant For a reaction at equilibrium, the equilibrium constant determines the relative concentrations of products and reactants.

where K is an equilibrium constant that defines the reaction’s equilibrium position. The equilibrium constant is just the numerical value obtained when substituting the concentrations of reactants and products at equilibrium into equation 6.4; thus, [C]c [D]d eq eq K = 6.5 [A]a [B]b eq eq where the subscript “eq” indicates a concentration at equilibrium. Although the subscript “eq” is usually omitted, it is important to remember that the value of K is determined by the concentrations of solutes at equilibrium. As written, equation 6.5 is a limiting law that applies only to infinitely dilute solutions, in which the chemical behavior of any species in the system is unaffected by all other species. Corrections to equation 6.5 are possible and are discussed in more detail at the end of the chapter.

6C Manipulating Equilibrium Constants
We will use two useful relationships when working with equilibrium constants. First, if we reverse a reaction’s direction, the equilibrium constant for the new reaction is simply the inverse of that for the original reaction. For example, the equilibrium constant for the reaction A + 2B

t AB2

K1 =

[AB2 ] [A][B]2

is the inverse of that for the reaction AB2

t A + 2B

K2 =

1 [A][B]2 = K1 [AB2 ]

Second, if we add together two reactions to obtain a new reaction, the equilibrium constant for the new reaction is the product of the equilibrium constants for the original reactions. A+C AC + C A + 2C

t AC t AC2

K1 = K2 =

[AC] [A][C] [AC 2 ] [AC][C]

t AC2

K3 = K1K2 =

[AC] [AC 2 ] [AC 2 ] × = [A][C] [AC][C] [A][C]2

Chapter 6 Equilibrium Chemistry EXAMPLE 6.1 Calculate the equilibrium constant for the reaction 2A + B given the following information Rxn 1: A + B Rxn 2: A + E Rxn 3: C + E Rxn 4: F + C SOLUTION The overall reaction is given as Rxn 1 + Rxn 2 – Rxn 3 + Rxn 4 If Rxn 3 is reversed, giving Rxn 5: B

139

t C + 3D
K1 = 0.40 K2 = 0.10 K3 = 2.0 K4 = 5.0

tD tC+D+F tB tD+B

tC+E

K5 =

1 1 = = 0.50 K3 2.0

then the overall reaction is Rxn 1 + Rxn 2 + Rxn 5 + Rxn 4 and the overall equilibrium constant is Koverall = K1 × K2 × K5 × K4 = 0.40 × 0.10 × 0.50 × 5.0 = 0.10

6D Equilibrium Constants for Chemical Reactions
Several types of reactions are commonly used in analytical procedures, either in preparing samples for analysis or during the analysis itself. The most important of these are precipitation reactions, acid–base reactions, complexation reactions, and oxidation–reduction reactions. In this section we review these reactions and their equilibrium constant expressions.

6D.1 Precipitation Reactions
A precipitation reaction occurs when two or more soluble species combine to form an insoluble product that we call a precipitate. The most common precipitation reaction is a metathesis reaction, in which two soluble ionic compounds exchange parts. When a solution of lead nitrate is added to a solution of potassium chloride, for example, a precipitate of lead chloride forms. We usually write the balanced reaction as a net ionic equation, in which only the precipitate and those ions involved in the reaction are included. Thus, the precipitation of PbCl2 is written as Pb2+(aq) + 2Cl–(aq) precipitate An insoluble solid that forms when two or more soluble reagents are combined.

t PbCl2(s)

In the equilibrium treatment of precipitation, however, the reverse reaction describing the dissolution of the precipitate is more frequently encountered. PbCl2(s)

t Pb2+(aq) + 2Cl–(aq)

140

Modern Analytical Chemistry The equilibrium constant for this reaction is called the solubility product, Ksp, and is given as Ksp = [Pb2+][Cl–]2 = 1.7 × 10–5 6.6

solubility product The equilibrium constant for a reaction in which a solid dissociates into its ions (Ksp).

Note that the precipitate, which is a solid, does not appear in the Ksp expression. It is important to remember, however, that equation 6.6 is valid only if PbCl2(s) is present and in equilibrium with the dissolved Pb2+ and Cl–. Values for selected solubility products can be found in Appendix 3A.

6D.2 Acid–Base Reactions acid A proton donor. base A proton acceptor.

A useful definition of acids and bases is that independently introduced by Johannes Brønsted (1879–1947) and Thomas Lowry (1874–1936) in 1923. In the Brønsted-Lowry definition, acids are proton donors, and bases are proton acceptors. Note that these definitions are interrelated. Defining a base as a proton acceptor means an acid must be available to provide the proton. For example, in reaction 6.7 acetic acid, CH3COOH, donates a proton to ammonia, NH3, which serves as the base. CH3COOH(aq) + NH3(aq)

t CH3COO–(aq) + NH4+(aq)

6.7

When an acid and a base react, the products are a new acid and base. For example, the acetate ion, CH3COO–, in reaction 6.7 is a base that reacts with the acidic ammonium ion, NH4+, to produce acetic acid and ammonia. We call the acetate ion the conjugate base of acetic acid, and the ammonium ion is the conjugate acid of ammonia. Strong and Weak Acids The reaction of an acid with its solvent (typically water) is called an acid dissociation reaction. Acids are divided into two categories based on the ease with which they can donate protons to the solvent. Strong acids, such as HCl, almost completely transfer their protons to the solvent molecules. HCl(aq) + H2O(l) → H3O+(aq) + Cl–(aq) In this reaction H2O serves as the base. The hydronium ion, H3O+, is the conjugate acid of H2O, and the chloride ion is the conjugate base of HCl. It is the hydronium ion that is the acidic species in solution, and its concentration determines the acidity of the resulting solution. We have chosen to use a single arrow (→) in place of the double arrows ( ) to indicate that we treat HCl as if it were completely dissociated in aqueous solutions. A solution of 0.10 M HCl is effectively 0.10 M in H3O+ and 0.10 M in Cl–. In aqueous solutions, the common strong acids are hydrochloric acid (HCl), hydroiodic acid (HI), hydrobromic acid (HBr), nitric acid (HNO3), perchloric acid (HClO4), and the first proton of sulfuric acid (H2SO4). Weak acids, of which aqueous acetic acid is one example, cannot completely donate their acidic protons to the solvent. Instead, most of the acid remains undissociated, with only a small fraction present as the conjugate base.

t

acid dissociation constant The equilibrium constant for a reaction in which an acid donates a proton to the solvent (Ka).

CH3COOH(aq) + H2O(l)

t H3O+(aq) + CH3COO–(aq)

The equilibrium constant for this reaction is called an acid dissociation constant, Ka, and is written as Ka = [H 3O + ][CH3COO – ] = 1.75 × 10 –5 [CH 3COOH]

Chapter 6 Equilibrium Chemistry Note that the concentration of H2O is omitted from the Ka expression because its value is so large that it is unaffected by the dissociation reaction.* The magnitude of Ka provides information about the relative strength of a weak acid, with a smaller Ka corresponding to a weaker acid. The ammonium ion, for example, with a Ka of 5.70 × 10–10, is a weaker acid than acetic acid. Monoprotic weak acids, such as acetic acid, have only a single acidic proton and a single acid dissociation constant. Some acids, such as phosphoric acid, can donate more than one proton and are called polyprotic weak acids. Polyprotic acids are described by a series of acid dissociation steps, each characterized by it own acid dissociation constant. Phosphoric acid, for example, has three acid dissociation reactions and acid dissociation constants. H 3 PO4 (aq) + H 2 O(l) Ka1 =

141

t H3O + (aq) + H2 PO4– (aq)

[H 2 PO4– ][H 3O + ] = 7.11 × 10 –3 [H 3 PO4 ]

H 2 PO4– (aq) + H 2 O(l) Ka2 =

t H3O + (aq) + HPO42 − (aq)

[HPO4 2 − ][H 3O + ] = 6.32 × 10 –8 [H 2 PO4– ]

HPO4 2 − (aq) + H 2 O(l) Ka3 =

t H3O + (aq) + PO43 − (aq)

[PO4 3 − ][H 3O + ] = 4.5 × 10 –13 [HPO4 2 − ]

The decrease in the acid dissociation constant from Ka1 to Ka3 tells us that each successive proton is harder to remove. Consequently, H3PO4 is a stronger acid than H2PO4–, and H2PO4– is a stronger acid than HPO42–. Strong and Weak Bases Just as the acidity of an aqueous solution is a measure of the concentration of the hydronium ion, H3O+, the basicity of an aqueous solution is a measure of the concentration of the hydroxide ion, OH–. The most common example of a strong base is an alkali metal hydroxide, such as sodium hydroxide, which completely dissociates to produce the hydroxide ion. NaOH(aq) → Na+(aq) + OH–(aq) Weak bases only partially accept protons from the solvent and are characterized by a base dissociation constant, Kb. For example, the base dissociation reaction and base dissociation constant for the acetate ion are CH 3COO – (aq) + H 2 O(l) Kb =

t OH – (aq) + CH3COOH(aq)

base dissociation constant The equilibrium constant for a reaction in which a base accepts a proton from the solvent (Kb).

[CH 3COOH][OH − ] = 5.71 × 10 –10 [CH 3COO – ]

Polyprotic bases, like polyprotic acids, also have more than one base dissociation reaction and base dissociation constant. Amphiprotic Species Some species can behave as either an acid or a base. For example, the following two reactions show the chemical reactivity of the bicarbonate ion, HCO3–, in water.
*The concentration of pure water is approximately 55.5 M

142

Modern Analytical Chemistry HCO3–(aq) + H2O(l)

t H3O+(aq) + CO32–(aq) HCO3–(aq) + H2O(l) t OH–(aq) + H2CO3(aq)

6.8 6.9

amphiprotic A species capable of acting as both an acid and a base.

A species that can serve as both a proton donor and a proton acceptor is called amphiprotic. Whether an amphiprotic species behaves as an acid or as a base depends on the equilibrium constants for the two competing reactions. For bicarbonate, the acid dissociation constant for reaction 6.8 Ka2 = 4.69 × 10–11 is smaller than the base dissociation constant for reaction 6.9. Kb2 = 2.25 × 10–8 Since bicarbonate is a stronger base than it is an acid (kb2 > ka2), we expect that aqueous solutions of HCO3– will be basic. Dissociation of Water Water is an amphiprotic solvent in that it can serve as an acid or a base. An interesting feature of an amphiprotic solvent is that it is capable of reacting with itself as an acid and a base. H2O(l) + H2O(l)

t H3O+(aq) + OH–(aq)
6.10

The equilibrium constant for this reaction is called water’s dissociation constant, Kw, Kw = [H3O+][OH–] which has a value of 1.0000 × 10–14 at a temperature of 24 °C. The value of Kw varies substantially with temperature. For example, at 20 °C, Kw is 6.809 × 10–15, but at 30 °C K w is 1.469 × 10 –14 . At the standard state temperature of 25 °C, K w is 1.008 × 10–14, which is sufficiently close to 1.00 × 10–14 that the latter value can be used with negligible error. The pH Scale An important consequence of equation 6.10 is that the concentrations of H3O+ and OH– are related. If we know [H3O+] for a solution, then [OH–] can be calculated using equation 6.10. EXAMPLE 6.2 What is the [OH–] if the [H3O+] is 6.12 × 10–5 M? SOLUTION [OH – ] = Kw 1.00 × 10 –14 = 1.63 × 10 –10 = 6.12 × 10 –5 [H 3 O + ]

pH Defined as pH = –log[H3O+].

Equation 6.10 also allows us to develop a pH scale that indicates the acidity of a solution. When the concentrations of H3O+ and OH– are equal, a solution is neither acidic nor basic; that is, the solution is neutral. Letting [H3O+] = [OH–] and substituting into equation 6.10 leaves us with Kw = [H3O+]2 = 1.00 × 10–14

Chapter 6 Equilibrium Chemistry Solving for [H3O+] gives [H 3 O+ ] = 1.00 × 10 –14 = 1.00 × 10 –7
1 2 3 4 5 6 pH 7 8 9 10 Milk of magnesia “Pure” rain Milk Neutral Blood Seawater Vinegar Gastric juice

143

A neutral solution has a hydronium ion concentration of 1.00 × 10–7 M and a pH of 7.00.* For a solution to be acidic, the concentration of H3O+ must be greater than that for OH–, or [H3O+] > 1.00 × 10–7 M The pH of an acidic solution, therefore, must be less than 7.00. A basic solution, on the other hand, will have a pH greater than 7.00. Figure 6.3 shows the pH scale along with pH values for some representative solutions. Tabulating Values for Ka and Kb A useful observation about acids and bases is that the strength of a base is inversely proportional to the strength of its conjugate acid. Consider, for example, the dissociation reactions of acetic acid and acetate. CH3COOH(aq) + H2O(l)

11 12 13 14 Household bleach

t H3O+(aq) + CH3COO–(aq) CH3COO–(aq) + H2O(l) t CH3COOH(aq) + OH–(aq)
2H2O(l)

6.11 6.12

Figure 6.3 pH scale showing values for representative solutions.

Adding together these two reactions gives

t H3O+(aq) + OH–(aq)

6.13

The equilibrium constant for equation 6.13 is Kw. Since equation 6.13 is obtained by adding together reactions 6.11 and 6.12, Kw may also be expressed as the product of Ka for CH3COOH and Kb for CH3COO–. Thus, for a weak acid, HA, and its conjugate weak base, A–, Kw = Ka × Kb 6.14

This relationship between Ka and Kb simplifies the tabulation of acid and base dissociation constants. Acid dissociation constants for a variety of weak acids are listed in Appendix 3B. The corresponding values of Kb for their conjugate weak bases are determined using equation 6.14.

EXAMPLE 6.3 Using Appendix 3B, calculate the following equilibrium constants (a) Kb for pyridine, C5H5N (b) Kb for dihydrogen phosphate, H2PO4– SOLUTION (a) K b, C 5H 5N = Kw Ka, C 5H 5NH + Kw Ka, H 3PO 4 = 1.00 × 10 –14 = 1.69 × 10 –9 5.90 × 10 –6 1.00 × 10 –14 = 1.41 × 10 –12 7.11 × 10 –3

(b ) K b , H

2PO 4

–

=

=

*The use of a p-function to express a concentration is covered in Chapter 2.

144

Modern Analytical Chemistry

6D.3 Complexation Reactions
A more general definition of acids and bases was proposed by G. N. Lewis (1875–1946) in 1923. The Brønsted–Lowry definition of acids and bases focuses on an acid’s proton-donating ability and a base’s proton-accepting ability. Lewis theory, on the other hand, uses the breaking and forming of covalent bonds to describe acid–base characteristics. In this treatment, an acid is an electron pair acceptor, and a base is an electron pair donor. Although Lewis theory can be applied to the treatment of acid–base reactions, it is more useful for treating complexation reactions between metal ions and ligands. The following reaction between the metal ion Cd2+ and the ligand NH3 is typical of a complexation reaction. Cd2+(aq) + 4(:NH3)(aq)

ligand A Lewis base that binds with a metal ion.

t Cd(:NH3)42+(aq)

6.15

formation constant The equilibrium constant for a reaction in which a metal and a ligand bind to form a metal–ligand complex (Kf).

The product of this reaction is called a metal–ligand complex. In writing the equation for this reaction, we have shown ammonia as :NH3 to emphasize the pair of electrons it donates to Cd2+. In subsequent reactions we will omit this notation. The formation of a metal–ligand complex is described by a formation constant, Kf. The complexation reaction between Cd2+ and NH3, for example, has the following equilibrium constant Kf = [Cd(NH 3 )42 + ] = 5.5 × 107 [Cd2 + ][NH 3 ]4 6.16

dissociation constant The equilibrium constant for a reaction in which a metal–ligand complex dissociates to form uncomplexed metal ion and ligand (Kd).

The reverse of reaction 6.15 is called a dissociation reaction and is characterized by a dissociation constant, Kd, which is the reciprocal of Kf. Many complexation reactions occur in a stepwise fashion. For example, the reaction between Cd2+ and NH3 involves four successive reactions

t Cd(NH3)2+(aq) Cd(NH3)2+(aq) + NH3(aq) t Cd(NH3)22+(aq) Cd(NH3)22+(aq) + NH3(aq) t Cd(NH3)32+(aq) Cd(NH3)32+(aq) + NH3(aq) t Cd(NH3)42+(aq)
Cd2+(aq) + NH3(aq)

6.17 6.18 6.19 6.20

stepwise formation constant The formation constant for a metal–ligand complex in which only one ligand is added to the metal ion or to a metal–ligand complex (Ki). cumulative formation constant The formation constant for a metal–ligand complex in which two or more ligands are simultaneously added to a metal ion or to a metal–ligand complex (βi).

This creates a problem since it no longer is clear what reaction is described by a formation constant. To avoid ambiguity, formation constants are divided into two categories. Stepwise formation constants, which are designated as Ki for the ith step, describe the successive addition of a ligand to the metal–ligand complex formed in the previous step. Thus, the equilibrium constants for reactions 6.17–6.20 are, respectively, K1, K2, K3, and K4. Overall, or cumulative formation constants, which are designated as βi, describe the addition of i ligands to the free metal ion. The equilibrium constant expression given in equation 6.16, therefore, is correctly identified as β4, where β4 = K1 × K2 × K3 × K4 In general βi = K1 × K2 × . . . × Ki Stepwise and cumulative formation constants for selected metal–ligand complexes are given in Appendix 3C.

Chapter 6 Equilibrium Chemistry Equilibrium constants for complexation reactions involving solids are defined by combining appropriate Ksp and Kf expressions. For example, the solubility of AgCl increases in the presence of excess chloride as the result of the following complexation reaction AgCl(s) + Cl–(aq)

145

t AgCl2–(aq)

6.21

This reaction can be separated into three reactions for which equilibrium constants are known—the solubility of AgCl, described by its Ksp AgCl(s)

t Ag+(aq) + Cl–(aq)

and the stepwise formation of AgCl2–, described by K1 and K2

t AgCl(aq) AgCl(aq) + Cl–(aq) t AgCl2–(aq)
Ag+(aq) + Cl–(aq) The equilibrium constant for reaction 6.21, therefore, is equal to Ksp × K1 × K2. EXAMPLE 6.4 Determine the value of the equilibrium constant for the reaction PbCl2(s) SOLUTION This reaction can be broken down into three reactions. The first of these reactions is the solubility of PbCl2, described by its Ksp PbCl2(s)

t PbCl2(aq)

t Pb2+(aq) + 2Cl–(aq)

and the second and third are the stepwise formation of PbCl2 (aq), described by K1 and K2

t PbCl+(aq) PbCl+(aq) + Cl–(aq) t PbCl2(aq)
Pb2+(aq) + Cl–(aq) Using values for Ksp, K1, and K2 from Appendices 3A and 3C, we find the equilibrium constant to be K = Ksp × K1 × K2 = (1.7 × 10–5)(38.9)(1.62) = 1.1 × 10–3

6D.4 Oxidation–Reduction Reactions
In a complexation reaction, a Lewis base donates a pair of electrons to a Lewis acid. In an oxidation–reduction reaction, also known as a redox reaction, electrons are not shared, but are transferred from one reactant to another. As a result of this electron transfer, some of the elements involved in the reaction undergo a change in oxidation state. Those species experiencing an increase in their oxidation state are oxidized, while those experiencing a decrease in their oxidation state are reduced. For example, in the following redox reaction between Fe3+ and oxalic acid, H2C2O4, iron is reduced since its oxidation state changes from +3 to +2. 2Fe3+(aq) + H2C2O4(aq) + 2H2O(l) redox reaction An electron-transfer reaction.

t 2Fe2+(aq) + 2CO2(g) + 2H3O+(aq)

6.22

146

Modern Analytical Chemistry Oxalic acid, on the other hand, is oxidized since the oxidation state for carbon increases from +3 in H2C2O4 to +4 in CO2. Redox reactions, such as that shown in equation 6.22, can be divided into separate half-reactions that individually describe the oxidation and the reduction processes. H2C2O4(aq) + 2H2O(l) → 2CO2(g) + 2H3O+(aq) + 2e– Fe3+(aq) + e– → Fe2+(aq) It is important to remember, however, that oxidation and reduction reactions always occur in pairs.* This relationship is formalized by the convention of calling the species being oxidized a reducing agent, because it provides the electrons for the reduction half-reaction. Conversely, the species being reduced is called an oxidizing agent. Thus, in reaction 6.22, Fe3+ is the oxidizing agent and H2C2O4 is the reducing agent. The products of a redox reaction also have redox properties. For example, the Fe2+ in reaction 6.22 can be oxidized to Fe3+, while CO2 can be reduced to H2C2O4. Borrowing some terminology from acid–base chemistry, we call Fe2+ the conjugate reducing agent of the oxidizing agent Fe3+ and CO2 the conjugate oxidizing agent of the reducing agent H2C2O4. Unlike the reactions that we have already considered, the equilibrium position of a redox reaction is rarely expressed by an equilibrium constant. Since redox reactions involve the transfer of electrons from a reducing agent to an oxidizing agent, it is convenient to consider the thermodynamics of the reaction in terms of the electron. The free energy, ∆G, associated with moving a charge, Q, under a potential, E, is given by ∆G = EQ Charge is proportional to the number of electrons that must be moved. For a reaction in which one mole of reactant is oxidized or reduced, the charge, in coulombs, is Q = nF where n is the number of moles of electrons per mole of reactant, and F is Faraday’s constant (96,485 C ⋅ mol–1). The change in free energy (in joules per mole; J/mol) for a redox reaction, therefore, is ∆G = –nFE 6.23 where ∆G has units of joules per mole. The appearance of a minus sign in equation 6.23 is due to a difference in the conventions for assigning the favored direction for reactions. In thermodynamics, reactions are favored when ∆G is negative, and redox reactions are favored when E is positive. The relationship between electrochemical potential and the concentrations of reactants and products can be determined by substituting equation 6.23 into equation 6.3 –nFE = –nFE° + RT ln Q

oxidation A loss of electrons. reduction A gain of electrons.

reducing agent A species that donates electrons to another species. oxidizing agent A species that accepts electrons from another species.

Nernst equation An equation relating electrochemical potential to the concentrations of products and reactants.

where E° is the electrochemical potential under standard-state conditions. Dividing through by –nF leads to the well-known Nernst equation.
*Separating a redox reaction into its half-reactions is useful if you need to balance the reaction. One method for balancing redox reactions is reviewed in Appendix 4.

Chapter 6 Equilibrium Chemistry E = Eo – RT ln Q nF

147

Substituting appropriate values for R and F, assuming a temperature of 25 °C (298 K), and switching from ln to log* gives the potential in volts as E = Eo – 0.05916 log Q n 6.24

The standard-state electrochemical potential, E°, provides an alternative way of expressing the equilibrium constant for a redox reaction. Since a reaction at equilibrium has a ∆G of zero, the electrochemical potential, E, also must be zero. Substituting into equation 6.24 and rearranging shows that Eo = RT log K nF 6.25

Standard-state potentials are generally not tabulated for chemical reactions, but are calculated using the standard-state potentials for the oxidation, E°ox, and reduction half-reactions, E°red. By convention, standard-state potentials are only listed for reduction half-reactions, and E° for a reaction is calculated as E°reac = E°red – E°ox where both E°red and E°ox are standard-state reduction potentials. Since the potential for a single half-reaction cannot be measured, a reference halfreaction is arbitrarily assigned a standard-state potential of zero. All other reduction potentials are reported relative to this reference. The standard half-reaction is 2H3O+(aq) + 2e–

t 2H2O(l) + H2(g)

Appendix 3D contains a listing of the standard-state reduction potentials for selected species. The more positive the standard-state reduction potential, the more favorable the reduction reaction will be under standard-state conditions. Thus, under standard-state conditions, the reduction of Cu2+ to Cu (E° = +0.3419) is more favorable than the reduction of Zn2+ to Zn (E° = –0.7618). EXAMPLE 6.5 Calculate (a) the standard-state potential, (b) the equilibrium constant, and (c) the potential when [Ag + ] = 0.020 M and [Cd 2+ ] = 0.050 M, for the following reaction taking place at 25 °C. Cd(s) + 2Ag+(aq) SOLUTION (a) In this reaction Cd is undergoing oxidation, and Ag+ is undergoing reduction. The standard-state cell potential, therefore, is Eo = EoAg + / Ag – EoCd 2 + /Cd = 0.7996 V – (–0.4030 V) = 1.2026 V (b) To calculate the equilibrium constant, we substitute the values for the standard-state potential and number of electrons into equation 6.25. 1.2026 =
*ln(x) = 2.303 log(x)

t Cd2+(aq) + 2Ag(s)

0.05916 log K 2

148

Modern Analytical Chemistry Solving for K gives the equilibrium constant as log K = 40.6558 K = 4.527 × 1040 (c) The potential when the [Ag+] is 0.020 M and the [Cd2+] is 0.050 M is calculated using equation 6.24 employing the appropriate relationship for the reaction quotient Q. E = E° – 0.05916 [Cd2 + ] log n [Ag + ]2 0.05916 (0.050) log 2 (0.020)2

= 1.2026 – = 1.14 V

6E Le Châtelier’s Principle
The equilibrium position for any reaction is defined by a fixed equilibrium constant, not by a fixed combination of concentrations for the reactants and products. This is easily appreciated by examining the equilibrium constant expression for the dissociation of acetic acid. Ka = [H 3O + ][CH3COO – ] = 1.75 × 10 –5 [CH 3COOH] 6.26

Le Châtelier’s principle When stressed, a system that was at equilibrium returns to its equilibrium state by reacting in a manner that relieves the stress.

As a single equation with three variables, equation 6.26 does not have a unique solution for the concentrations of CH3COOH, CH3COO–, and H3O+. At constant temperature, different solutions of acetic acid may have different values for [H3O+], [CH3COO–] and [CH3COOH], but will always have the same value of Ka. If a solution of acetic acid at equilibrium is disturbed by adding sodium acetate, the [CH3COO–] increases, suggesting an apparent increase in the value of Ka. Since Ka must remain constant, however, the concentration of all three species in equation 6.26 must change in a fashion that restores Ka to its original value. In this case, equilibrium is reestablished by the partial reaction of CH3COO– and H3O+ to produce additional CH3COOH. The observation that a system at equilibrium responds to a stress by reequilibrating in a manner that diminishes the stress, is formalized as Le Châtelier’s principle. One of the most common stresses that we can apply to a reaction at equilibrium is to change the concentration of a reactant or product. We already have seen, in the case of sodium acetate and acetic acid, that adding a product to a reaction mixture at equilibrium converts a portion of the products to reactants. In this instance, we disturb the equilibrium by adding a product, and the stress is diminished by partially reacting the excess product. Adding acetic acid has the opposite effect, partially converting the excess acetic acid to acetate. In our first example, the stress to the equilibrium was applied directly. It is also possible to apply a concentration stress indirectly. Consider, for example, the following solubility equilibrium involving AgCl AgCl(s)

t Ag+(aq) + Cl–(aq)

6.27

Chapter 6 Equilibrium Chemistry The effect on the solubility of AgCl of adding AgNO3 is obvious,* but what is the effect of adding a ligand that forms a stable, soluble complex with Ag+? Ammonia, for example, reacts with Ag+ as follows Ag+(aq) + 2NH3(aq)

149

t Ag(NH3)2+(aq)
Ag+
+

6.28

Adding ammonia decreases the concentration of as the Ag(NH3)2 complex forms. In turn, decreasing the concentration of Ag+ increases the solubility of AgCl as reaction 6.27 reestablishes its equilibrium position. Adding together reactions 6.27 and 6.28 clarifies the effect of ammonia on the solubility of AgCl, by showing that ammonia is a reactant. AgCl(s) + 2NH3(aq)

t Ag(NH3)2+(aq) + Cl–(aq)

6.29

EXAMPLE 6.6 What is the effect on the solubility of AgCl if HNO3 is added to the equilibrium solution defined by reaction 6.29? SOLUTION Nitric acid is a strong acid that reacts with ammonia as shown here HNO3(aq) + NH3(aq)

t NH4+(aq) + NO3–(aq)

Adding nitric acid lowers the concentration of ammonia. Decreasing ammonia’s concentration causes reaction 6.29 to move from products to reactants, decreasing the solubility of AgCl.

Increasing or decreasing the partial pressure of a gas is the same as increasing or decreasing its concentration.† The effect on a reaction’s equilibrium position can be analyzed as described in the preceding example for aqueous solutes. Since the concentration of a gas depends on its partial pressure, and not on the total pressure of the system, adding or removing an inert gas has no effect on the equilibrium position of a gas-phase reaction. Most reactions involve reactants and products that are dispersed in a solvent. If the amount of solvent is changed, either by diluting or concentrating the solution, the concentrations of all reactants and products either decrease or increase. The effect of these changes in concentration is not as intuitively obvious as when the concentration of a single reactant or product is changed. As an example, let’s consider how dilution affects the equilibrium position for the formation of the aqueous silver-amine complex (reaction 6.28). The equilibrium constant for this reaction is β2 = [Ag(NH 3 )2+ ]eq
2 [Ag + ]eq[NH 3 ]eq

6.30

*Adding AgNO3 decreases the solubility of AgCl. †The relationship between pressure and concentration can be deduced from the ideal gas law. Starting with PV = nRT, we solve for the molar concentration n P Molar concentration = = V RT Of course, this assumes an ideal gas (which is usually a reasonable assumption under normal laboratory conditions).

150

Modern Analytical Chemistry where the subscript “eq” is included for clarification. If a portion of this solution is diluted with an equal volume of water, each of the concentration terms in equation 6.30 is cut in half. Thus, the reaction quotient becomes Q = which we can rewrite as  0.5   [Ag(NH 3 )2+ ]eq  = 4 × β2 Q =  2   (0.5)3   [Ag + ][NH 3 ]eq  Since Q is greater than β2, equilibrium must be reestablished by shifting the reaction to the left, decreasing the concentration of Ag(NH3)2+. Furthermore, this new equilibrium position lies toward the side of the equilibrium reaction with the greatest number of solutes (one Ag+ ion and two molecules of NH3 versus the single metal–ligand complex). If the solution of Ag(NH3)2+ is concentrated, by evaporating some of the solvent, equilibrium is reestablished in the opposite direction. This is a general conclusion that can be applied to any reaction, whether gas-phase, liquid-phase, or solid-phase. Increasing volume always favors the direction producing the greatest number of particles, and decreasing volume always favors the direction producing the fewest particles. If the number of particles is the same on both sides of the equilibrium, then the equilibrium position is unaffected by a change in volume. (0.5)[Ag(NH 3 )2+ ]eq
2 (0.5)[Ag + ]eq (0.5)2 [NH 3 ]eq

6F Ladder Diagrams
When developing or evaluating an analytical method, we often need to understand how the chemistry taking place affects our results. We have already seen, for example, that adding NH3 to a solution of Ag+ is a poor idea if we intend to isolate the Ag + as a precipitate of AgCl (reaction 6.29). One of the primary sources of determinate method errors is a failure to account for potential chemical interferences. In this section we introduce the ladder diagram as a simple graphical tool for evaluating the chemistry taking place during an analysis.1 Using ladder diagrams, we will be able to determine what reactions occur when several reagents are combined, estimate the approximate composition of a system at equilibrium, and evaluate how a change in solution conditions might affect our results.

ladder diagram A visual tool for evaluating systems at equilibrium.

6F.1 Ladder Diagrams for Acid–Base Equilibria
To see how a ladder diagram is constructed, we will use the acid–base equilibrium between HF and F– HF(aq) + H2O(l)

t H3O+(aq) + F–(aq)
[H 3O + ][F – ] [HF] [F – ] [HF]

for which the acid dissociation constant is Ka, HF =

Taking the log of both sides and multiplying through by –1 gives – log Ka, HF = – log [H 3O + ] – log

Chapter 6 Equilibrium Chemistry Finally, replacing the negative log terms with p-functions and rearranging leaves us with pH = pKa + log [F – ] [HF] 6.31
F–

151

Examining equation 6.31 tells us a great deal about the relationship between pH and the relative amounts of F– and HF at equilibrium. If the concentrations of F– and HF are equal, then equation 6.31 reduces to pH = pKa,HF = –log(Ka,HF) = –log(6.8 × 10–4) = 3.17 For concentrations of F– greater than that of HF, the log term in equation 6.31 is positive and
HF pH pH = pKa,HF = 3.17

pH > pKa,HF

or

pH > 3.17

This is a reasonable result since we expect the concentration of hydrofluoric acid’s conjugate base, F–, to increase as the pH increases. Similar reasoning shows that the concentration of HF exceeds that of F– when pH < pKa,HF or pH < 3.17 Figure 6.4
Ladder diagram for HF, showing areas of Now we are ready to construct the ladder diagram for HF (Figure 6.4). predominance for HF and F–. The ladder diagram consists of a vertical scale of pH values oriented so that smaller (more acidic) pH levels are at the bottom and larger (more basic) pH levels are at the top. A horizontal line is drawn at a pH equal to pKa,HF. This line, or step, separates the solution into regions where each of the two conjugate forms of HF predominate. By referring to the ladder diagram, we see that at a pH of 2.5 hydrofluoric acid will exist predominately as HF. If we add sufficient base to the solution such that the pH increases to 4.5, the predominate form becomes F–. NH3 Figure 6.5 shows a second ladder diagram containing information about HF/F– and NH4+/NH3. From this ladder diagram we see that if the pH is less than 3.17, the predominate species are HF and NH4+. For pH’s between 3.17 and 9.24 the predominate species are F– and NH4+, whereas above a pH of pH = pKa,NH3 = 9.24 9.24 the predominate species are F– and NH3. Ladder diagrams are particularly useful for evaluating the reactivity of acids and bases. An acid and a base cannot coexist if their respective areas of F– predominance do not overlap. If we mix together solutions of NH3 and HF, NH4+ the reaction

HF(aq) + NH3(aq)

t NH4+(aq) + F–(aq)

6.32

pH

pH = pKa,HF = 3.17

occurs because the predominance areas for HF and NH3 do not overlap. Before continuing, let us show that this conclusion is reasonable by calculating the equilibrium constant for reaction 6.32. To do so we need the following three reactions and their equilibrium constants. HF(aq) + H 2 O(l) NH 3 (aq) + H 2 O(l)

HF

t H3O + (aq) + F – (aq) t OH – (aq) + NH4+ (aq) t 2H2 O(l)
K =

Ka = 6.8 × 10 –4 K b = 1.75 × 10 –5 1 1 = Kw 1.00 × 10 –14

Figure 6.5
Ladder diagram for HF and NH3.

H 3O + (aq) + OH – (aq)

152

Modern Analytical Chemistry Adding together these reactions gives us reaction 6.32, for which the equilibrium constant is K = Ka K b (6.8 × 10 –4 )(1.75 × 10 –5 ) = 1.19 × 106 = Kw (1.00 × 10 –14 )

Since the equilibrium constant is significantly greater than 1, the reaction’s equilibrium position lies far to the right. This conclusion is general and applies to all ladder diagrams. The following example shows how we can use the ladder diagram in Figure 6.5 to evaluate the composition of any solution prepared by mixing together solutions of HF and NH3. EXAMPLE

6.7

Predict the pH and composition of a solution prepared by adding 0.090 mol of HF to 0.040 mol of NH3. SOLUTION Since HF is present in excess and the reaction between HF and NH 3 is favorable, the NH3 will react to form NH4+. At equilibrium, essentially no NH3 remains and Moles NH4+ = 0.040 mol Converting NH3 to NH4+ consumes 0.040 mol of HF; thus Moles HF = 0.090 – 0.040 = 0.050 mol Moles F– = 0.040 mol According to the ladder diagram for this system (see Figure 6.5), a pH of 3.17 results when there is an equal amount of HF and F–. Since we have more HF than F–, the pH will be slightly less than 3.17. Similar reasoning will show you that mixing together 0.090 mol of NH3 and 0.040 mol of HF will result in a solution whose pH is slightly larger than 9.24.

If the areas of predominance for an acid and a base overlap each other, then practically no reaction occurs. For example, if we mix together solutions of NaF and NH4Cl, we expect that there will be no significant change in the moles of F– and NH4+. Furthermore, the pH of the mixture must be between 3.17 and 9.24. Because F– and NH4+ can coexist over a range of pHs we cannot be more specific in estimating the solution’s pH. The ladder diagram for HF/F – also can be used to evaluate the effect of pH on other equilibria that include either HF or F–. For example, the solubility of CaF2 CaF2(s)

t Ca2+(aq) + 2F–(aq)

is affected by pH because F– is a weak base. Using Le Châtelier’s principle, if F– is converted to HF, the solubility of CaF2 will increase. To minimize the solubility of CaF2 we want to control the solution’s pH so that F– is the predominate species. From the ladder diagram we see that maintaining a pH of more than 3.17 ensures that solubility losses are minimal.

Chapter 6 Equilibrium Chemistry

153

6F.2 Ladder Diagrams for Complexation Equilibria
The same principles used in constructing and interpreting ladder diagrams for acid–base equilibria can be applied to equilibria involving metal–ligand complexes. For complexation reactions the ladder diagram’s scale is defined by the concentration of uncomplexed, or free ligand, pL. Using the formation of Cd(NH3)2+ as an example Cd2+(aq) + NH3(aq)

t Cd(NH3)2+(aq)
Cd2+ log K1 = 2.55 Cd(NH3)2+ log K2 = 2.01 p NH3 Cd(NH3)22+ log K3 = 1.34 Cd(NH3)32+

we can easily show that the dividing line between the predominance regions for Cd2+ and Cd(NH3)2+ is log(K1). [Cd(NH 3 )2 + ] K1 = [Cd2 + ][NH 3 ] log(K1 ) = log [Cd(NH 3 )2 + ] – log[NH 3 ] [Cd2 + ] [Cd(NH 3 )2 + ] + pNH3 [Cd2 + ] [Cd2 + ] [Cd(NH 3 )2 + ]

log(K1 ) = log

pNH3 = log(K1 ) + log

log K4 = 0.84 Cd(NH3)42+

Since K1 for Cd(NH3)2+ is 3.55 × 102, log(K1) is 2.55. Thus, for a pNH3 greater than 2.55 (concentrations of NH3 less than 2.8 × 10–3 M), Cd2+ is the predominate species. A complete ladder diagram for the metal–ligand complexes of Cd 2+ and NH3 is shown in Figure 6.6.

Figure 6.6
Ladder diagram for metal–ligand complexes of Cd2+ and NH3.

EXAMPLE 6.8 Using the ladder diagram in Figure 6.7, predict the result of adding 0.080 mol of Ca2+ to 0.060 mol of Mg(EDTA)2–. EDTA is an abbreviation for the ligand ethylenediaminetetraacetic acid. SOLUTION The predominance regions for Ca 2+ and Mg(EDTA) 2– do not overlap, therefore, the reaction Ca2+ + Mg(EDTA)2–

t Mg2+ + Ca(EDTA)2–

will take place. Since there is an excess of Ca2+, the composition of the final solution is approximately Moles Ca2+ = 0.080 – 0.060 = 0.020 mol Moles Ca(EDTA)2– = 0.060 mol

154

Modern Analytical Chemistry Moles Mg2+ = 0.060 mol Moles Mg(EDTA)2– = 0 mol
Ca2+

log Kf,Ca(EDTA)2– = 10.69

p EDTA Ca(EDTA)2–

Mg2+

log Kf,Mg(EDTA)2– = 8.79

Mg(EDTA)2–

Figure 6.7
Ladder diagram for metal–ligand complexes of ethylenediaminetetraacetic acid (EDTA) with Ca2+ and Mg2+.

We can also construct ladder diagrams using cumulative formation constants in place of stepwise formation constants. The first three stepwise formation constants for the reaction of Zn2+ with NH3

t Zn(NH3)2+(aq) K1 = 1.6 × 102 Zn(NH3)2+(aq) + NH3(aq) t Zn(NH3)22+(aq) K2 = 1.95 × 102 Zn(NH3)22+(aq) + NH3(aq) t Zn(NH3)32+(aq) K3 = 2.3 × 102
Zn2+(aq) + NH3(aq) show that the formation of Zn(NH3)32+ is more favorable than the formation of Zn(NH3)2+ or Zn(NH3)22+. The equilibrium, therefore, is best represented by the cumulative formation reaction Zn2+(aq) + 3NH3(aq) for which β3 = [Zn(NH 3 )32 + ] = 7.2 × 106 [Zn2 + ][NH 3 ]3 [Zn(NH 3 )32 + ] – 3 log [NH 3 ] [Zn2 + ]

t Zn(NH3)32+(aq)

Taking the log of each side gives log β3 = log

Chapter 6 Equilibrium Chemistry or pNH3 = 1 1 [Zn2 + ] log β3 + log 3 3 [Zn(NH 3 )32 + ]

155

Zn2+

The concentrations of Zn2+ and Zn(NH3)32+, therefore, are equal when pNH3 = 1 1 log β3 = log(7.2 × 106 ) = 2.29 3 3 pNH3 3 log β3 = 2.29

A complete ladder diagram for the Zn2+–NH3 system is shown in Figure 6.8.

Zn(NH3)32+

6F.3 Ladder Diagram for Oxidation–Reduction Equilibria
Ladder diagrams can also be used to evaluate equilibrium reactions in redox systems. Figure 6.9 shows a typical ladder diagram for two half-reactions in which the scale is the electrochemical potential, E. Areas of predominance are defined by the Nernst equation. Using the Fe3+/Fe2+ half-reaction as an example, we write E = E ° – 0.05916 log [Fe2 + ] [Fe2 + ] = +0.771V – 0.05916 log 3+] [Fe3 + ] [Fe Figure 6.8

log K4 = 2.03

Zn(NH3)42+

Ladder diagram for Zn2+, Zn(NH3)32+, and Zn(NH3)42+, showing how cumulative For potentials more positive than the standard-state potential, the predominate formation constants are included. 3+, whereas Fe2+ predominates for potentials more negative than E°. species is Fe When coupled with the step for the Sn4+/Sn2+ half-reaction, we see that Sn2+ can be used to reduce Fe3+. If an excess of Sn2+ is added, the potential of the resulting solution will be near +0.154 V. Using standard-state potentials to construct a ladder diagram can present problems if solutes are not at their standard-state concentraFe3+ tions. Because the concentrations of the reduced and oxidized species are in a logarithmic term, deviations from standard-state concentraE ° 3+/Fe2+ = +0.771 Fe tions can usually be ignored if the steps being compared are separated E 1b A trickier problem occurs when a half-reaction’s poby at least 0.3 V. tential is affected by the concentration of another species. For example, Fe2+ the potential for the following half-reaction

UO22+(aq) + 4H3O+(aq) + 2e–

t U4+(aq) + 6H2O(l)

Sn4+

depends on the pH of the solution. To define areas of predominance in this case, we begin with the Nernst equation E = 0.327 – 0.05916 [U 4 + ] log 2 [UO22 + ][H 3O + ]4

E ° 4+/Sn2+ = +0.154 Sn

Sn2+

and factor out the concentration of H3O+. E = 0.327 + 0.05916 0.05916 [U 4 + ] log [H 3O + ]4 – log 2 2 [UO22 + ] Figure 6.9
Ladder diagram for the Fe3+/Fe2+ and Sn4+/SN2+ halfreactions.

From this equation we see that the areas of predominance for UO22+ and U4+ are defined by a step whose potential is E = 0.327 + 0.05916 log [H 3O + ]4 = 0.327 – 0.1183 pH 2

Figure 6.10 shows how a change in pH affects the step for the UO22+/U4+ half-reaction.

156

Modern Analytical Chemistry

6G Solving Equilibrium Problems
UO2
2+

+0.327 V (pH = 0)

E

+0.209 V (pH = 1)

Ladder diagrams are a useful tool for evaluating chemical reactivity, usually providing a reasonable approximation of a chemical system’s composition at equilibrium. When we need a more exact quantitative description of the equilibrium condition, a ladder diagram may not be sufficient. In this case we can find an algebraic solution. Perhaps you recall solving equilibrium problems in your earlier coursework in chemistry. In this section we will learn how to set up and solve equilibrium problems. We will start with a simple problem and work toward more complex ones.

6G.1 A Simple Problem: Solubility of Pb(IO3)2 in Water
+0.090 V (pH = 2) U4+

Figure 6.10
Ladder diagram showing the effect of a change in pH on the areas of predominance for the UO22+/U4+ half-reaction.

When an insoluble compound such as Pb(IO3)2 is added to a solution a small portion of the solid dissolves. Equilibrium is achieved when the concentrations of Pb2+ and IO3– are sufficient to satisfy the solubility product for Pb(IO3)2. At equilibrium the solution is saturated with Pb(IO3)2. How can we determine the concentrations of Pb2+ and IO3–, and the solubility of Pb(IO3)2 in a saturated solution prepared by adding Pb(IO3)2 to distilled water? We begin by writing the equilibrium reaction Pb(IO3)2(s) and its equilibrium constant Ksp = [Pb2+][IO3–]2 = 2.5 × 10–13 6.33

t Pb2+(aq) + 2IO3–(aq)

As equilibrium is established, two IO3– ions are produced for each ion of Pb2+. If we assume that the molar concentration of Pb2+ at equilibrium is x then the molar concentration of IO3– is 2x. To help keep track of these relationships, we can use the following table.
PbI2(s)
Initial concentration Change in concentration Equilibrium concentration solid solid solid

t

Pb2+(aq)
0 +x 0+x=x

+

2IO3–(aq)
0 +2x 0 + 2x = 2x

Substituting the equilibrium concentrations into equation 6.33 (x)(2x)2 = 2.5 × 10–13 and solving gives 4x3 = 2.5 × 10–13 x = 3.97 × 10–5 The equilibrium concentrations of Pb2+ and IO3–, therefore, are [Pb2+] = x = 4.0 × 10–5 M [I–] = 2x = 7.9 × 10–5 M Since one mole of Pb(IO3)2 contains one mole of Pb2+, the solubility of Pb(IO3)2 is the same as the concentration of Pb2+ ; thus, the solubility of Pb(IO3 ) 2 is 4.0 × 10–5 M.

Chapter 6 Equilibrium Chemistry

157

6G.2 A More Complex Problem: The Common Ion Effect
Calculating the solubility of Pb(IO3)2 in distilled water is a straightforward problem since the dissolution of the solid is the only source of Pb2+ or IO3–. How is the solubility of Pb(IO 3 ) 2 affected if we add Pb(IO 3 ) 2 to a solution of 0.10 M Pb(NO3)2? Before we set up and solve the problem algebraically, think about the chemistry occurring in this system, and decide whether the solubility of Pb(IO3)2 will increase, decrease, or remain the same. This is a good habit to develop. Knowing what answers are reasonable will help you spot errors in your calculations and give you more confidence that your solution to a problem is correct. We begin by setting up a table to help us keep track of the concentrations of Pb2+ and IO3– in this system.
PbI2(s)
Initial concentration Change in concentration Equilibrium concentration solid solid solid

t

Pb2+(aq)
0.10 +x 0.10 + x = x

+

2IO3–(aq)
0 +2x 0 + 2x = 2x

Substituting the equilibrium concentrations into the solubility product expression (equation 6.33) (0.10 + x)(2x)2 = 2.5 × 10–13 and multiplying out the terms on the left leaves us with 4x3 + 0.40x2 = 2.5 × 10–13 6.34

This is a more difficult equation to solve than that for the solubility of Pb(IO3)2 in distilled water, and its solution is not immediately obvious. A rigorous solution to equation 6.34 can be found using available computer software packages and spreadsheets. How might we solve equation 6.34 if we do not have access to a computer? One possibility is that we can apply our understanding of chemistry to simplify the algebra. From Le Châtelier’s principle, we expect that the large initial concentration of Pb2+ will significantly decrease the solubility of Pb(IO3)2. In this case we can reasonably expect the equilibrium concentration of Pb2+ to be very close to its initial concentration; thus, the following approximation for the equilibrium concentration of Pb2+ seems reasonable [Pb2+] = 0.10 + x ≈ 0.10 M Substituting into equation 6.34 (0.10)(2x)2 = 2.5 × 10–13 and solving for x gives 0.40x2 = 2.5 × 10–13 x = 7.91 × 10–7 Before accepting this answer, we check to see if our approximation was reasonable. In this case the approximation 0.10 + x ≈ 0.10 seems reasonable since the difference between the two values is negligible. The equilibrium concentrations of Pb2+ and IO3–, therefore, are [Pb2+] = 0.10 + x ≈ 0.10 M [I–] = 2x = 1.6 × 10–6 M

158

Modern Analytical Chemistry The solubility of Pb(IO3)2 is equal to the additional concentration of Pb2+ in solution, or 7.9 × 10–7 mol/L. As expected, the solubility of Pb(IO3)2 decreases in the presence of a solution that already contains one of its ions. This is known as the common ion effect. As outlined in the following example, the process of making and evaluating approximations can be extended if the first approximation leads to an unacceptably large error. EXAMPLE 6.9 Calculate the solubility of Pb(IO3)2 in 1.0 × 10–4 M Pb(NO3)2. SOLUTION Letting x equal the change in the concentration of Pb 2+ , the equilibrium concentrations are [Pb2+] = 1.0 × 10–4 + x and (1.0 × 10–4 + x)(2x)2 = 2.5 × 10–13 We start by assuming that [Pb2+] = 1.0 × 10–4 + x ≈ 1.0 × 10–4 M and solve for x, obtaining a value of 2.50 × 10–5. Substituting back gives the calculated concentration of Pb2+ at equilibrium as [Pb2+] = 1.0 × 10–4 + 2.50 × 10–5 = 1.25 × 10–4 M a value that differs by 25% from our approximation that the equilibrium concentration is 1.0 × 10–4 M. This error seems unreasonably large. Rather than shouting in frustration, we make a new assumption. Our first assumption that the concentration of Pb 2+ is 1.0 × 10 –4 M was too small. The calculated concentration of 1.25 × 10–4 M, therefore, is probably a little too large. Let us assume that [Pb2+] = 1.0 × 10–4 + x ≈1.2 × 10–4 M Substituting into the solubility product equation and solving for x gives us x = 2.28 × 10–5 or a concentration of Pb2+ at equilibrium of [Pb2+] = 1.0 × 10–4 + (2.28 × 10–5) = 1.23 × 10–4 M which differs from our assumed concentration of 1.2 × 10–4 M by 2.5%. This seems to be a reasonable error since the original concentration of Pb2+ is given to only two significant figures. Our final solution, to two significant figures, is [Pb2+] = 1.2 × 10–4 M [IO3–] = 4.6 × 10–5 M and the solubility of Pb(IO3)2 is 2.3 × 10–5 mol/L. This iterative approach to solving an equation is known as the method of successive approximations. [IO3–] = 2x

common ion effect The solubility of an insoluble salt decreases when it is placed in a solution already containing one of the salt’s ions.

Chapter 6 Equilibrium Chemistry

159

6G.3 Systematic Approach to Solving Equilibrium Problems
Calculating the solubility of Pb(IO3)2 in a solution of Pb(NO3)2 was more complicated than calculating its solubility in distilled water. The necessary calculations, however, were still relatively easy to organize, and the assumption used to simplify the problem was fairly obvious. This problem was reasonably straightforward because it involved only a single equilibrium reaction, the solubility of Pb(IO3)2. Calculating the equilibrium composition of a system with multiple equilibrium reactions can become quite complicated. In this section we will learn how to use a systematic approach to setting up and solving equilibrium problems. As its name implies, a systematic approach involves a series of steps: 1. Write all relevant equilibrium reactions and their equilibrium constant expressions. 2. Count the number of species whose concentrations appear in the equilibrium constant expressions; these are your unknowns. If the number of unknowns equals the number of equilibrium constant expressions, then you have enough information to solve the problem. If not, additional equations based on the conservation of mass and charge must be written. Continue to add equations until you have the same number of equations as you have unknowns. 3. Decide how accurate your final answer needs to be. This decision will influence your evaluation of any assumptions you use to simplify the problem. 4. Combine your equations to solve for one unknown (usually the one you are most interested in knowing). Whenever possible, simplify the algebra by making appropriate assumptions. 5. When you obtain your final answer, be sure to check your assumptions. If any of your assumptions prove invalid, then return to the previous step and continue solving. The problem is complete when you have an answer that does not violate any of your assumptions. Besides equilibrium constant equations, two other types of equations are used in the systematic approach to solving equilibrium problems. The first of these is a mass balance equation, which is simply a statement of the conservation of matter. In a solution of a monoprotic weak acid, for example, the combined concentrations of the conjugate weak acid, HA, and the conjugate weak base, A–, must equal the weak acid’s initial concentration, CHA.* The second type of equation is a charge balance equation. A charge balance equation is a statement of solution electroneutrality. Total positive charge from cations = total negative charge from anions Mathematically, the charge balance expression is expressed as i =1 n m

mass balance equation An equation stating that matter is conserved, and that the total amount of a species added to a solution must equal the sum of the amount of each of its possible forms present in solution. charge balance equation An equation stating that the total concentration of positive charge in a solution must equal the total concentration of negative charge.

∑ (z + ) i × [M z + ] i = ∑ (z – ) j × [A z – ]j j =1

where i and j are, respectively, the concentrations of the ith cation and the jth anion, and (z+)i and (z –)j are the charges of the ith cation and the jth anion. Note that the concentration terms are multiplied by the absolute values of each ion’s charge, since electroneutrality is a conservation of charge, not concentration. Every ion in solution, even those not involved in any equilibrium
*You may recall that this is the difference between a formal concentration and a molar concentration.

[Mz+]

[Az–]

160

Modern Analytical Chemistry reactions, must be included in the charge balance equation. The charge balance equation for an aqueous solution of Ca(NO3)2 is 2 × [Ca2+] + [H3O+] = [OH–] + [NO3–] Note that the concentration of Ca2+ is multiplied by 2, and that the concentrations of H3O+ and OH– are also included. Charge balance equations must be written carefully since every ion in solution must be included. This presents a problem when the concentration of one ion in solution is held constant by a reagent of unspecified composition. For example, in many situations pH is held constant using a buffer. If the composition of the buffer is not specified, then a charge balance equation cannot be written. EXAMPLE 6.10 Write a mass balance and charge balance equations for a 0.10 M solution of NaHCO3. SOLUTION It is easier to keep track of what species are in solution if we write down the reactions that control the solution’s composition. These reactions are the dissolution of a soluble salt NaHCO3(s) → Na+(aq) + HCO3–(aq) and the acid–base dissociation reactions of HCO3– and H2O HCO3–(aq) + H2O(l)

t H3O+(aq) + CO32–(aq) HCO3–(aq) + H2O(l) t OH–(aq) + H2CO3(aq) 2H2O(l) t H3O+(aq) + OH–(aq)
0.10 M = [H2CO3] + [HCO3–] + [CO32–] 0.10 M = [Na+]

The mass balance equations are

The charge balance equation is [Na+] + [H3O+] = [OH–] + [HCO3–] + 2 × [CO32–]

6G.4 pH of a Monoprotic Weak Acid
To illustrate the systematic approach, let us calculate the pH of 1.0 M HF. Two equilbria affect the pH of this system. The first, and most obvious, is the acid dissociation reaction for HF HF(aq) + H2O(l)

t H3O+(aq) + F–(aq)
6.35

for which the equilibrium constant expression is Ka = [H 3O + ][F – ] = 6.8 × 10 –4 [HF]

Chapter 6 Equilibrium Chemistry The second equilibrium reaction is the dissociation of water, which is an obvious yet easily disregarded reaction 2H2O(l)

161

t H3O+(aq) + OH–(aq)
6.36 [OH–]). [F–], O+],

Kw = [H3O+][OH–] = 1.00 × 10–14

Counting unknowns, we find four ([HF], [H3 and To solve this problem, therefore, we need to write two additional equations involving these unknowns. These equations are a mass balance equation CHF = [HF] + [F–] and a charge balance equation [H3O+] = [F–] + [OH–] 6.38 6.37

We now have four equations (6.35, 6.36, 6.37, and 6.38) and four unknowns ([HF], [F–], [H3O+], and [OH–]) and are ready to solve the problem. Before doing so, however, we will simplify the algebra by making two reasonable assumptions. First, since HF is a weak acid, we expect the solution to be acidic; thus it is reasonable to assume that [H3O+] >> [OH–] simplifying the charge balance equation (6.38) to [H3O+] = [F–] [HF] >> [F–] Thus, the mass balance equation (6.36) simplifies to CHF = [HF] 6.40 6.39

Second, since HF is a weak acid we expect that very little dissociation occurs, and

For this exercise we will accept our assumptions if the error introduced by each assumption is less than ±5%. Substituting equations 6.39 and 6.40 into the equilibrium constant expression for the dissociation of HF (equation 6.35) and solving for the concentration of H3O+ gives us Ka = [H 3 O + ] = Ka CHF = [H 3O + ][H 3O + ] CHF (6.8 × 10 –4 )(1.0) = 2.6 × 10 –2 M

Before accepting this answer, we must verify that our assumptions are acceptable. The first assumption was that the [OH–] is significantly smaller than the [H3O+]. To calculate the concentration of OH– we use the Kw expression (6.36) [OH – ] = Kw 1.00 × 10 –14 = 3.8 × 10 –13 M = +] 2.6 × 10 –2 [H 3 O

Clearly this assumption is reasonable. The second assumption was that the [F–] is significantly smaller than the [HF]. From equation 6.39 we have [F–] = 2.6 × 10–2 M

162

Modern Analytical Chemistry Since the [F–] is 2.6% of CHF, this assumption is also within our limit that the error be no more than ±5%. Accepting our solution for the concentration of H3O+, we find that the pH of 1.0 M HF is 1.59. How does the result of this calculation change if we require our assumptions to have an error of less than ±1%. In this case we can no longer assume that [HF] >> [F–]. Solving the mass balance equation (6.37) for [HF] [HF] = CHF – [F–] and substituting into the Ka expression along with equation 6.39 gives Ka = [H 3O + ]2 CHF – [H 3O + ]

Rearranging leaves us with a quadratic equation [H3O+]2 = KaCHF – Ka[H3O+] [H3O+]2 + Ka[H3O+] – KaCHF = 0 which we solve using the quadratic formula x = –b ± b 2 – 4ac 2a

where a, b, and c are the coefficients in the quadratic equation ax2 + bx + c = 0. Solving the quadratic formula gives two roots, only one of which has any chemical significance. For our problem the quadratic formula gives roots of x = –6.8 × 10 –4 ± (6.8 × 10 –4 )2 – (4)(1)(–6.8 × 10 –4 )(1.0) 2(1) –6.8 × 10 –4 ± 5.22 × 10 –2 2

=

= 2.57 × 10 –2 or – 2.63 × 10 –2 Only the positive root has any chemical significance since the negative root implies that the concentration of H3O+ is negative. Thus, the [H3O+] is 2.6 × 10–2 M, and the pH to two significant figures is still 1.59. This same approach can be extended to find the pH of a monoprotic weak base, replacing Ka with Kb, CHF with the weak base’s concentration, and solving for the [OH–] in place of [H3O+]. EXAMPLE 6.11 Calculate the pH of 0.050 M NH3. State any assumptions made in simplifying the calculation, and verify that the error is less than 5%. SOLUTION Since NH3 is a weak base (Kb = 1.75 × 10–5), we assume that [OH–] >> [H3O+] and CNH3 = 0.050 M With these assumptions, we find (be sure to check the derivation) [OH – ] = K b CNH 3 = (1.75 × 10 –5 )(0.050) = 9.35 × 10 –4 M

Chapter 6 Equilibrium Chemistry Both assumptions are acceptable (again, verify that this is true). The concentration of H3O+ is calculated using Kw [H 3 O + ] = giving a pH of 10.97. Kw 1.00 × 10 –14 = 1.07 × 10 –11 = –] 9.35 × 10 –4 [OH

163

CH3
+H 3N

pKa1 = 2.348 COOH
+H 3N

CH3 C H
HL

pKa2 = 9.867 COO – H2N

CH3 C H
L–

C H
H2L+

COO –

Figure 6.11
Acid–base equilibria for the amino acid alanine.

6G.5 pH of a Polyprotic Acid or Base
A more challenging problem is to find the pH of a solution prepared from a polyprotic acid or one of its conjugate species. As an example, we will use the amino acid alanine whose structure and acid dissociation constants are shown in Figure 6.11.
CH3

pH of 0.10 M Alanine hydrochloride is a salt consisting of the diprotic weak acid H2L+ and Cl–. Because H2L+ has two acid dissociation reactions, a complete systematic solution to this problem will be more complicated than that for a monoprotic weak acid. Using a ladder diagram (Figure 6.12) can help us simplify the problem. Since the areas of predominance for H2L+ and L– are widely separated, we can assume that any solution containing an appreciable quantity of H2L+ will contain essentially no L–. In this case, HL is such a weak acid that H2L+ behaves as if it were a monoprotic weak acid. To find the pH of 0.10 M H2L+, we assume that [H3O+] >> [OH–] Because H2L+ is a relatively strong weak acid, we cannot simplify the problem further, leaving us with [H 3O + ]2 Ka = CH 2L+ – [H 3O + ] Solving the resulting quadratic equation gives the [H3O+] as 1.91 × 10–2 M or a pH of 1.72. Our assumption that [H3O+] is significantly greater than [OH–] is acceptable. pH of 0.10 M L– The alaninate ion is a diprotic weak base, but using the ladder diagram as a guide shows us that we can treat it as if it were a monoprotic weak base. Following the steps in Example 6.11 (which is left as an exercise), we find that the pH of 0.10 M alaninate is 11.42.

H2L+

H2N

C H
L–

COO –

9.867 CH3 pH
+H 3N

C H
HL

COO –

2.348 CH3
+H 3N

C H
H2L+

COOH

Figure 6.12
Ladder diagram for the amino acid alanine.

164

Modern Analytical Chemistry pH of 0.1 M HL Finding the pH of a solution of alanine is more complicated than that for H2L+ or L– because we must consider two equilibrium reactions involving HL. Alanine is an amphiprotic species, behaving as an acid HL(aq) + H2O(l) and a base HL(aq) + H2O(l) 2H2O(l)

t H3O+(aq) + L–(aq)

t OH–(aq) + H2L+(aq)

As always, we must also consider the dissociation of water

t H3O+(aq) + OH–(aq)
[H 3O + ][L– ] [HL]

This leaves us with five unknowns ([H2L+], [HL], [L–], [H3O+], and [OH–]), for which we need five equations. These equations are Ka2 and Kb2 for HL, Ka2 = K b2 = the Kw equation, Kw = [H3O+][OH–] a mass balance equation on HL, CHL = [H2L+] + [HL] + [L–] and a charge balance equation [H2L+] + [H3O+] = [OH–] + [L–] From the ladder diagram it appears that we may safely assume that the concentrations of H2L+ and L– are significantly smaller than that for HL, allowing us to simplify the mass balance equation to CHL = [HL] Next we solve Kb2 for [H2 [H 2 L+ ] = and Ka2 for [L–] [L– ] = Ka2 [HL] K C = a2 HL +] [H 3 O [H 3 O + ] L+] K w[HL] [HL][H 3O + ] CHL[H 3O + ] = = –] Ka1 Ka1 Ka1[OH

[OH – ][H 2 L+ ] Kw = [HL] Ka1

Substituting these equations, along with the equation for Kw, into the charge balance equation gives us CHL[H 3O + ] Kw K C + [H 3 O + ] = + a2 HL + Ka1 [H 3 O ] [H 3 O + ] which simplifies to C  1 [H 3O + ] HL + 1 = (K w + Ka2 CHL )  Ka1  [H 3 O + ] [H 3O + ]2 = Ka2 CHL + K w (CHL / Ka1 ) + 1 Ka1Ka2 CHL + Ka1K w CHL + Ka1 6.41

[H 3 O + ] =

Chapter 6 Equilibrium Chemistry We can simplify this equation further if Ka1Kw Ba2+ > Pb2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+ > Ag+ > K+ > NH4+ > Na+ > H+ > Li+ Note that highly charged ions bind more strongly than ions of lower charge. Within a group of ions of similar charge, those ions with a smaller hydrated radius (Table 6.1 in Chapter 6) or those that are more polarizable bind more strongly. For a strong base anion exchanger the general order is SO42– > I– > HSO4– > NO3– > Br– > NO2– > Cl– > HCO3– > CH3COO– > OH– > F– Again, ions of higher charge and smaller hydrated radius bind more strongly than ions with a lower charge and a larger hydrated radius. The mobile phase in IEC is usually an aqueous buffer, the pH and ionic composition of which determines a solute’s retention time. Gradient elutions are possible in which the ionic strength or pH of the mobile phase is changed with time. For example, an IEC separation of cations might use a dilute solution of HCl as the mobile phase. Increasing the concentration of HCl speeds the elution rate for more strongly retained cations, since the higher concentration of H+ allows it to compete more successfully for the ion-exchange sites. Ion-exchange columns can be substituted into the general HPLC instrument shown in Figure 12.26. The most common detector measures the conductivity of the mobile phase as it elutes from the column. The high concentration of electrolyte in the mobile phase is a problem, however, because the mobile-phase ions dominate the conductivity. For example, if a dilute solution of HCl is used as the mobile phase, the presence of large concentrations of H3O+ and Cl– produces a background conductivity that may prevent the detection of analytes eluting from the column. To minimize the mobile phase’s contribution to conductivity, an ion-suppressor column is placed between the analytical column and the detector. This column selectively removes mobile-phase electrolyte ions without removing solute ions. For example, in cation ion-exchange chromatography using a dilute solution of HCl as

ion-suppressor column A column used to minimize the conductivity of the mobile phase in ionexchange chromatography.

Chapter 12 Chromatographic and Electrophoretic Methods the mobile phase, the suppressor column contains an anion-exchange resin. The exchange reaction H+(aq) + Cl–(aq) + Resin+–OH–

593

t Resin+–Cl– + H2O(l)

replaces the ionic HCl with H2O. Analyte cations elute as hydroxide salts instead of as chloride salts. A similar process is used in anion ion-exchange chromatography in which a cation ion-exchange resin is placed in the suppressor column. If the mobile phase contains Na2CO3, the exchange reaction 2Na+(aq) + CO32–(aq) + 2Resin––H+

t 2Resin––Na+ + H2CO3(aq) single-column ion chromatography Ion-exchange chromatography in which conditions are adjusted so that an ionsuppressor column is not needed.

replaces a strong electrolyte, Na2CO3, with a weak electrolyte, H2CO3. Ion suppression is necessary when using a mobile phase containing a high concentration of ions. Single-column ion chromatography, in which an ion-suppressor column is not needed, is possible if the concentration of ions in the mobile phase can be minimized. Typically this is done by using a stationary phase resin with a low capacity for ion exchange and a mobile phase with a small concentration of ions. Because the background conductivity due to the mobile phase is sufficiently small, it is possible to monitor a change in conductivity as the analytes elute from the column. A UV/Vis absorbance detector can also be used if the solute ions absorb ultraviolet or visible radiation. Alternatively, solutions that do not absorb in the UV/Vis range can be detected indirectly if the mobile phase contains a UV/Vis-absorbing species. In this case, when a solute band passes through the detector, a decrease in absorbance is measured at the detector. Ion-exchange chromatography has found important applications in water analysis and in biochemistry. For example, Figure 12.34a shows how ion-exchange chromatography can be used for the simultaneous analysis of seven common anions in approximately 12 min. Before IEC, a complete analysis of the same set of anions required 1–2 days. Ion-exchange chromatography also has been used for the analysis of proteins, amino acids, sugars, nucleotides, pharmaceuticals, consumer products, and clinical samples. Several examples are shown in Figure 12.34.

12H Size-Exclusion Chromatography
Two classes of micron-sized stationary phases have been encountered in this section: silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 Å for silica particles and from 50 to 1,000,000 Å for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute’s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. The size selectivity of a particular packing is not infinite, but is limited to a moderate range. All solutes significantly smaller than the pores move through the column’s entire volume and elute simultaneously, with a retention volume, Vr, of Vr = Vi + Vo 12.32

size-exclusion chromatography A form of liquid chromatography in which the stationary phase is a porous material and in which separations are based on the size of the solutes.

where Vi is the volume of mobile phase occupying the packing material’s pore space, and Vo is volume of mobile phase in the remainder of the column. The maximum size for which equation 12.32 holds is the packing material’s inclusion limit,

inclusion limit In size-exclusion chromatography, the smallest solute that can be separated from other solutes; all smaller solutes elute together.

Monovalent and divalent cations, and transition metals Anion standards 2 1 5 1. Fluoride, (2 ppm) 2. Chloride, (4 ppm) 3. Nitrite, (4 ppm) 4. Bromide, (4 ppm) 5. Nitrate, (4 ppm) 6. Phosphate, (6 ppm) 7. Sulfate, (6 ppm) 2 3 4 7 8 9 6

CHROM 6355

3

4 5 6

7

1

1. Lithium (0.5 ppm) 2. Sodium (0.5 ppm) 3. Ammonium (0.5 ppm) 4. Potassium (0.8 ppm) 5. Nickel (5 ppm) 6. Zinc (5 ppm) 7. Cobalt (5 ppm) 8. Magnesium (0.35 ppm) and Manganese (0.35 ppm) 9. Calcium (0.7 ppm)

0

4

8

12 Min. 0 5 10 15 20 25 Min. Column: Mobile Phase: Flowrate: Detector: (b) Antifreeze analysis 10 1. Glycolate 2. Phosphate 3. Formate 4. Chloride 5. Nitrite 6. Bromide 7. Chlorate 8. Nitrate 9. Benzoate 10. Sulfate CHROM 5885 1 Universal cation 100 × 4.6 mm 2 mM Tartaric acid/1 mM oxalic acid 1.0 mL/min Conductivity Allsep™ Anion, 100 × 4.6 mm 0.7 mM NaHCO3:1.2 mM Na2CO3 1.0 mL/min 40°C Suppressed conductivity

Column: Mobile Phase: Flowrate: Temperature: Detector: (a)

Carnitine and choline in vitamins

CHROM 6291

4 1 5 2 3 6

7

8

1. Sodium (1 ppm) 2. L-Carnitine (3 ppm) 3. Choline (3 ppm) 4. Calcium, trace

9

2

3 4

0

2

4

6

8

10

12

14 Min.

0

10

20

30 Min. Universal cation, 100 × 4.6 mm 5 mM HCl 1.0 mL/min Conductivity

Column: Mobile Phase: Flowrate: Detector: (c)

Wescan Anion/S, 250 × 4.6 mm 4 mM Phthalic Acid, pH3.9 3.4 mL/min Conductivity

Column: Mobile Phase: Flowrate: Detector: (d)

Figure 12.34
Examples of the application of ion-exchange chromatography to the analysis of (a) inorganic anions, (b) inorganic cations, (c) antifreeze, and (d) vitamins. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL).

594

Chapter 12 Chromatographic and Electrophoretic Methods
Proteins 6 Proteins 3 1. Thyroglobulin 2. Holo-transferrin 3. Lysozyme 1 2 2 3 1 CHROM 8273 4 5

595
CHROM 5192

1. Thyroglobulin 2. Ferritin 3. Transferrin 4. b -Lactoglobulin 5. Myoglobin 6. Glycyl-L-Tyrosine

0

2

4

6

8

10 Min.

0

5 Column:

10

15

20

25 Min.

Column: Macrosphere GPC, 7.0µm, 300Å, 250 × 4.6mm Mobile Phase: 0.05M Potassium Phosphate Dibasic & 0.15M Sodium Sulfate, pH7.0 Flowrate: 0.5mL/min Detector: UV at 280nm (a) (b)

Macrosphere GPC 150/300/500Å, 7µm, 250 × 4.6mm (3) Mobile Phase: 0.05M KH2PO4 & 0.15M Na2SO4, pH7.0 Flowrate: 0.3mL/min Detector: UV at 280nm

Figure 12.35
Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column; that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL).

or permeation limit. All solutes too large to enter the pores elute simultaneously with a retention volume of Vr = Vo 12.33 exclusion limit In size-exclusion chromatography, the largest solute that can be separated from other solutes; all larger solutes elute together.

Equation 12.33 defines the packing material’s exclusion limit. In between the inclusion limit and the exclusion limit, each solute spends an amount of time in the pore space proportional to its size. The retention volume for a solute is Vr = Vo + DVi 12.34

where D is the solute’s distribution ratio, which ranges from 0 at the exclusion limit to 1 at the inclusion limit. The validity of equation 12.34 requires that size exclusion be the only interaction between the solute and the stationary phase responsible for the separation. To this end, silica particles used for size exclusion are deactivated as described earlier, and polymer resins are synthesized without exchange sites. Size-exclusion chromatography provides a rapid means for separating larger molecules, including polymers and biomolecules. Figure 12.35 shows the application of size-exclusion chromatography for the analysis of protein mixtures. In Figure 12.35a, a column packing with 300 Å pores, with an inclusion limit of 7500 g/mol and an exclusion limit of 1.2 × 106 g/mol, is used to separate a mixture of three proteins. Mixtures containing a wider range of formula weights can be separated by joining together several columns in series. Figure 12.35b shows an example spanning an inclusion limit of 4000 g/mol and an exclusion limit of 7.5 × 106 g/mol. Another important application is for the determination of formula weights. Calibration curves of log(formula weight) versus Vr are prepared between the exclusion limit and inclusion limit (Figure 12.36). Since the retention volume is, to some

596

Modern Analytical Chemistry degree, a function of a solute’s size and shape, reasonably accurate determinations of formula weight are possible only if the standards are carefully chosen to minimize the effect of shape. Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram.

log(Formula weight)

Exclusion limit

Inclusion limit

Retention volume

12I Supercritical Fluid Chromatography

Despite their importance, gas chromatography and liquid chromatography cannot be used to separate and analyze all types of samples. Gas chromatography, particuCalibration curve for the determination of larly when using capillary columns, provides for rapid separations with excellent formula weight by size-exclusion chromatography. resolution. Its application, however, is limited to volatile analytes or those analytes that can be made volatile by a suitable derivatization. Liquid chromatography can be used to separate a wider array of solutes; however, the most commonly used detectors (UV, fluorescence, and electrochemical) do not respond as universally as the flame ionization detector commonly used in gas chromatography. supercritical fluid chromatography Supercritical fluid chromatography (SFC) provides a useful alternative to gas A separation technique in which the chromatography and liquid chromatography for some samples. The mobile phase mobile phase is a supercritical fluid. in supercritical fluid chromatography is a gas held at a temperature and pressure exceeding its critical point (Figure 12.37). Under these conditions the mobile phase is neither a gas nor a liquid. Instead, the mobile phase is a supercritical fluid whose properties are intermediate between those of a gas and a liquid (Table 12.6). Specifically, supercritical fluids have viscosities that are similar to those of gases, which means that they can move through either capillary or packed columns without the need for the high pressures encountered in HPLC. Analysis time and resolution, although not as good as in GC, are usually better than that obtainable with conventional HPLC. The density of a supercritical fluid, however, is much closer to that of Supercritical Critical point a liquid, accounting for its ability to function as a solvent. The mobile phase in SFC, fluid therefore, behaves more like the liquid mobile phase in HPLC than the gaseous mobile phase in GC. Solid The most common mobile phase for supercritical fluid chromatography is Liquid CO2. Its low critical temperature, 31 °C, and critical pressure, 72.9 atm, are relatively easy to achieve and maintain. Although supercritical CO2 is a good solvent for Gas nonpolar organics, it is less useful for polar solutes. The addition of an organic Triple point modifier, such as methanol, improves the mobile phase’s elution strength. Other common mobile phases and their critical temperatures and pressures are listed in Temperature Table 12.7. Figure 12.37 Figure 12.36
Phase diagram for a supercritical fluid.

Pressure

Table 12.6
Colorplate 11 shows the phase transition of liquid CO2 to supercritical CO2.

Typical Properties of Gases, Liquids, and Supercritical Fluidsa
Density (g cm–3)
≈ 10–3 ≈ 0.1–1 ≈1

Phase gas supercritical fluid liquid a Viscosity (g cm–1 s–1)
≈ 10–4 ≈ 10–4–10–3 ≈ 10–2

Diffusion coefficient (cm2 s–1)
≈ 10–1 ≈ 10–4–10–3 < 10–5

Values are reported to the nearest factor of 10.

Chapter 12 Chromatographic and Electrophoretic Methods The instrumentation necessary for supercritical fluid chromatography is essentially the same as that for a standard GC or HPLC. The only important addition is the need for a pressure restrictor to maintain the critical pressure. Gradient elutions, similar to those in HPLC, are accomplished by changing the applied pressure over time. The resulting change in the density of the mobile phase affects its solvent strength. Detection can be accomplished using standard GC detectors or HPLC detectors. Supercritical fluid chromatography has found many applications in the analysis of polymers, fossil fuels, waxes, drugs, and food products. Its application in the analysis of triglycerides is shown in Figure 12.38.

597

Table 12.7
Compound
carbon dioxide ethane nitrous oxide ammonia diethyl ether isopropanol methanol ethanol water

Critical Point Properties for Selected Supercritical Fluids
Critical Temperature (°C)
31.3 32.4 36.5 132.3 193.6 235.3 240.5 243.4 374.4

Critical Pressure (atm)
72.9 48.3 71.4 111.3 36.3 47.0 78.9 63.0 226.8

12J Electrophoresis
Thus far all the separations we have considered involve a mobile phase and a stationary phase. Separation of a comTriglycerides by SFC CHROM plex mixture of analytes occurs because each analyte has a 8139 different ability to partition between the two phases. An 2 analyte whose distribution ratio favors the stationary 1 3 1. iso-Octane 5 2. Tripalmitin (16:0) phase is retained on the column for a longer time, thereby 3. Tristearin (18:0) 4 eluting with a longer retention time. Although the meth4. Triarachidin (20:0) 6 ods described in the preceding sections involve different 5. Tierucin (22:1) 6. Tribehenin (22:0) types of stationary and mobile phases, all are forms of 8 Min. 0 2 4 6 chromatography. Electrophoresis is another class of separation techColumn: Deltabond C8, 250 × 4.6 niques in which analytes are separated based on their Mobile Phase: 7% (v/v) Methanol-Modified CO2 ability to move through a conductive medium, usually an Flowrate: 2.0mL/min aqueous buffer, in response to an applied electric field. In Column Temp: 40°C Detector: ELSD the absence of other effects, cations migrate toward the electric field’s negatively charged cathode, and anions migrate toward the positively charged anode. More highly charged ions and ions of Figure 12.38 Example of the application of supercritical smaller size, which means they have a higher charge-to-size ratio, migrate at a fluid chromatography to the analysis of faster rate than larger ions, or ions of lower charge. Neutral species do not experitriglycerides. (Chromatogram courtesy of ence the electric field and remain stationary. As we will see shortly, under normal Alltech Associates, Inc. Deerfield, IL). conditions even neutral species and anions migrate toward the cathode. In either case, differences in their rate of migration allow for the separation of complex mixelectrophoresis A separation technique based on a tures of analytes. solute’s ability to move through a There are several forms of electrophoresis. In slab gel electrophoresis the conconductive medium under the influence ducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs of an electric field. are formed by pouring the gel between two glass plates separated by spacers. Typical thicknesses are 0.25–1 mm. Gel electrophoresis is an important technique in biochemistry, in which it is frequently used for DNA sequencing. Although it is a powerful tool for the qualitative analysis of complex mixtures, it is less useful for quantitative work. capillary electrophoresis In capillary electrophoresis the conducting buffer is retained within a capillary Electrophoresis taking place in a capillary tube. tube whose inner diameter is typically 25–75 µm. Samples are injected into one end of the capillary tube. As the sample migrates through the capillary, its components electropherogram separate and elute from the column at different times. The resulting electropheroThe equivalent of a chromatogram in electrophoresis. gram looks similar to the chromatograms obtained in GC or HPLC and provides

598

Modern Analytical Chemistry both qualitative and quantitative information. Only capillary electrophoretic methods receive further consideration in this text.

12J.1 Theory of Capillary Electrophoresis
In capillary electrophoresis the sample is injected into a buffered solution retained within a capillary tube. When an electric field is applied to the capillary tube, the sample’s components migrate as the result of two types of mobility: electrophoretic mobility and electroosmotic mobility. Electrophoretic mobility is the solute’s response to the applied electric field. As described earlier, cations move toward the negatively charged cathode, anions move toward the positively charged anode, and neutral species, which do not respond to the electric field, remain stationary. The other contribution to a solute’s migration is electroosmotic flow, which occurs when the buffer solution moves through the capillary in response to the applied electric field. Under normal conditions the buffer solution moves toward the cathode, sweeping most solutes, even anions, toward the negatively charged cathode. Electrophoretic Mobility The velocity with which a solute moves in response to the applied electric field is called its electrophoretic velocity, νep; it is defined as νep = µepE 12.35 where µep is the solute’s electrophoretic mobility, and E is the magnitude of the applied electric field. A solute’s electrophoretic mobility is defined as µ ep = q 6 πηr 12.36

electrophoretic mobility A measure of a solute’s ability to move through a conductive medium in response to an applied electric field (µep). electroosmotic flow The movement of the conductive medium in response to an applied electric field.

electrophoretic velocity The velocity with which a solute moves through the conductive medium due to its electrophoretic mobility (νep).

where q is the solute’s charge, η is the buffer solvent’s viscosity, and r is the solute’s radius. Using equations 12.35 and 12.36, we can make several important conclusions about a solute’s electrophoretic velocity. Electrophoretic mobility, and, therefore, electrophoretic velocity, is largest for more highly charged solutes and solutes of smaller size. Since q is positive for cations and negative for anions, these species migrate in opposite directions. Neutral species, for which q is 0, have an electrophoretic velocity of 0. Electroosmotic Mobility When an electric field is applied to a capillary filled with an aqueous buffer, we expect the buffer’s ions to migrate in response to their electrophoretic mobility. Because the solvent, H2O, is neutral, we might reasonably expect it to remain stationary. What is observed under normal conditions, however, is that the buffer solution moves toward the cathode. This phenomenon is called the electroosmotic flow. Electroosmosis occurs because the walls of the capillary tubing are electrically charged. The surface of a silica capillary contains large numbers of silanol groups (Si–OH). At pH levels greater than approximately 2 or 3, the silanol groups ionize to form negatively charged silanate ions (Si–O–). Cations from the buffer are attracted to the silanate ions. As shown in Figure 12.39, some of these cations bind tightly to the silanate ions, forming an inner, or fixed, layer. Other cations are more loosely bound, forming an outer, or mobile, layer. Together these two layers are called the double layer. Cations in the outer layer migrate toward the cathode. Because these cations are solvated, the solution is also pulled along, producing the electroosmotic flow.

Chapter 12 Chromatographic and Electrophoretic Methods
– + + – – – Double layer + – + – + + – +– + – – – + – + + – + – + – + + + – + + + – + + + – – + – + – + + + + – + – Bulk solution

599

–

+

+ –

Mobile layer Fixed layer

–

+–

Capillary wall + Anode Direction of electroosmotic flow – Cathode

Figure 12.39
Schematic diagram showing the origin of electroosmotic flow.

Electroosmotic flow velocity, νeof, is a function of the magnitude of the applied electric field and the buffer solution’s electroosmotic mobility, µeof. νeof = µeofE Electroosmotic mobility is defined as µ eof = εζ 4 πη 12.38 12.37

electroosmotic flow velocity The velocity with which the solute moves through the capillary due to the electroosmotic flow (νeof).

where ε is the buffer solution’s dielectric constant, ζ is the zeta potential, and η is the buffer solution’s viscosity. Examining equations 12.37 and 12.38 shows that the zeta potential plays an important role in determining the electroosmotic flow velocity. Two factors determine the zeta potential and, therefore, the electroosmotic velocity. First, the zeta potential is directly proportional to the charge on the capillary walls, with a greater density of silanate ions corresponding to a larger zeta potential. Below a pH of 2, for example, there are few silanate ions; thus, the zeta potential and electroosmotic flow velocity are 0. As the pH level is increased, both the zeta potential and the electroosmotic flow velocity increase. Second, the zeta potential is proportional to the thickness of the double layer. Increasing the buffer solution’s ionic strength provides a higher concentration of cations, decreasing the thickness of the double layer. The electroosmotic flow profile is very different from that for a phase moving under forced pressure. Figure 12.40 compares the flow profile for electroosmosis with that for hydrodynamic pressure. The uniform, flat profile for electroosmosis helps to minimize band broadening in capillary electrophoresis, thus improving separation efficiency. Total Mobility A solute’s net, or total velocity, νtot, is the sum of its electrophoretic velocity and the electroosmotic flow velocity; thus, νtot = νep + νeof and µtot = µep + µeof
+ Anode

zeta potential The change in potential across a double layer (ζ).

Hydrodynamic flow profile

(a)

(b) – Cathode Electroosmotic flow profile

Figure 12.40
Schematic showing a comparison of the flow profiles for (a) GC and HPLC, and (b) electrophoresis.

600

Modern Analytical Chemistry Under normal conditions the following relationships hold (νtot)cations > νeof (νtot)anions < νeof (νtot)neutrals = νeof Thus, cations elute first in an order corresponding to their electrophoretic mobilities, with small, highly charged cations eluting before larger cations of lower charge. Neutral species elute as a single band, with an elution rate corresponding to the electroosmotic flow velocity. Finally, anions are the last components to elute, with smaller, highly charged anions having the longest elution time. Migration Time A solute’s total velocity is given by ν tot = l tm

where l is the distance the solute travels between its point of injection and the detector, and tm is the migration time. Since νtot = µtotE = (µep + µeof)E we have, after rearranging, tm = l (µ ep + µ eof )E 12.39

Finally, the magnitude of the electric field is E = V L 12.40

where V is the applied potential, and L is the length of the capillary tube. Substituting equation 12.40 into equation 12.39 gives tm = (µ ep lL + µ eof )V 12.41

Examining equation 12.41 shows that we can decrease a solute’s migration time (and thus the total analysis time) by applying a higher voltage or by using a shorter capillary tube. Increasing the electroosmotic flow also shortens the analysis time, but, as we will see shortly, at the expense of resolution. Efficiency The efficiency of capillary electrophoresis is characterized by the number of theoretical plates, N, just as it is in GC or HPLC. In capillary electrophoresis, the number of theoretic plates is determined by N = (µ ep + µ eof )V 2D 12.42

where D is the solute’s diffusion coefficient. From equation 12.42 it is easy to see that the efficiency of a capillary electrophoretic separation increases with higher voltages. Again, increasing the electroosmotic flow velocity improves efficiency, but at the expense of resolution. Two additional observations deserve comment.

Chapter 12 Chromatographic and Electrophoretic Methods First, solutes with larger electrophoretic mobilities (in the same direction as the electroosmotic flow) have greater efficiencies; thus, smaller, more highly charged solutes are not only the first solutes to elute, but do so with greater efficiency. Second, efficiency in capillary electrophoresis is independent of the capillary’s length. Typical theoretical plate counts are approximately 100,000–200,000 for capillary electrophoresis. Selectivity In chromatography, selectivity is defined as the ratio of the capacity factors for two solutes (equation 12.11). In capillary electrophoresis, the analogous expression for selectivity is µ ep,1 α = µ ep,2 where µep,1 and µep,2 are the electrophoretic mobilities for solutes 1 and 2, respectively, chosen such that α ≥ 1. Selectivity often can be improved by adjusting the pH of the buffer solution. For example, NH4+ is a weak acid with a pKa of 9.24. At a pH of 9.24 the concentrations of NH4+ and NH3 are equal. Decreasing the pH below 9.24 increases its electrophoretic mobility because a greater fraction of the solute is present as the cation NH4+. On the other hand, raising the pH above 9.24 increases the proportion of the neutral NH3, decreasing its electrophoretic mobility. Resolution The resolution between two solutes is R= 0.177(µ ep,2 − µ ep,1 )V 1 / 2 (µ avg + µ eof )D Figure 12.41 12.43
Schematic diagram for capillary electrophoresis. The sample and source reservoir are switched when making injections.

601

Capillary tube Detector Cathode (–)

Anode (+)

Source Destination reservoir Sample reservoir Power supply

where µavg is the average electrophoretic mobility for the two solutes. Examining equation 12.43 shows that increasing the applied voltage and decreasing the electroosmotic flow velocity improves resolution. The latter effect is particularly important because increasing electroosmotic flow improves analysis time and efficiency while decreasing resolution.

Capillary opening

12J.2 Instrumentation
The basic instrumentation for capillary electrophoresis is shown in Figure 12.41 and includes a power supply for applying the electric field, anode and cathode compartments containing reservoirs of the buffer solution, a sample vial containing the sample, the capillary tube, and a detector. Each part of the instrument receives further consideration in this section. Capillary Tubes Figure 12.42 shows a cross section of a typical capillary tube. Most capillary tubes are made from fused silica coated with a 20–35-µm layer of polyimide to give it mechanical strength. The inner diameter is typically 25–75 µm, which is smaller than that for a capillary GC column, with an outer diameter of 200–375 µm. The narrow bore of the capillary column and the relative thickness of the capillary’s walls are important. When an electric field is applied to a capillary containing a conductive medium, such as a buffer solution, current flows through the capillary. This current leads to Joule heating, the extent of which is proportional to the capillary’s radius and the magnitude of the electric field. Joule heating is a problem because it changes the buffer solution’s viscosity, with the solution at the center of the
Polyimide coating Fused silica capillary

Figure 12.42
Schematic diagram showing a cross section of a capillary column for capillary electrophoresis.

Joule heating The heating of a conductive solution due to the passage of an electric current through the solution.

602

Modern Analytical Chemistry capillary being less viscous than that near the capillary walls. Since the solute’s electrophoretic mobility depends on the buffer’s viscosity (see equation 12.36), solutes in the center of the capillary migrate at a faster rate than solutes near the capillary walls. The result is additional band broadening that degrades the separation. Capillaries with smaller inner diameters generate less Joule heating, and those with larger outer diameters are more effective at dissipating the heat. Capillary tubes may be placed inside a thermostated jacket to control heating, in which case smaller outer diameters allow a more rapid dissipation of thermal energy. Injecting the Sample The mechanism by which samples are introduced in capillary electrophoresis is quite different from that used in GC or HPLC. Two types of injection are commonly used: hydrodynamic injection and electrokinetic injection. In both cases the capillary tube is filled with buffer solution. One end of the capillary tube is placed in the destination reservoir, and the other is placed in the sample vial. Hydrodynamic injection uses pressure to force a small portion of the sample into the capillary tubing. To inject a sample hydrodynamically a difference in pressure is applied across the capillary by either pressurizing the sample vial or by applying a vacuum to the destination reservoir. The volume of sample injected, in liters, is given by the following equation Vinj = ∆Pd 4 πt × 103 128 ηL 12.44

hydrodynamic injection An injection technique in capillary electrophoresis in which pressure is used to inject sample into the capillary column.

where ∆P is the pressure difference across the capillary in pascals, d is the capillary’s inner diameter in meters, t is the amount of time that the pressure is applied in seconds, η is the buffer solution’s viscosity in kilograms per meter per second (kg m–1 s–1), and L is the length of the capillary tubing in meters. The factor of 103 changes the units from cubic meters to liters. EXAMPLE 12.9 A hydrodynamic injection is made by applying a pressure difference of 2.5 × 103 Pa (approximately 0.02 atm) for 2 s to a 75-cm long capillary tube with an internal diameter of 50 µm. Assuming that the buffer solution’s viscosity is 10–3 kg m–1 s–1, what volume of sample is injected? SOLUTION Making appropriate substitutions into equation 12.44 gives the volume of injected sample as Vinj = (2.5 × 103 Pa)(50 × 10 −6 m)4 (3.14)(2 s) × 103 = 1 × 10 −9 L = 1 nL (128)(0.001 kg m −1 s −1 )(0.75 m)

Since the injected sample plug is cylindrical, its length, lplug, is easily calculated using the equation for the volume of a cylinder. V = πr2lplug Thus, lplug = (1 × 10 −9 L)(10 −3 m 3 / L) V = 5 × 10 −4 m = 0.5 mm = (3.14)(25 × 10 −6 m )2 πr 2

Chapter 12 Chromatographic and Electrophoretic Methods

603

+ Anode

Sample plug

– Cathode

+ Anode

Sample plug

– Cathode

Stacked cations

+ Anode

Sample plug

– Cathode

Figure 12.43
Schematic diagram demonstrating stacking.

Electrokinetic injections are made by placing both the capillary and the anode into the sample vial and briefly applying an electric field. The moles of solute injected into the capillary, ninj, are determined using ninj = πCtr 2 (µ ep + µ eof )E κ buf κ samp 12.45

electrokinetic injection An injection technique in capillary electrophoresis in which an electric field is used to inject sample into the capillary column.

where C is the solute’s concentration in the sample, t is the amount of time that the electric field is applied, r is the capillary’s radius, µep is the solute’s electrophoretic mobility, µeof is the electroosmotic mobility, E is the applied electric field, and κbuf and κsamp are the conductivities of the buffer solution and sample, respectively. An important consequence of equation 12.45 is that it is inherently biased toward sampling solutes with larger electrophoretic mobilities. Those solutes with the largest electrophoretic mobilities (smaller, more positively charged ions) are injected in greater numbers than those with the smallest electrophoretic mobilities (smaller, more negatively charged ions). When a solute’s concentration in the sample is too small to reliably analyze, it may be possible to inject the solute in a manner that increases its concentration in the capillary tube. This method of injection is called stacking. Stacking is accomplished by placing the sample in a solution whose ionic strength is significantly less than that of the buffering solution. Because the sample plug has a lower concentration of ions than the buffering solution, its resistance is greater. Since the electric current passing through the capillary is fixed, we know from Ohm’s law E = iR that the electric field in the sample plug is greater than that in the buffering solution. Electrophoretic velocity is directly proportional to the electric field (see equation 12.35); thus, ions in the sample plug migrate with a greater velocity. When the solutes reach the boundary between the sample plug and the buffering solution, the electric field decreases and their electrophoretic velocity slows down, “stacking” together in a smaller sampling zone (Figure 12.43). Applying the Electric Field Migration in electrophoresis occurs in response to the applied electric field. The ability to apply a large electric field is important because

stacking A means of concentrating solutes in capillary electrophoresis after their injection onto the capillary column.

604

Modern Analytical Chemistry hν hν
Polyimide coating Reflective coating Fused silica capillary Reflective coating Polyimide coating (a) (b) Capillary opening

Figure 12.44
Schematic diagrams of two approaches to on-column detection using UV/Vis absorption spectroscopy.

higher voltages lead to shorter analysis times (see equation 12.41), more efficient separations (see equation 12.42), and better resolution (see equation 12.43). Because narrow-bore capillary tubes dissipate Joule heating so efficiently, voltages of up to 40 kV can be applied. Detectors Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrometry. Whenever possible, detection is done “on-column” before the solutes elute from the capillary tube and additional band broadening occurs. UV/Vis detectors are among the most popular. Because absorbance is directly proportional to path length, the capillary tubing’s small diameter leads to signals that are smaller than those obtained in HPLC. Several approaches have been used to increase the path length, including a Z-shaped sample cell or multiple reflections (Figure 12.44). Detection limits are about 10–7 M. Better detection limits are obtained using fluorescence, particularly when using a laser as an excitation source. When using fluorescence detection, a small portion of the capillary’s protective coating is removed and the laser beam is focused on the inner portion of the capillary tubing. Emission is measured at an angle of 90° to the laser. Because the laser provides an intense source of radiation that can be focused to a narrow spot, detection limits are as low as 10–16 M. Solutes that do not absorb UV/Vis radiation or undergo fluorescence can be detected by other detectors. Table 12.8 provides a list of detectors used in capillary electrophoresis along with some of their important characteristics.

12J.3 Capillary Electrophoresis Methods
There are several different forms of capillary electrophoresis, each of which has its particular advantages. Several of these methods are briefly described in this section. capillary zone electrophoresis A form of capillary electrophoresis in which separations are based on differences in the solutes’ electrophoretic mobilities.

Capillary Zone Electrophoresis The simplest form of capillary electrophoresis is capillary zone electrophoresis (CZE). In CZE the capillary tube is filled with a buffer solution and, after loading the sample, the ends of the capillary tube are placed in reservoirs containing additional buffer solution. Under normal conditions, the end of the capillary containing the sample is the anode, and solutes migrate toward

Chapter 12 Chromatographic and Electrophoretic Methods

605

Table 12.8
Detector

Characteristics of Selected Detectors for Capillary Electrophoresis
Selectivity
solute must have UV/Vis absorbing chromophore universal solute must have favorable fluorescent quantum efficiency solute must have favorable fluorescent quantum efficiency universal when monitoring all ions; selective when monitoring single ion solute must undergo oxidation or reduction universal solutes must be radioactive

Detection Limit Moles Injected Molaritya
10–13–10–16 10–12–10–15 10–15–10–17 10–18–10–20 10–16–10–17 10–18–10–19 10–15–10–16 10–17–10–19 10–5–10–7 10–4–10–6 10–7–10–9 10–13–10–16 10–8–10–10 10–7–10–10 10–7–10–9 10–10–10–12

On-Column Detection? yes yes yes yes no no no yes

UV/Vis absorbance indirect absorbance fluorescence laser fluorescence mass spectrometer amperometry conductivity radiometric

aConcentration

Source: Adapted from Baker, D. R. Capillary Electrophoresis. Wiley-Interscience: New York, 1995.16 depends on the volume of sample injected.

– + + – – – +

+ + – – – – – +

+ – + – +

– – + +

– + + – + – + – +

+ Bulk solution – – – Mobile layer +

+ + – +

– +

– +

– – + – – + + +

+ –

–

+

+ + – –

+ –

+ –

–

+

+ –

Capillary wall + Anode Direction of electroosmotic flow – Cathode

Figure 12.45
Schematic diagram showing the reversal of electroosmotic flow.

the cathode at a velocity determined by their electrophoretic mobility and the electroosmotic flow. Cations elute first, with smaller, more highly charged cations eluting before larger cations with smaller charges. Neutral species elute as a single band. Finally, anions are the last species to elute, with smaller, more negatively charged anions being the last to elute. The direction of electroosmotic flow and, therefore, the order of elution in CZE can be reversed. This is accomplished by adding an alkylammonium salt to the buffer solution. As shown in Figure 12.45, the positively charged end of the alkylammonium ion binds to the negatively charged silanate ions on the capillary’s walls. The alkylammonium ion’s “tail” is hydrophobic and associates with the tail of another alkylammonium ion. The result is a layer of positive charges to which anions in the buffer solution are attracted. The migration of these solvated anions toward

606

Modern Analytical Chemistry the anode reverses the electroosmotic flow’s direction. The order of elution in this case is exactly the opposite of that observed under normal conditions. Capillary zone electrophoresis also can be accomplished without an electroosmotic flow by coating the capillary’s walls with a nonionic reagent. In the absence of electroosmotic flow only cations migrate from the anode to the cathode. Anions elute into the source reservoir while neutral species remain stationary. Capillary zone electrophoresis provides effective separations of any charged species, including inorganic anions and cations, organic acids and amines, and large biomolecules such as proteins. For example, CZE has been used to separate a mixture of 36 inorganic and organic ions in less than 3 minutes.17 Neutral species, of course, cannot be separated. Micellar Electrokinetic Capillary Chromatography One limitation to CZE is its inability to separate neutral species. Micellar electrokinetic chromatography (MEKC) overcomes this limitation by adding a surfactant, such as sodium dodecylsulfate (Figure 12.46a) to the buffer solution. Sodium dodecylsulfate, (SDS) has a long-chain hydrophobic “tail” and an ionic functional group, providing a negatively charged “head.” When the concentration of SDS is sufficiently large, a micelle forms. A micelle consists of an agglomeration of 40–100 surfactant molecules in which the hydrocarbon tails point inward, and the negatively charged heads point outward (Figure 12.46b). Because micelles are negatively charged, they migrate toward the cathode with a velocity less than the electroosmotic flow velocity. Neutral species partition themselves between the micelles and the buffer solution in much the same manner as they do in HPLC. Because there is a partitioning between two phases, the term “chromatography” is used. Note that in MEKC both phases are “mobile.” The elution order for neutral species in MEKC de– pends on the extent to which they partition into the mi– celles. Hydrophilic neutrals are insoluble in the micelle’s hydrophobic inner environment and elute as a single – band as they would in CZE. Neutral solutes that are ex– tremely hydrophobic are completely soluble in the mi– celle, eluting with the micelles as a single band. Those neutral species that exist in a partition equilibrium be(b) tween the buffer solution and the micelles elute between the completely hydrophilic and completely hydrophobic neutrals. Those neutral species favoring the buffer solution elute before those favoring the micelles. Micellar electrokinetic chromatography has been used to separate a wide variety of samples, including mixtures of pharmaceutical compounds, vitamins, and explosives.
–
– – –

micellar electrokinetic capillary chromatography A form of capillary electrophoresis in which neutral solutes are separated based on their ability to partition into a charged micelle. micelle An agglomeration of molecules containing ionic “heads” and hydrophobic “tails,” which form into a structure with a hydrophobic interior and a hydrophilic exterior.

–

O H3C (CH2)11 O S O Represented as – (a) O–Na+

–

–

–

Figure 12.46
(a) Structure of sodium dodecylsulfate; (b) structure of a micelle.

capillary gel electrophoresis A form of capillary electrophoresis in which the capillary column contains a gel enabling separations based on size.

Capillary Gel Electrophoresis In capillary gel electrophoresis (CGE) the capillary tubing is filled with a polymeric gel. Because the gel is porous, solutes migrate through the gel with a velocity determined both by their electrophoretic mobility and their size. The ability to effect a separation based on size is useful when the solutes have similar electrophoretic mobilities. For example, fragments of DNA of varying length have similar charge-to-size ratios, making their separation by CZE difficult. Since the DNA fragments are of different size, a CGE separation is possible. The capillary used for CGE is usually treated to eliminate electroosmotic flow, thus preventing the gel’s extrusion from the capillary tubing. Samples are injected

–

–

–

–

–

–

–

–

–

– –

Chapter 12 Chromatographic and Electrophoretic Methods electrokinetically because the gel provides too much resistance for hydrodynamic sampling. The primary application of CGE is the separation of large biomolecules, including DNA fragments, proteins, and oligonucleotides. Capillary Electrochromatography Another approach to separating neutral species is capillary electrochromatography (CEC). In this technique the capillary tubing is packed with 1.5–3-µm silica particles coated with a bonded, nonpolar stationary phase. Neutral species separate based on their ability to partition between the stationary phase and the buffer solution (which, due to electroosmotic flow, is the mobile phase). Separations are similar to the analogous HPLC separation, but without the need for high-pressure pumps. Furthermore, efficiency in CEC is better than in HPLC, with shorter analysis times.

607

capillary electrochromatography A form of capillary electrophoresis in which a stationary phase is included within the capillary column.

12J.4 Representative Method
Although each capillary electrophoretic method has its own unique considerations, the following description of the determination of a vitamin B complex provides an instructive example of a typical procedure.

Representative Methods

Method 12.3

Determination of a Vitamin B Complex by CZE or MEKC18

Description of Method. The water-soluble vitamins B1 (thiamine hydrochloride),
B2 (riboflavin), B3 (niacinamide), and B6 (pyridoxine hydrochloride) may be determined by CZE using a pH 9 sodium tetraborate/sodium dihydrogen phosphate buffer or by MEKC using the same buffer with the addition of sodium dodecylsulfate. Detection is by UV absorption at 200 nm. An internal standard of o-ethoxybenzamide is used to standardize the method.

Procedure. A vitamin B complex tablet is crushed and placed in a beaker with
20.00 mL of a 50% v/v methanol solution that is 20 mM in sodium tetraborate and contains 100.0 ppm of o-ethoxybenzamide. After mixing for 2 min to ensure that the B vitamins are dissolved, a 5.00-mL portion is passed through a 0.45-µm filter to remove insoluble binders. An approximately 4-nL sample is loaded into a 50-µm internal diameter capillary column. For CZE the capillary column contains a 20 mM pH 9 sodium tetraborate/sodium dihydrogen phosphate buffer. For MEKC the buffer is also 150 mM in sodium dodecylsulfate. A 40-kV/m electric field is used to effect both the CZE and MEKC separations.

Questions
1. Methanol, which elutes at 4.69 min, is included as a neutral species to indicate the electroosmotic flow. When using standard solutions of each vitamin, CZE peaks are found at 3.41 min, 4.69 min, 6.31 min, and 8.31 min. Examine the structures and pKa information in Figure 12.47, and determine the order in which the four B vitamins elute. Vitamin B1 is a cation and must, therefore, elute before the neutral species methanol; thus it elutes first at 3.41 min. Vitamin B3 is a neutral species and should elute with methanol at 4.69 min. The remaining two B vitamins are weak acids that partially ionize in the pH 9 buffer. Of the two, vitamin B6 is the stronger acid and is ionized (as the anion) to a greater extent. Vitamin B6, therefore, is the last of the vitamins to elute. —Continued

608

Modern Analytical Chemistry

Continued from page 607 O CH3 N H3C N NH2
B1

H3C

N+ S CH2CH2OH

N NH N CH2 HO CH CH CH CH2OH
B2

H3C

N

O

CONH2 HOH2C N
B3

CH2OH OH HO HO N
B6

CH3

pKa = 9.0

pKa = 9.7

Figure 12.47
Structures of the vitamins B1, B2, B3, and B6.

2. The order of elution when using MEKC is vitamin B3 (5.58 min), vitamin B6 (6.59 min), vitamin B2 (8.81 min), and vitamin B1 (11.21 min). What conclusions can you make about the solubility of the B vitamins in the sodium dodecylsulfate micelles? The elution time for vitamin B1 shows the greatest change, increasing from 3.41 min to 11.21 min. Clearly vitamin B1 has the greatest solubility in the micelles. Vitamins B2 and B3 have a more limited solubility in the micelles, showing slightly longer elution times. Interestingly, the elution time for vitamin B6 decreases in the presence of the micelles. 3. A quantitative analysis for vitamin B1 was carried out using this procedure. When a solution of 100.0 ppm B1 and 100.0 ppm o-ethoxybenzamide was analyzed, the peak area for vitamin B1 was 71% of that for the internal standard. The analysis of a 0.125-g vitamin B complex tablet gave a peak area for vitamin B1 that was 1.82 times as great as that for the internal standard. How many milligrams of vitamin B1 are in the tablet? For an internal standardization the relevant equation is SA C = k A SIS C IS where SA and SIS are, respectively, the signals for the analyte and internal standard, and CA and CIS are their respective concentrations. Making appropriate substitutions for the standard solution 71 100.0 ppm = k × 100 100.0 ppm gives k as 0.71. Substituting values for the sample 1.82 CA = 0.71 × 1 100.0 ppm gives the concentration of vitamin B1 as 256 ppm. This is the concentration in the sample as injected. To determine the number of milligrams of vitamin B1, we must account for the sample’s dissolution; thus 256 mg × 0.0200 L = 5.1 mg vitamin B1 L

Chapter 12 Chromatographic and Electrophoretic Methods

609

12J.5 Evaluation
When compared with GC and HPLC, capillary electrophoresis provides similar levels of accuracy, precision, and sensitivity and a comparable degree of selectivity. The amount of material injected into a capillary electrophoretic column is significantly smaller than that for GC and HPLC; typically 1 nL versus 0.1 µL for capillary GC and 1–100 µL for HPLC. Detection limits for capillary electrophoresis, however, are 100–1000 times poorer than those for GC and HPLC. The most significant advantages of capillary electrophoresis are improvements in separation efficiency, time, and cost. Capillary electrophoretic columns contain substantially more theoretical plates (≈106 plates/m) than that found in HPLC (≈105 plates/m) and capillary GC columns (≈103 plates/m), providing unparalleled resolution and peak capacity. Separations in capillary electrophoresis are fast and efficient. Furthermore, the capillary column’s small volume means that a capillary electrophoresis separation requires only a few microliters of buffer solution, compared with 20–30 mL of mobile phase for a typical HPLC separation.

12K KEY TERMS adjusted retention time (p. 551) band broadening (p. 553) baseline width (p. 548) bleed (p. 566) bonded stationary phase (p. 580) capacity factor (p. 551) capillary column (p. 562) capillary electrochromatography (p. 607) capillary electrophoresis (p. 597) capillary gel electrophoresis (p. 606) capillary zone electrophoresis (p. 604) chromatogram (p. 548) chromatography (p. 546) column chromatography (p. 546) countercurrent extraction (p. 546) cryogenic focusing (p. 568) electrokinetic injection (p. 603) electron capture detector (p. 570) electroosmotic flow (p. 598) electroosmotic flow velocity (p. 599) electropherogram (p. 597) electrophoresis (p. 597) electrophoretic mobility (p. 598) electrophoretic velocity (p. 598) exclusion limit (p. 595) flame ionization detector (p. 570) fronting (p. 555) gas chromatography (p. 563) gas–liquid chromatography (p. 564) gradient elution (p. 558) guard column (p. 579) headspace sampling (p. 567) high-performance liquid chromatography (p. 578) hydrodynamic injection (p. 602) inclusion limit (p. 593) ion-exchange chromatography (p. 590) ion-suppressor column (p. 592) isocratic elution (p. 582) Joule heating (p. 601) Kovat’s retention index (p. 575) liquid–solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554) planar chromatography (p. 546) polarity index (p. 580) resolution (p. 549) retention time (p. 548) retention volume (p. 548) reverse-phase chromatography (p. 580) selectivity factor (p. 552) single-column ion chromatography (p. 593) size-exclusion chromatography (p. 593) solid-phase microextraction (p. 567) split injection (p. 568) splitless injection (p. 568) stacking (p. 603) stationary phase (p. 546) supercritical fluid chromatography (p. 596) support-coated open tubular column (p. 565) tailing (p. 555) temperature programming (p. 558) theoretical plate (p. 553) thermal conductivity detector (p. 569) van Deemter equation (p. 561) void time (p. 549) void volume (p. 549) wall-coated open tubular column (p. 565) zeta potential (p. 599)

610

Modern Analytical Chemistry

12L SUMMARY
Chromatography and electrophoresis are powerful analytical techniques that can separate a sample into its components while providing a means for determining their concentration. Chromatographic separations utilize the selective partitioning of the sample’s components between a stationary phase that is immobilized within a column and a mobile phase that passes through the column. The effectiveness of a separation is described by the resolution between the chromatographic bands for two components and is a function of the component’s capacity factor, the column’s efficiency, and the column’s selectivity. A component’s capacity factor is a measure of the degree to which it successfully partitions into the stationary phase, with larger capacity factors corresponding to more strongly retained components. The column’s selectivity for two components is the ratio of the component’s capacity factors, providing a relative measure of the column’s ability to retain the two components. Column efficiency accounts for those factors that cause a component’s chromatographic band to increase in width during the separation. Column efficiency is defined in terms of the number of theoretical plates and the height of a theoretical plate, the latter of which is a function of a number of parameters, most notably the mobile phase’s flow rate. Chromatographic separations are optimized by increasing the number of theoretical plates, increasing the column’s selectivity, or increasing the component’s capacity factors. In gas chromatography (GC) the mobile phase is an inert gas, and the stationary phase is a nonpolar or polar organic liquid that is either coated on a particulate material and packed into a widebore column or coated on the walls of a narrow-bore capillary column. Gas chromatography is useful for the analysis of volatile components. In high-performance liquid chromatography (HPLC) the mobile phase is either a nonpolar solvent (normal phase) or a polar solvent (reverse phase). A stationary phase of opposite polarity, which is bonded to a particulate material, is packed into a widebore column. HPLC can be applied to a wider range of samples than GC; however, the separation efficiency for HPLC is not as good as that for GC. Together, GC and HPLC account for the largest number of chromatographic separations. Other separation techniques, however, find specialized applications. Of particular importance are: ion-exchange chromatography, which is useful for separating anions and cations; size-exclusion chromatography, which is useful for separating large molecules; and supercritical fluid chromatography, which combines many of the advantages of GC and HPLC for the analysis of materials that are not easily analyzed by either of these methods. In capillary zone electrophoresis a sample’s components are separated based on their ability to move through a conductive medium under the influence of an applied electric field. Because of the effect of electroosmotic flow, positively charged solutes elute first, with smaller, more highly charged cationic solutes eluting before larger cations of lower charge. Neutral species elute without undergoing further separation. Finally, anions elute last, with smaller, more negatively charged anions being the last to elute. By adding a surfactant, neutral species also can be separated by micellar electrokinetic capillary chromatography. Electrophoretic separations also can take advantage of the ability of polymeric gels to separate solutes by size (capillary gel electrophoresis) and the ability of solutes to partition into a stationary phase (capillary electrochromatography). In comparison to GC and HPLC, capillary electrophoresis provides faster and more efficient separations.

12M Suggested EXPERIMENTS

Experiments

The following experiments may be used to illustrate the application of chromatography and electrophoresis to a number of different types of samples. Experiments are grouped by the type of technique, and each is briefly annotated. The first set of experiments describes the application of gas chromatography. These experiments encompass a variety of different types of samples, columns, and detectors. Most experiments may be easily modified to use available equipment and detectors. Elderd, D. M.; Kildahl, N. K.; Berka, L. H. “Experiments for Modern Introductory Chemistry: Identification of Arson Accelerants by Gas Chromatography,” J. Chem. Educ. 1996, 73, 675–677. Although aimed at the introductory class, this simple experiment provides a nice demonstration of the use of GC for a qualitative analysis. Students obtain chromatograms for several possible accelerants using headspace sampling and then analyze the headspace over a sealed sample of charred wood to determine the accelerant used in burning the wood. Separations are carried out using a wide-bore capillary column with a stationary phase of methyl 50% phenyl silicone and a flame ionization detector. —Continued

Chapter 12 Chromatographic and Electrophoretic Methods

611

Experiments

Graham, R. C.; Robertson, J. K. “Analysis of Trihalomethanes in Soft Drinks,” J. Chem. Educ. 1988, 65, 735–737. Trihalomethanes are extracted from soft drinks using a liquid–liquid extraction with pentane. Samples are analyzed using a packed column containing 20% OV-101 on 80/100 mesh Gaschrom Q equipped with an electron capture detector. Kegley, S. E.; Hansen, K. J.; Cunningham, K. L. “Determination of Polychlorinated Biphenyls (PCBs) in River and Bay Sediments,” J. Chem. Educ. 1996, 73, 558–562. This somewhat lengthy experiment provides a thorough introduction to the use of GC for the analysis of trace-level environmental pollutants. Sediment samples are extracted by sonicating with 3 × 100-mL portions of 1:1 acetone:hexane. The extracts are then filtered and concentrated before bringing to a final volume of 10 mL. Samples are analyzed with a capillary column using a stationary phase of 5% phenylmethyl silicone, a splitless injection, and an ECD detector. Quach, D. T.; Ciszkowski, N. A.; Finlayson-Pitts, B. J. “A New GC-MS Experiment for the Undergraduate Instrumental Analysis Laboratory in Environmental Chemistry: Methyl-tbutyl Ether and Benzene in Gasoline,” J. Chem. Educ. 1998, 75, 1595–1598. This experiment describes the determination of methyl-tbutyl ether and benzene in gasoline using the method of standard additions. Two compounds naturally present at high concentration (o-xylene and toluene) are used as internal standards to correct for variations in the amount of sample injected into the GC. Because of the complexity of gasoline, single-ion monitoring is used to determine the signals for the analytes and internal standards. Separations are carried out using a capillary column with a stationary phase of 5% diphenyl/95% dimethylsiloxane. Rice, G. W. “Determination of Impurities in Whiskey Using Internal Standard Techniques,” J. Chem. Educ. 1987, 64, 1055–1056. An internal standard of 1-butanol is used to determine the concentrations of one or more of the following impurities commonly found in whiskey: acetaldehyde, methanol, ethyl acetate, 1-propanol, 2-methyl-1-propanol, acetic acid, 2-methyl-1-butanol and 3-methyl-1-butanol. A packed column using 5% Carbowax 20m on 80/120 Carbopak B and an FID detector were used. Rubinson, J. F.; Neyer-Hilvert, J. “Integration of GC-MS Instrumentation into the Undergraduate Laboratory: Separation and Identification of Fatty Acids in Commercial Fats and Oils,” J. Chem. Educ. 1997, 74, 1106–1108.

Fatty acids from commercial fats and oils, such as peanut oil, are extracted with methanolic NaOH and made volatile by derivatizing with a solution of methanol/BF3. Separations are carried out using a capillary 5% phenylmethyl silicone column with MS detection. By searching the associated spectral library students are able to identify the fatty acids present in their sample. Quantitative analysis is by external standards. Rudzinski, W. E.; Beu, S. “Gas Chromatographic Determination of Environmentally Significant Pesticides,” J. Chem. Educ. 1982, 59, 614–615. Students analyze samples of orange juice that have been spiked with diazinon, malathion, and ethion. Samples are extracted with acetonitrile and then extracted with pet ether. The pesticide residues are then purified using an activated magnesium silicate (Florisil) column, eluting the pesticides with mixtures of pet ether and ethyl ether. After removing most of the solvent, samples are analyzed by GC using a packed glass column containing 1.5% SP-2250/1.95% SP2401 on 100/120 Supelcoport. Both electron capture and flame ionization detection are used. Welch, W. C.; Greco, T. G. “An Experiment in Manual Multiple Headspace Extraction for Gas Chromatography,” J. Chem. Educ. 1993, 70, 333–335. The principle of headspace sampling is introduced in this experiment using a mixture of methanol, chloroform, 1,2dichloroethane, 1,1,1-trichloroethane, benzene, toluene, and p-xylene. Directions are given for evaluating the distribution coefficient for the partitioning of a volatile species between the liquid and vapor phase and for its quantitative analysis in the liquid phase. Both packed (OV-101) and capillary (5% phenyl silicone) columns were used. The GC is equipped with a flame ionization detector. Another experiment with the same focus is Ramachandran, B. R.; Allen, J. M.; Halpern, A. M. “Air-Water Partitioning of Environmentally Important Organic Compounds,” J. Chem. Educ. 1996, 73, 1058–1061. This experiment provides an alternative approach to measuring the partition coefficient (Henry’s law constant) for volatile organic compounds in water. A OV-101 packed column and flame ionization detector are used. Williams, K. R.; Pierce, R. E. “The Analysis of Orange Oil and the Aqueous Solubility of d-Limonene,” J. Chem. Educ. 1998, 75, 223–226. Two experiments are described in this paper. In the first experiment students determine the %w/w orange oil in a prepared sample by analyzing for d-limonene using anisole as an internal standard. Separations are accomplished using —Continued

612

Modern Analytical Chemistry

Experiments

Continued from page 611 a megabore open tubular column with a 5% phenylmethyl silicone bonded stationary phase and a thermal conductivity detector. In the second experiment the solubility of d-limonene is determined by equilibrating different volumes of d-limonene with water and measuring the amount of d-limonene in the overlying vapor phase using headspace sampling. Wong, J. W.; Ngim, K. K.; Shibamoto, T.; et al. “Determination of Formaldehyde in Cigarette Smoke,” J. Chem. Educ. 1997, 74, 1100–1103. Formaldehyde from cigarette smoke is collected by trapping the smoke in a 1-L separatory funnel and extracting into an aqueous solution. To aid in its detection, cysteamine is included in the aqueous extracting solution, leading to the formation of a thiazolidine derivative. Samples are analyzed using a DB-1 capillary column with a thermionic or flame photometric detector. Directions also are given for using an HPLC. Formaldehyde is derivatized using 2,4dinitrophenylhydrazine, and samples are analyzed using a C18 column with a UV detector set to 365 nm. Yang, M. J.; Orton, M. L., Pawliszyn, J. “Quantitative Determination of Caffeine in Beverages Using a Combined SPME-GC/MS Method,” J. Chem. Educ. 1997, 74, 1130–1132. Caffeine in coffee, tea, and soda is determined by a solidphase microextraction using an uncoated silica fiber, followed by a GC analysis using a capillary SPB-5 column with an MS detector. Standard solutions are spiked with 13C3 caffeine as an internal standard.

The second set of experiments describes the application of high-performance liquid chromatography. These experiments encompass a variety of different types of samples and a variety of common detectors. Bidlingmeyer, B. A.; Schmitz, S. “The Analysis of Artificial Sweeteners and Additives in Beverages by HPLC,” J. Chem. Educ. 1991, 68, A195–A200. The concentrations of benzoic acid, aspartame, caffeine, and saccharin in a variety of beverages are determined in this experiment. A C18 column and a mobile phase of 80% v/v acetic acid (pH = 4.2) and 20% v/v methanol are used to effect the separation. A UV detector set to 254 nm is used to measure the eluent’s absorbance. The ability to adjust retention times by changing the mobile phase’s pH is also explored. DiNunzio, J. E. “Determination of Caffeine in Beverages by High Performance Liquid Chromatography,” J. Chem. Educ. 1985, 62, 446–447. The concentration of caffeine in a typical serving of coffee and soda is determined in this experiment. Separations are achieved using a C18 column with a mobile phase of 30% v/v methanol in water, with UV detection at a wavelength of 254 nm. Ferguson, G. K. “Quantitative HPLC Analysis of an Analgesic/Caffeine Formulation: Determination of Caffeine,” J. Chem. Educ. 1998, 75, 467–469. The %w/w caffeine in an analgesic formulation is determined in this experiment. The separation uses a C18 column with a mobile phase of 94.1% v/v water, 5.5% v/v acetonitrile, 0.2% v/v triethylamine, and 0.2% v/v acetic acid. A UV detector is set to 254 nm. Ferguson, G. K. “Quantitative HPLC Analysis of a Psychotherapeutic Medication: Simultaneous Determination of Amitriptyline Hydrochloride and Perphenazine,” J. Chem. Educ. 1998, 75, 1615–1618. This experiment describes a quantitative analysis for the active ingredients in a prescription antipsychotic medication. The separation makes use of a cyanopropyl derivatized column and a mobile phase of 70% v/v acetonitrile, 5% v/v methanol, and 25% v/v 0.1 M aqueous KH2PO4. A UV detector set to 215 nm is used to measure the eluent’s absorbance. Haddad, P.; Hutchins, S.; Tuffy, M. “High Performance Liquid Chromatography of Some Analgesic Compounds,” J. Chem. Educ. 1983, 60, 166–168. This experiment focuses on developing an HPLC separation capable of distinguishing acetylsalicylic acid, paracetamol, salicylamide, caffeine, and phenacetin. A C18 column and UV detection are used to obtain chromatograms. Solvent parameters used to optimize the separation include the pH of the buffered aqueous mobile phase, the %v/v methanol added to the aqueous mobile phase, and the use of tetrabutylammonium phosphate as an ion-pairing reagent. Mueller, B. L.; Potts, L. W. “HPLC Analysis of an Asthma Medication,” J. Chem. Educ. 1988, 65, 905–906. This experiment describes the quantitative analysis of the asthma medication Quadrinal for the active ingredients theophylline, salicylic acid, phenobarbital, ephedrine HCl, and potassium iodide. Separations are carried out using a C18 column with a mobile phase of 19% v/v acetonitrile, 80% v/v water, and 1% acetic acid. A small amount of triethylamine (0.03% v/v) is included to ensure the elution of ephedrine HCl. A UV detector set to 254 nm is used to record the chromatogram. —Continued

Chapter 12 Chromatographic and Electrophoretic Methods

613

Experiments

Remcho, V. T.; McNair, H. M.; Rasmussen, H. T. “HPLC Method Development with the Photodiode Array Detector,” J. Chem. Educ. 1992, 69, A117–A119. A mixture of methyl paraben, ethyl paraben, propyl paraben, diethyl phthalate, and butyl paraben is separated by HPLC. This experiment emphasizes the development of a mobilephase composition capable of separating the mixture. A photodiode array detector demonstrates the coelution of the two compounds. Siturmorang, M.; Lee, M. T. B.; Witzeman, L. K.; et al. “Liquid Chromatography with Electrochemical Detection (LC-EC): An Experiment Using 4-Aminophenol,” J. Chem. Educ. 1998, 75, 1035–1038. The use of an amperometric detector is emphasized in this experiment. Hydrodynamic voltammetry (see Chapter 11) is first performed to identify a potential for the oxidation of 4-aminophenol without an appreciable background current due to the oxidation of the mobile phase. The separation is then carried out using a C18 column and a mobile phase of 50% v/v pH 5, 20 mM acetate buffer with 0.02 M MgCl2, and 50% v/v methanol. The analysis is easily extended to a mixture of 4-aminophenol, ascorbic acid, and catechol, and to the use of a UV detector. Tran, C. D.; Dotlich, M. “Enantiomeric Separation of BetaBlockers by High Performance Liquid Chromatography,” J. Chem. Educ. 1995, 72, 71–73. This experiment introduces the use of a chiral column (a β-cyclodextrin-bonded C18 column) to separate the beta-blocker drugs Inderal LA (S-propranolol and

R-propranolol), Tenormim (DL-atenolol) and Lopressor (DL-metaprolol). The mobile phase was 90:10 (v/v) acetonitrile and water. A UV detector set to 254 nm is used to obtain the chromatogram. Van Arman, S. A.; Thomsen, M. W. “HPLC for Undergraduate Introductory Laboratories,” J. Chem. Educ. 1997, 74, 49–50. In this experiment students analyze an artificial RNA digest consisting of cytidine, uridine, thymidine, guanosine, and adenosine using a C18 column and a mobile phase of 0.4% v/v triethylammonium acetate, 5% v/v methanol, and 94.6% v/v water. The chromatogram is recorded using a UV detector at a wavelength of 254 nm. Wingen, L. M.; Low, J. C.; Finlayson-Pitts, B. J. “Chromatography, Absorption, and Fluorescence: A New Instrumental Analysis Experiment on the Measurement of Polycyclic Aromatic Hydrocarbons in Cigarette Smoke,” J. Chem. Educ. 1998, 75, 1599–1603. The analysis of cigarette smoke for 16 different polyaromatic hydrocarbons is described in this experiment. Separations are carried out using a polymeric bonded silica column with a mobile phase of 50% v/v water, 40% v/v acetonitrile, and 10% v/v tetrahydrofuran. A notable feature of this experiment is the evaluation of two means of detection. The ability to improve sensitivity by selecting the optimum excitation and emission wavelengths when using a fluorescence detector is demonstrated. A comparison of fluorescence detection with absorbance detection shows that better detection limits are obtained when using fluorescence.

The third set of experiments provides a few representative applications of ion chromatography. Bello, M. A.; Gustavo González, A. “Determination of Phosphate in Cola Beverages Using Nonsuppressed Ion Chromatography,” J. Chem. Educ. 1996, 73, 1174–1176. In this experiment phosphate is determined by singlecolumn, or nonsuppressed, ion chromatography using an anionic column and a conductivity detector. The mobile phase is a mixture of n-butanol, acetonitrile, and water (containing sodium gluconate, boric acid, and sodium tetraborate). Kieber, R. J.; Jones, S. B. “An Undergraduate Laboratory for the Determination of Sodium, Potassium, and Chloride,” J. Chem. Educ. 1994, 71, A218–A222. Three techniques, one of which is ion chromatography, are used to determine the concentrations of three ions in solution. The combined concentrations of Na+ and K+ are determined by an ion exchange with H+, the concentration of which is subsequently determined by an acid–base titration using NaOH. Flame atomic absorption is used to measure the concentration of Na+, and K+ is determined by difference. The concentration of Cl– is determined by ionexchange chromatography on an anionic column using a mobile phase of HCO3– and CO32– with ion suppression. A conductivity detector is used to record the chromatogram. Koubek, E.; Stewart, A. E. “The Analysis of Sulfur in Coal,” J. Chem. Educ. 1992, 69, A146–A148. Sulfur in coal is converted into a soluble sulfate by heating to 800 °C in the presence of MgO and Na2CO3. After dissolving in water, the concentration of sulfate is determined by single-column ion chromatography, using an anionic column and a mobile phase of 1 mM potassium hydrogen phthalate. A conductivity detector is used to record the chromatogram. Luo, P.; Luo, M. A.; Baldwin, R. P. “Determination of Sugars in Food Products,” J. Chem. Educ., 1993, 70, 679–681. —Continued

614

Modern Analytical Chemistry

Experiments

Continued from page 613 The concentrations of nine sugars (fucose, methylglucose, arabinose, glucose, fructose, lactose, sucrose, cellobiose, and maltose) in beer, milk, and soda are determined using an anionic column and a mobile phase of 0.1 M NaOH. Detection is by amperometry at a Cu working electrode.

The last set of experiments provides examples of the application of capillary electrophoresis. These experiments encompass a variety of different types of samples and include examples of capillary zone electrophoresis and micellar electrokinetic chromatography. Conradi, S.; Vogt, C.; Rohde, E. “Separation of Enatiomeric Barbiturates by Capillary Electrophoresis Using a Cyclodextrin-Containing Run Buffer,” J. Chem. Educ. 1997, 74, 1122–1125. In this experiment the enantiomers of cyclobarbital and thiopental, and phenobarbital are separated using MEKC with cyclodextran as a chiral selector. By adjusting the pH of the buffer solution and the concentration and type of cyclodextran, students are able to find conditions in which the enantiomers of cyclobarbital and thiopental are resolved. Conte, E. D.; Barry, E. F.; Rubinstein, H. “Determination of Caffeine in Beverages by Capillary Zone Electrophoresis,” J. Chem. Educ. 1996, 73, 1169–1170. Caffeine in tea and coffee is determined by CZE using nicotine as an internal standard. The buffer solution is 50 mM sodium borate adjusted to pH 8.5 with H3PO4. A UV detector set to 214 nm is used to record the electropherograms. Hage, D. S.; Chattopadhyay, A.; Wolfe, C. A. C.; et al. “Determination of Nitrate and Nitrite in Water by Capillary Electrophoresis,” J. Chem. Educ. 1998, 75, 1588–1590. In this experiment the concentrations of NO2 and NO3 are determined by CZE using IO4– as an internal standard. The buffer solution is 0.60 M sodium acetate buffer adjusted to a pH of 4.0. A UV detector set to 222 nm is used to record the electropherogram. Janusa, M. A.; Andermann, L. J.; Kliebert, N. M.; et al. “Determination of Chloride Concentration Using Capillary Electrophoresis,” J. Chem. Educ. 1998, 75, 1463–1465. Directions are provided for the determination of chloride in samples using CZE. The buffer solution includes pyromellitic acid which allows the indirect determination of chloride by monitoring absorbance at 250 nm. McDevitt, V. L.; Rodríguez, A.; Williams, K. R. “Analysis of Soft Drinks: UV Spectrophotometry, Liquid Chromatography, and Capillary Electrophoresis,” J. Chem. Educ. 1998, 75, 625–629.
– –

Caffeine, benzoic acid, and aspartame in soft drinks are analyzed by three methods. Using several methods to analyze the same sample provides students with the opportunity to compare results with respect to accuracy, volume of sample required, ease of performance, sample throughput, and detection limit. Thompson, L.; Veening, H.; Strain, T. G. “Capillary Electrophoresis in the Undergraduate Instrumental Analysis Laboratory: Determination of Common Analgesic Formulations,” J. Chem. Educ. 1997, 74, 1117–1121. Students determine the concentrations of caffeine, acetaminophen, acetylsalicylic acid, and salicylic acid in several analgesic preparations using both CZE (70 mM borate buffer solution, UV detection at 210 nm) and HPLC (C18 column with 3% v/v acetic acid mixed with methanol as a mobile phase, UV detection at 254 nm). Vogt, C.; Conradi, S.; Rhode, E. “Determination of Caffeine and Other Purine Compounds in Food and Pharmaceuticals by Micellar Electrokinetic Chromatography,” J. Chem. Educ. 1997, 74, 1126–1130. This experiment describes a quantitative analysis for caffeine, theobromine, and theophylline in tea, pain killers, and cocoa. Separations are accomplished by MEKC using a pH 8.25 borate–phosphate buffer with added SDS. A UV detector set to 214 nm is used to record the electropherogram. An internal standard of phenobarbital is included for quantitative work. Weber, P. L.; Buck, D. R. “Capillary Electrophoresis: A Fast and Simple Method for the Determination of the Amino Acid Composition of Proteins,” J. Chem. Educ. 1994, 71, 609–612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of α-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate.

Chapter 12 Chromatographic and Electrophoretic Methods

615

12N PROBLEMS
1. The following data were obtained for four compounds separated on a 20-m capillary column.
Compound
A B C

6. Complete the following table.
NB
100,000 10,000 10,000

α
1.05 1.10 1.05

k′ B
0.50 4.0 3.0

R

tr (min)
8.04 8.26 8.43

w (min)
0.15 0.15 0.16

1.50 1.00 1.75

7.

(a) Calculate the number of theoretical plates for each compound and the average number of theoretical plates for the column. (b) Calculate the average height of a theoretical plate. (c) Explain why it is possible for each compound to have a different number of theoretical plates. 2. Using the data from Problem 1, calculate the resolution and selectivity factors for each pair of adjacent compounds. For resolution, use both equations 12.1 and 12.21, and compare your results. Discuss how you might improve the resolution between compounds B and C. The retention time for an unretained solute is 1.19 min. 3. Using the chromatogram shown here, which was obtained on a 2-m column, determine values for tr, w, tr k′, N, and H. ′,

efficiency of a GC separation of 2butanone on a dinonyl phthalate column. Evaluating the plate height as a function of flow rate gave a van Deemter equation for which A is 1.65 mm, B is 25.8 mm ⋅ mL min–1, and C is 0.0236 mm ⋅ min mL–1. (a) Prepare a graph of H versus u for flow rates in the range of 5–120 mL/min. (b) For what range of flow rates does each term in the van Deemter equation have the greatest effect? (c) What are the optimum flow rate and the height of a theoretical plate at that flow rate? (d) For open tubular columns the A term is no longer needed. If the B and C terms remain unchanged, what are the optimum flow rate and the height of a theoretical plate at that flow rate? (e) How many more theoretical plates will there be in the open tubular column compared with the packed column? (f ) Equation 12.28 is written in terms of the linear velocity (centimeters per second), yet we have evaluated it in this problem using the flow rate (milliliters per minute). Why can we do this?

Moody19 studied the

Nonretained solutes

8. Hsieh and Jorgenson20 prepared 12–33-µm HPLC columns packed with 5.44-µm spherical stationary phase particles. To evaluate these columns they measured reduced plate height, h, h = H dp

Signal

0

100

200

300

400

500

Retention time (s)

as a function of reduced flow rate, v, v = udp Dm

4. Using the partial chromatogram shown here, determine the resolution between the two solute bands.

Signal

where dp is the particle diameter, and Dm is the solute’s diffusion coefficient in the mobile phase. The data were analyzed using van Deemter plots, with a portion of their results summarized in the following table for the solute norepinephrine.
Column Internal Diameter (lm)
33 33 23 23 17 17 12 12

A
0.63 0.67 0.40 0.58 0.31 0.40 0.22 0.19

B
1.32 1.30 1.34 1.11 1.47 1.41 1.53 1.27

C
0.10 0.08 0.09 0.09 0.09 0.11 0.11 0.12

300 325 350 375 400 425 450 Retention time (s)

5. The chromatogram in Problem 4 was obtained on a 2-m column with a column dead time of 50 s. How long a column is needed to achieve a resolution of 1.5? What height of a theoretical plate is needed to achieve a resolution of 1.5 without increasing the length of the column?

616

Modern Analytical Chemistry
(a) Construct separate van Deemter plots using the data in the first and last rows for flow rates in the range 0.7–15. Determine the optimum flow rate and plate height for each case, given dp = 5.44 µm and Dm = 6.23 × 10–6 cm2 s–1. (b) The A term in the van Deemter equation appears to be strongly correlated with the column’s inner diameter, with smaller diameter columns providing smaller values of A. Explain why this effect is seen (Hint: Consider how many particles can fit across a capillary of each diameter). 13. Loconto and co-workers describe a method for determining trace levels of water in soil.22 The method takes advantage of the reaction of water with calcium carbide, CaC2, to produce acetylene gas, C2H2. By carrying out the reaction in a sealed vial, the amount of acetylene produced may be determined by sampling the headspace. In a typical analysis a sample of soil is placed in a sealed vial with CaC2. Analysis of the headspace gave a blank-corrected signal of 2.70 × 105. A second sample is prepared in the same manner except that a standard addition of 5.0 mg H2O/g solid is added, giving a blank-corrected signal of 1.06 × 106. Determine the number of milligrams of H2O/g soil in the soil sample. 14. Van Atta and Van Atta used gas chromatography to determine the %v/v methyl salicylate in rubbing alcohol.23 A set of standard additions was prepared by transferring 20.00 mL of rubbing alcohol to separate 25-mL volumetric flasks and pipeting 0.00 mL, 0.20 mL, and 0.50 mL of methyl salicylate to the flasks. All three flasks were then diluted to volume using isopropanol. Analysis of the three samples gave peak heights for methyl salicylate of 57.00 mm, 88.5 mm, and 132.5 mm, respectively. Determine the %v/v methyl salicylate in the rubbing alcohol. 15. The amount of camphor in an analgesic ointment can be determined by GC using the method of internal standards.24 A standard sample was prepared by placing 45.2 mg of camphor and 2.00 mL of a 6.00 mg/mL internal standard solution of terpene hydrate in a 25-mL volumetric flask and diluting to volume with CCl4. When an approximately 2-µL sample of the standard was injected, the FID signals for the two components were measured (in arbitrary units) as 67.3 for camphor and 19.8 for terpene hydrate. A 53.6-mg sample of an analgesic ointment was prepared for analysis by placing it in a 50-mL Erlenmeyer flask along with 10 mL of CCl4. After heating to 50 °C in a water bath, the sample was cooled to below room temperature and filtered. The residue was washed with two 5-mL portions of CCl4, and the combined filtrates were collected in a 25-mL volumetric flask. After adding 2.00 mL of the internal standard solution, the contents of the flask were diluted to volume with CCl4. Analysis of an approximately 2-µL sample gave FID signals of 13.5 for the terpene hydrate and 24.9 for the camphor. Report the %w/w camphor in the analgesic ointment. 16. The concentration of pesticide residues on agricultural products, such as oranges, may be determined by GC-MS.25 Pesticide residues are extracted from the sample using methylene chloride, and the concentrations of the extracted pesticides are concentrated by evaporating the methylene chloride to a smaller volume. Calibration is accomplished using anthracene-d10 as an internal standard. In a study to determine the parts per billion of heptachlor epoxide on oranges, a 50.0-g sample of orange rinds was chopped and extracted with 50.00 mL of methylene chloride. After removing any insoluble material by filtration, the methylene chloride was reduced in volume, spiked with a known amount of the internal standard, and diluted to 10 mL in a volumetric flask. Analysis of the sample gives a peak–area ratio (Aanal/ Aint stan) of 0.108. A series of calibration standards, each

9. Method 12.1 describes the analysis of the trihalomethanes CHCl3, CHBr3, CHCl2Br, and CHClBr2 in drinking water using a packed column with a nonpolar stationary phase. Predict the order in which these four trihalomethanes will elute. 10. A mixture of n-heptane, tetrahydrofuran, 2-butanone, and n-propanol elutes in this order when using a polar stationary phase such as Carbowax. The elution order is exactly the opposite when using a nonpolar stationary phase such as polydimethyl siloxane. Explain the order of elution in each case. 11. The analysis of trihalomethanes in drinking water is described in Method 12.1. A single standard gives the following results when carried through the described procedure.
Trihalomethane
CHCl3 CHCl2Br CHClBr2 CHBr3

Concentration (ppb)
1.30 0.90 4.00 1.20

Peak Area
1.35 × 104 6.12 × 104 1.71 × 104 1.52 × 104

Analysis of water from a drinking fountain gives areas of 1.56 × 104, 5.13 × 104, 1.49 × 104, and 1.76 × 104 for CHCl3, CHCl2Br, CHClBr2, and CHBr3, respectively. Determine the concentration of each of the trihalomethanes in the sample of water. 12. Zhou and colleagues determined the %w/w H2O in methanol by GC, using a capillary column coated with a nonpolar stationary phase and a thermal conductivity detector.21 A series of calibration standards gave the following results.
% w/w H2O
0.00 0.0145 0.0472 0.0951 0.1757 0.2901

Peak Height (arb. units)
1.15 2.74 6.33 11.58 20.43 32.97

(a) What is the %w/w H2O in a sample giving a peak height of 8.63? (b) The %w/w H2O in a freeze-dried antibiotic is determined in the following manner. A 0.175-g sample is placed in a vial along with 4.489 g of methanol. Water in the vial extracts into the methanol. Analysis of the sample gave a peak height of 13.66. What is the %w/w H2O in the antibiotic?

Chapter 12 Chromatographic and Electrophoretic Methods containing the same amount of anthracene-d10 as the sample, give the following results. ppb Heptachlor Epoxide
20.0 60.0 200.0 500.0 1000.0

617

Aanal/Aint stan
0.065 0.153 0.637 1.554 3.198

21. Haddad and associates report the following capacity factors ′ for the reverse-phase separation of salicylamide (ksal) and ′ ).27 caffeine (Kcaff
%v/v methanol ksal ′ kcaff ′ 30% 2.4 4.3 35% 1.6 2.8 40% 1.6 2.3 45% 1.0 1.4 50% 0.7 1.1 55% 0.7 0.9

Report the concentration of heptachlor epoxide residue (in nanograms per gram) on the oranges. 17. The adjusted retention times for octane, toluene, and nonane on a particular GC column are 15.98 min, 17.73 min, and 20.42 min, respectively. What is the retention index for all three compounds? 18. The following data were collected for a series of normal alkanes using a stationary phase of Carbowax 20M.
Alkane
pentane hexane heptane octane nonane

Explain the changes in capacity factor. What is the advantage of using a mobile phase with a smaller %v/v methanol? Are there any disadvantages? 22. Suppose that you are to separate a mixture of benzoic acid, aspartame, and caffeine in a diet soda. The following information is available to you.
Compound benzoic acid aspartame caffeine tr in Aqueous Mobile Phase Buffered to a pH of 3.0 3.5 4.0 4.5 7.4 5.9 3.6 7.0 6.0 3.7 6.9 7.1 4.1 4.4 8.1 4.4

tr ′ (min)
0.79 1.99 4.47 14.12 33.11

(a) Explain the change in retention time for each compound. (b) Plot retention time versus pH for each compound on the same graph, and identify a pH level that will yield an acceptable separation. 23. The composition of a multivitamin tablet is conveniently determined using an HPLC with a diode array UV/Vis detector. A 5-µL standard sample containing 170 ppm vitamin C, 130 ppm niacin, 120 ppm niacinamide, 150 ppm pyridoxine, 60 ppm thiamine, 15 ppm folic acid, and 10 ppm riboflavin is injected into the HPLC, giving signals (in arbitrary units) of, respectively, 0.22, 1.35, 0.90, 1.37, 0.82, 0.36, and 0.29. The multivitamin tablet is prepared for analysis by grinding into a powder and transferring to a 125-mL Erlenmeyer flask containing 10 mL of 1% v/v NH3 in dimethyl sulfoxide. After sonicating in an ultrasonic bath for 2 min, 90 mL of 2% acetic acid is added, and the mixture is stirred for 1 min and sonicated at 40 °C for 5 min. The extract is then filtered through a 0.45-µm membrane filter. Injection of a 5-µL sample into the HPLC gives signals of 0.87 for vitamin C, 0.00 for niacin, 1.40 for niacinamide, 0.22 for pyridoxine, 0.19 for thiamine, 0.11 for folic acid, and 0.44 for riboflavin. Report the number of milligrams of each vitamin present in the tablet. 24. The amount of caffeine in an analgesic tablet was determined by HPLC using a normal calibration curve. Standard solutions of caffeine were prepared and analyzed using a 10-µL fixed-volume injection loop. Results for the standards are summarized in the following table.
Concentration of Standards (ppm)
50.0 100.0 150.0 200.0 250.0

What is the retention index for a compound whose adjusted retention time is 9.36 min? 19. The following data have been reported for the gas chromatographic analysis of p-xylene and methylisobutylketone (MIBK) on a capillary column.8
Injection Mode split splitless

Compound
MIBK p-xylene MIBK p-xylene

tr (min)
1.878 5.234 3.420 5.795

Peak Area
54285 123483 2493005 3396656

Peak Width (min)
0.028 0.044 1.057 1.051

Explain the difference in the retention times, the peak areas, and the peak widths when switching from a split injection to a splitless injection. 20. Otto and Wegscheider report the following capacity factors for the reverse phase separation of 2-aminobenzoic acid on a C18 column when using 10% v/v methanol as a mobile phase.26 pH 2.0 3.0 4.0 5.0 6.0 7.0

k′

10.5 16.7 15.8 8.0 2.2 1.8

Signal (arbitrary units)
8354 16925 25218 33584 42002

Explain the changes in capacity factor.

618

Modern Analytical Chemistry
The sample was prepared by placing a single analgesic tablet in a small beaker and adding 10 mL of methanol. After allowing the sample to dissolve, the contents of the beaker, including the insoluble binder, were quantitatively transferred to a 25-mL volumetric flask and diluted to volume with methanol. The sample was then filtered, and a 1.00-mL aliquot was transferred to a 10-mL volumetric flask and diluted to volume with methanol. When analyzed by HPLC, the signal for the caffeine was found to be 21469. Report the number of milligrams of caffeine in the analgesic tablet. standard addition is prepared in a similar manner using a 10.093-g sample of the cereal and spiking it with 0.0200 mg of vitamin A. Injecting the sample and standard addition into the HPLC gives peak areas of 6.77 × 103 and 1.32 × 104, respectively. Report the vitamin A content of the sample in milligrams/100 g cereal. 27. Ohta and Tanaka reported a method for the simultaneous analysis of several inorganic anions and the cations Mg2+ and Ca2+ in water by ion-exchange chromatography.30 The mobile phase includes 1,2,4-benzenetricarboxylate, which absorbs strongly at 270 nm. Indirect detection of the analytes is possible because their presence in the detector leads to a decrease in absorbance. Unfortunately, Ca2+ and Mg2+, which are present at high concentrations in many environmental waters, form stable complexes with 1,2,4benzenetricarboxylate that interfere with the analysis. (a) Adding EDTA to the mobile phase eliminates the interference caused by Ca2+ and Mg2+; explain why. (b) A standard solution containing 1.0 M NaHCO3, 0.20 mM NaNO2, 0.20 mM MgSO4, 0.10 mM CaCl2, and 0.10 mM Ca(NO3)2 gives the following typical peak areas (arbitrary units).
Ion Peak Area Ion Peak Area HCO3– 373.5 Ca2+ 458.9 Cl– 322.5 Mg2+ 352.0 NO2– 264.8 SO42– 341.3 NO3– 262.7

25. Kagel and Farwell report a reverse-phase HPLC method for determining the concentration of acetylsalicylic acid (ASA) and caffeine (CAF) in analgesic tablets using salicylic acid (SA) as an internal standard.28 A series of standards was prepared by adding known amounts of acetylsalicylic acid and caffeine to 250-mL Erlenmeyer flasks and adding 100 mL of methanol. A 10.00-mL aliquot of a standard solution of salicylic acid was then added to each. The following results are obtained for a typical set of standard solutions.
Milligrams ASA Milligrams CAF Peak Height Ratio ASA/SA Peak Height Ratio CAF/SA

Standard

1 2 3

200.0 250.0 300.0

20.0 40.0 60.0

20.5 25.1 30.9

10.6 23.0 36.8

A sample of an analgesic tablet was placed in a 250-mL Erlenmeyer flask and dissolved in 100 mL of methanol. After adding a 10.00-mL portion of the internal standard, the solution was filtered. Analysis of the sample gave a peak height ratio of 23.2 for ASA and 17.9 for CAF. (a) Determine the number of milligrams ASA and CAF in the tablet. (b) Why was it necessary to filter the sample? (c) The directions indicate that approximately 100 mL of methanol is used to dissolve the standards and samples. Why is it not necessary to measure this volume more precisely? (d) In the presence of moisture, ASA decomposes to SA and acetic acid. What complication might this present for this analysis? How might you evaluate whether this is a problem? 26. Bohman and colleagues described a reverse-phase HPLC method for the quantitative analysis of vitamin A in food using the method of standard additions.29 In a typical example, a 10.067-g sample of cereal is placed in a 250-mL Erlenmeyer flask along with 1 g of sodium ascorbate, 40 mL of ethanol, and 10 mL of 50% w/v KOH. After refluxing for 30 min, 60 mL of ethanol is added, and the solution is cooled to room temperature. Vitamin A is extracted using three 100-mL portions of hexane. The combined portions of hexane are evaporated, and the residue containing vitamin A is transferred to a 5-mL volumetric flask and diluted to volume with methanol. A

Analysis of a river water sample (pH of 7.49) gives the following results.
Ion Peak Area Ion Peak Area HCO3– 310.0 Ca2+ 734.3 Cl– 403.1 Mg2+ 193.6 NO2– 3.97 SO42– 324.3 NO3– 157.6

Determine the concentration of each ion in the sample of rain water. (c) The detection of HCO3– actually gives the total concentration of carbonate in solution ([CO32–] + [HCO3–] + [H2CO3]). Given that the pH of the water is 7.49, what is the actual concentration of HCO3–? (d) An independent analysis gives the following additional concentrations. [Na+] = 0.60 mM [NH4+] = 0.014 mM [K+] = 0.046 mM

A solution’s ionic balance is defined as the ratio of the total cation charge to the total anion charge. Determine the ion balance for this sample of water, and comment on whether the result is reasonable. 28. The concentrations of Cl–, NO3–, and SO42– may be determined by ion chromatography. A 50-µL standard sample of 10.0-ppm Cl–, 2.00-ppm NO3–, and 5.00-ppm SO42– gave signals (in arbitrary units) of 59.3, 16.1, and 6.08, respectively. A sample of effluent from a wastewater treatment plant was diluted tenfold, and a 50-µL portion gave signals of 44.2 for

Chapter 12 Chromatographic and Electrophoretic Methods
Cl–, 2.73 for NO3–, and 5.04 for SO42–. Report the parts per million of each anion in the effluent sample. 29. A series of polyvinylpyridine standards of different molecular weight were analyzed by size-exclusion chromatography, yielding the following results.
Formula Weight
600,000 100,000 20,000 3,000

619

Retention Volume (mL)
6.42 7.98 9.30 10.94

32. The analysis of NO3– in aquarium water was carried out by CZE using IO4– as an internal standard. A standard solution of 15.0-ppm NO3– and 10.0-ppm IO4– gives peak heights (arbitrary units) of 95.0 and 100.1, respectively. A sample of water from an aquarium is diluted 1:100, and sufficient internal standard added to make its concentration 10.0 ppm. Analysis gives signals of 29.2 and 105.8 for NO3– and IO4–, respectively. Report the parts per million of NO3– in the sample of aquarium water. 33. Suggest conditions for separating a mixture of 2-aminobenzoic acid (pKa1 = 2.08, pKa2 = 4.96), benzylamine (pKa = 9.35), and 4-methylphenol (pKa = 10.26) by capillary zone electrophoresis.

34. McKillop and associates have examined the electrophoretic separation of alkylpyridines by CZE.32 Separations were carried out using either 50-µm or 75-µm inner diameter capillaries, with a total length of 57 cm and a length of 50 cm 30. Diet soft drinks contain appreciable quantities of aspartame, from the point of injection to the detector. The run buffer was benzoic acid, and caffeine. What is the expected order of a pH 2.5 lithium phosphate buffer. Separations were achieved elution for these compounds in a capillary zone using an applied voltage of 15 kV. The electroosmotic flow electrophoresis separation using a pH 9.4 buffer solution, velocity, as measured using a neutral marker, was found given that aspartame has pKa values of 2.964 and 7.37, benzoic to be 6.398 × 10–5 cm2 V–1 s–1. The diffusion coefficient, acid’s pKa is 4.2, and the pKa for caffeine is less than 0. D, for the alkylpyridines may be taken to be 1.0 × 10–5 cm2 s–1.(a) Calculate the electrophoretic mobility for O O COOH 2-ethylpyridine, given that its elution time is 8.20 min. +H N 3 (b) How many theoretical plates are there for OCH3 N 2-ethylpyridine? (c) The electrophoretic mobilities for H 3-ethylpyridine and 4-ethylpyridine are 3.366 × 10–4 cm2 V–1 s–1 and 3.397 × 10–4 cm2 V–1 s–1, respectively. What is COOH benzoic acid the expected resolution between these two alkylpyridines? (d) Explain the trends in electrophoretic mobility shown aspartame in the following table.
O H3C N O N CH3

When a preparation of polyvinylpyridine of unknown formula weight was analyzed the retention volume was found to be 8.45. Report the average formula weight for the preparation.

Alkylpyridine
2-methylpyridine 2-ethylpyridine 2-propylpyridine 2-pentylpyridine 2-hexylpyridine

lep (cm2 V–1 s–1)
3.581 × 10–4 3.222 × 10–4 2.923 × 10–4 2.534 × 10–4 2.391 × 10–4

N CH3 caffeine N

31. Janusa and co-workers report the determination of chloride by CZE.31 Analysis of a series of external standards gives the following calibration curve. Area = -883 + 5590(ppm Cl– Cl–)

(e) Explain the trends in electrophoretic mobility shown in the following table.
Alkylpyridine
2-ethylpyridine 3-ethylpyridine 4-ethylpyridine

lep (cm2 V–1 s–1)
3.222 × 10–4 3.366 × 10–4 3.397 × 10–4

was analyzed by placing A standard sample of 57.22% w/w 0.1011 g in a 100-mL volumetric flask and diluting to volume. Three unknowns were prepared by pipeting 0.250 mL, 0.500 mL, and 0.750 mL of the bulk unknown into separate 50-mL volumetric flasks and diluting to volume. Analysis of the three unknowns gave areas of 15310, 31546, and 47582, respectively. Evaluate the accuracy of this analysis.

(f) The pKa for pyridine is 5.229. At a pH of 2.5 the electrophoretic mobility of pyridine is 4.176 × 10–4 cm2 V–1 s–1. What is the expected electrophoretic mobility if the run buffer’s pH is 7.5?

620

Modern Analytical Chemistry

12O SUGGESTED READINGS
The following texts provide a good introduction to the broad field of separations, including chromatography and electrophoresis. Giddings, J. C. Unified Separation Science. Wiley-Interscience: New York, 1991. Karger, B. L.; Snyder, L. R.; Harvath, C. An Introduction to Separation Science. Wiley-Interscience: New York, 1973. Miller, J. M. Separation Methods in Chemical Analysis. WileyInterscience: New York, 1975. A more recent discussion of peak capacity is presented in the following paper. Shen, Y.; Lee, M. “General Equation for Peak Capacity in Column Chromatography,” Anal. Chem. 1998, 70, 3853–3856. The following references may be consulted for more information on gas chromatography. Grob, R. L., ed. Modern Practice of Gas Chromatography. WileyInterscience: New York, 1972. Hinshaw, J. V. “A Compendium of GC Terms and Techniques,” LC•GC 1992, 10, 516–522. Ioffe, B. V.; Vitenberg, A. G. Head-Space Analysis and Related Methods in Gas Chromatography. Wiley-Interscience: New York, 1982. Kitson, F. G.; Larsen, B. S.; McEwen, C. N. Gas Chromatography and Mass Spectrometry: A Practical Guide. Academic Press: San Diego, 1996. The following references may be consulted for more information on high-performance liquid chromatography. Dorschel, C. A.; Ekmanis, J. L.; Oberholtzer, J. E.; et al. “LC Detectors,” Anal. Chem. 1989, 61, 951A–968A. Simpson, C. F., ed. Techniques in Liquid Chromatography. WileyHayden: Chichester, England, 1982. Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. Practical HPLC Method Development. Wiley-Interscience: New York, 1988. The following references may be consulted for more information on ion chromatography. Shpigun, O. A.; Zolotov, Y. A. Ion Chromatography in Water Analysis. Ellis Horwood: Chichester, England, 1988. Smith, F. C. Jr.; Chang, R. C. The Practice of Ion Chromatography. Wiley-Interscience: New York, 1983. The following references may be consulted for more information on supercritical fluid chromatography. Palmieri, M. D. “An Introduction to Supercritical Fluid Chromatography. Part I: Principles and Applications,” J. Chem. Educ. 1988, 65, A254–A259. Palmieri, M. D. “An Introduction to Supercritical Fluid Chromatography. Part II: Applications and Future Trends,” J. Chem. Educ. 1989, 66, A141–A147. The following references may be consulted for more information on capillary electrophoresis. Baker, D. R. Capillary Electrophoresis. Wiley-Interscience: New York, 1995. Copper, C. L. “Capillary Electrophoresis: Part I. Theoretical and Experimental Background,” J. Chem. Educ. 1998, 75, 343–347. Copper, C. L.; Whitaker, K. W. “Capillary Electrophoresis: Part II. Applications,” J. Chem. Educ. 1998, 75, 347–351. The application of spreadsheets and computer programs for modeling chromatography is described in the following papers. Abbay, G. N.; Barry, E. F.; Leepipatpiboon, S.; et al. “Practical Applications of Computer Simulation for Gas Chromatography Method Development,” LC•GC 1991, 9, 100–114. Drouen, A.; Dolan, J. W.; Snyder, L. R.; et al. “Software for Chromatographic Method Development,” LC•GC 1991, 9, 714–724. Kevra, S. A.; Bergman, D. L.; Maloy, J. T. “A Computational Introduction to Chromatographic Bandshape Analysis,” J. Chem. Educ. 1994, 71, 1023–1028. Sundheim, B. R. “Column Operations: A Spreadsheet Model.” J. Chem. Educ. 1992, 69, 1003–1005.

12P REFERENCES
1. Craig, L. C. J. Biol. Chem. 1944, 155, 519. 2. Martin, A. J. P.; Synge, R. L. M. Biochem. J. 1941, 35, 1358. 3. Giddings, J. C. Unified Separation Science. Wiley-Interscience: New York, 1991. 4. Hawkes, S. J. J. Chem. Educ. 1983, 60, 393–398. 5. Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 1128–1135. 6. Hinshaw, J. V. LC•GC, 1993, 11, 644–648. 7. Grob, K. Anal. Chem. 1994, 66, 1009A–1019A. 8. Marriott, P. J.; Carpenter, P. D. J. Chem. Educ. 1996, 73, 96–99. 9. Method 6232B as published in Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Health Association: Washington, D.C., 1992. 10. Novotny, M. Science 1989, 246, 51–57. 11. (a) Jorgenson, J. W.; Guthrie, E. J. J. Chromatog. 1983, 255, 335; (b) Kennedy, R. T.; Oates, M. D.; Cooper, B. R.; et al. Science 1989, 246, 57–63.

Chapter 12 Chromatographic and Electrophoretic Methods
12. Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. Practical HPLC Method Development. Wiley-Interscience: New York, 1988. 13. Foley, J. P. Chromatography 1987, 2(6), 43–51. 14. Yeung, E. S. LC•GC 1989, 7, 118–128. 15. Smyth, W. F. Analytical Chemistry of Complex Matrices. Wiley Teubner: Chichester, England, 1996, pp. 187–189. 16. Baker, D. R. Capillary Electrophoresis. Wiley-Interscience: New York, 1995. 17. Jones, W. R.; Jandik, P. J. Chromatog. 1992, 608, 385–393. 18. Smyth, W. F. Analytical Chemistry of Complex Matrices. Wiley Teubner: Chichester, England, 1996, pp. 154–156. 19. Moody, H. W. J. Chem. Educ. 1982, 59, 218–219. 20. Hsieh, S.; Jorgenson, J. W. Anal. Chem. 1996, 68, 1212–1217. 21. Zhou, X.; Hines, P. A.; White, K. C.; et al. Anal. Chem. 1998, 70, 390–394. 22. Loconto, P. R.; Pan, Y. L.; Voice, T. C. LC•GC 1996, 14, 128–132.

621

23. Van Atta, R. E.; Van Atta, R. L. J. Chem. Educ. 1980, 57, 230–231. 24. Pant, S. K.; Gupta, P. N.; Thomas, K. M.; et al. LC•GC 1990, 8, 322–325. 25. Feigel, C. Varian GC/MS Application Note, Number 22. 26. Otto, M.; Wegscheider, W. J. Chromatogr. 1983, 258, 11–22. 27. Haddad, P.; Hutchins, S.; Tuffy, M. J. Chem. Educ. 1983, 60, 166–168. 28. Kagel, R. A.; Farwell, S. O. J. Chem. Educ. 1983, 60, 163–166. 29. Bohman, O.; Engdahl, K. A.; Johnsson, H. J. Chem. Educ. 1982, 59, 251–252. 30. Ohta, K.; Tanaka, K. Anal. Chim. Acta 1998, 373, 189–195. 31. Janusa, M. A.; Andermann, L. J.; Kliebert, N. M.; et al. J. Chem. Educ. 1998, 75, 1463–1465. 32. McKillop, A. G.; Smith, R. M.; Rowe, R. C.; et al. Anal. Chem. 1999, 71, 497–503.

3 \Chapter 1
Kinetic Methods of Analysis system under thermodynamic control is in a state of equilibrium, and its signal has a constant, or steady-state value (Figure 13.1a). When a system is under kinetic control, however, its signal changes with time (Figure 13.1b) until equilibrium is established. Thus far, the techniques we have considered have involved measurements made when the system is at equilibrium. By changing the time at which measurements are made, an analysis can be carried out under either thermodynamic control or kinetic control. For example, one method for determining the concentration of NO2– in groundwater involves the diazotization reaction shown in Figure 13.2.1 The final product, which is a reddish-purple azo dye, absorbs visible light at a wavelength of 543 nm. Since the concentration of dye is determined by the amount of NO2– in the original sample, the solution’s absorbance can be used to determine the concentration of NO2–. The reaction in the second step, however, is not instantaneous. To achieve a steady-state signal, such as that in Figure 13.1a, the absorbance is measured following a 10-min delay. By measuring the signal during the 10-min development period, information about the rate of the reaction is obtained. If the reaction’s rate is a function of the concentration of NO2–, then the rate also can be used to determine its concentration in the sample.2 There are many potential advantages to kinetic methods of analysis, perhaps the most important of which is the ability to use chemical reactions that are slow to reach equilibrium. In this chapter we examine three techniques that rely on measurements made while the analytical system is under kinetic rather than thermodynamic control: chemical kinetic techniques, in which the rate of a chemical reaction is measured; radiochemical techniques, in which a radioactive element’s rate of nuclear decay is measured; and flow injection analysis, in which the analyte is injected into a continuously flowing carrier stream, where its mixing and reaction with reagents in the stream are controlled by the kinetic processes of convection and diffusion.

A

622

Chapter 13 Kinetic Methods of Analysis

623

13A Methods Based on Chemical Kinetics
The earliest examples of analytical methods based on chemical kinetics, which date from the late nineteenth century, took advantage of the catalytic activity of enzymes. Typically, the enzyme was added to a solution containing a suitable substrate, and the reaction between the two was monitored for a fixed time. The enzyme’s activity was determined by measuring the amount of substrate that had reacted. Enzymes also were used in procedures for the quantitative analysis of hydrogen peroxide and carbohydrates. The application of catalytic reactions continued in the first half of the twentieth century, and developments included the use of nonenzymatic catalysts, noncatalytic reactions, and differences in reaction rates when analyzing samples with several analytes.

Signal

Signal

Figure 13.1
Time (a) (b) Time
Plot of signal versus time for an analytical system that is under (a) thermodynamic control; and (b) under kinetic control.

Step 1
+

H2NO3S sulfanilamide Step 2

NH2 + NO2– + 2H+

H2NO3S diazonium ion

N

N + 2H2O

H2NO3S diazonium ion

N

+

N +
+

N -(1-naphthyl)-ethylenediamine dihydrochloride
NH2 C2H4NH3
+

H2NO3S

N

N
+

+ H+

NH2 azo dye C2H4NH3
+

Figure 13.2
Analytical scheme for the analysis of NO2– in groundwater.

624

Modern Analytical Chemistry Despite the variety of methods that had been developed, by 1960 kinetic methods were no longer in common use. The principal limitation to a broader acceptance of chemical kinetic methods was their greater susceptibility to errors from uncontrolled or poorly controlled variables, such as temperature and pH, and the presence of interferents that activate or inhibit catalytic reactions. Many of these limitations, however, were overcome during the 1960s, 1970s, and 1980s with the development of improved instrumentation and data analysis methods compensating for these errors.3

13A.1 Theory and Practice rate The change in a property’s value per unit change in time; the rate of a reaction is a change in concentration per unit change in time.

rate law An equation relating a reaction’s rate at a given time to the concentrations of species affecting the rate.

Every chemical reaction occurs at a finite rate and, therefore, can potentially serve as the basis for a chemical kinetic method of analysis. To be effective, however, the chemical reaction must meet three conditions. First, the rate of the chemical reaction must be fast enough that the analysis can be conducted in a reasonable time, but slow enough that the reaction does not approach its equilibrium position while the reagents are mixing. As a practical limit, reactions reaching equilibrium within 1 s are not easily studied without the aid of specialized equipment allowing for the rapid mixing of reactants. A second requirement is that the rate law for the chemical reaction must be known for the period in which measurements are made. In addition, the rate law should allow the kinetic parameters of interest, such as rate constants and concentrations, to be easily estimated. For example, the rate law for a reaction that is first order in the concentration of the analyte, A, is expressed as rate = − d[A] = k[A] dt 13.1

rate constant In a rate law, the proportionality constant between a reaction’s rate and the concentrations of species affecting the rate (k).

where k is the reaction’s rate constant. As shown in Appendix 5,* the integrated form of this rate law ln [A]t = ln [A]0 – kt or [A]t = [A]0e–kt 13.2

provides a simple mathematical relationship between the rate constant, the reaction’s elapsed time, t, the initial concentration of analyte, [A]0, and the analyte’s concentration at time t, [A]t. Unfortunately, most reactions of analytical interest do not follow the simple rate laws shown in equations 13.1 and 13.2. Consider, for example, the following reaction between an analyte, A, and a reagent, R, to form a product, P A+R

tP k b kf

where kf is the rate constant for the forward reaction, and kb is the rate constant for the reverse reaction. If the forward and reverse reactions occur in single steps, then the rate law is Rate = kf[A][R] – kb[P] 13.3

Although the rate law for the reaction is known, there is no simple integrated form. We can simplify the rate law for the reaction by restricting measurements to the

*Appendix 5 provides a general review of kinetics.

Chapter 13 Kinetic Methods of Analysis beginning of the reaction when the product’s concentration is negligible. Under these conditions, the second term in equation 13.3 can be ignored; thus Rate = kf[A][R] 13.4

625

The integrated form of the rate law for equation 13.4, however, is still too complicated to be analytically useful. We can simplify the kinetics, however, by carefully adjusting the reaction conditions.4 For example, pseudo-first-order kinetics can be achieved by using a large excess of R (i.e. [R]0 >> [A]0), such that its concentration remains essentially constant. Under these conditions Rate = − d[A] = k[R]0[A] = k ′[A] dt or [A]t = [A]0e–k′t 13.5 13.6

ln [A]t = ln [A]0 – k′t

It may even be possible to adjust conditions such that measurements are made under pseudo-zero-order conditions where Rate = − d[A] = k[A]0[R]0 = k ′′ dt 13.7 13.8

[A]t = [A]0 – k″t

A final requirement for a chemical kinetic method of analysis is that it must be possible to monitor the reaction’s progress by following the change in concentration for one of the reactants or products as a function of time. Which species is used is not important; thus, in a quantitative analysis the rate can be measured by monitoring the analyte, a reagent reacting with the analyte, or a product. For example, the concentration of phosphate can be determined by monitoring its reaction with Mo(VI) to form 12-molybdophosphoric acid (12-MPA). H3PO4 + 6Mo(VI) + 9H2O → 12-MPA + 9H3O+ 13.9

We can monitor the progress of this reaction by coupling it to a second reaction in which 12-MPA is reduced to form heteropolyphosphomolybdenum blue, PMB, 12-MPA + nRed → PMB + nOx where Red is a suitable reducing agent, and Ox is its conjugate form.5,6 The rate of formation of PMB is measured spectrophotometrically and is proportional to the concentration of 12-MPA. The concentration of 12-MPA, in turn, is proportional to the concentration of phosphate. Reaction 13.9 also can be followed spectrophotometrically by monitoring the formation of 12-MPA.6,7

Classifying Chemical Kinetic Methods A useful scheme for classifying chemical kinetic methods of analysis is shown in Figure 13.3.3 Methods are divided into two main categories. For those methods identified as direct-computation methods, the concentration of analyte, [A]0, is calculated using the appropriate rate law. Thus, for a first-order reaction in A, equation 13.2 is used to determine [A]0, provided that values for k, t, and [A]t are known. With a curve-fitting method, regression is used to find the best fit between the data (e.g., [A]t as a function of time) and the known mathematical model for the rate law. In this case, kinetic parameters, such as k and [A]0, are adjusted to find the best fit. Both categories are further subdivided into rate methods and integral methods.

626

Modern Analytical Chemistry
Chemical kinetic methods

Direct-computation methods

Curve-fitting methods

Integral methods

Rate methods

Integral methods

Rate methods

Fixed-time One-point Two-point

Initial rate

Linear response

Figure 13.3
Classification of chemical kinetic methods of analysis.

Variable-time One-point Two-point

Intermediate rate

Nonlinear response

Direct-Computation Integral Methods Integral methods for analyzing kinetic data make use of the integrated form of the rate law. In the one-point fixed-time integral method, the concentration of analyte is determined at a single time. The initial concentration of analyte, [A]0, is calculated using equation 13.2, 13.6, or 13.8, depending on whether the reaction follows first-order, pseudo-first-order, or pseudo-zeroorder kinetics. The rate constant for the reaction is determined in a separate experiment using a standard solution of analyte. Alternatively, the analyte’s initial concentration can be determined using a calibration curve consisting of a plot of [A]t for several standard solutions of known [A]0. EXAMPLE

13.1

The concentration of nitromethane, CH3NO2, can be determined from the kinetics of its decomposition in basic solution. In the presence of excess base the reaction is pseudo-first-order in nitromethane. For a standard solution of 0.0100 M nitromethane, the concentration of nitromethane after 2.00 s was found to be 4.24 × 10–4 M. When a sample containing an unknown amount of nitromethane was analyzed, the concentration remaining after 2.00 s was found to be 5.35 × 10–4 M. What is the initial concentration of nitromethane in the sample? SOLUTION The value for the pseudo-first-order rate constant is determined by solving equation 13.6 for k′ and making appropriate substitutions; thus k′ = ln[A]0 − ln[A]t ln(0.0100) − ln(4.24 × 10 −4 ) = = 1.58 s −1 t 2.00 s

Equation 13.6 can then be solved for the initial concentration of nitromethane. This is easiest to do using the exponential form of equation 13.6. [A]t = [A]0 e − k ′t [A]0 = [A]t 5.35 × 10 −4 M = = 0.0126 M −1 e − k ′t e −(1.58 s )(2.00 s)

Chapter 13 Kinetic Methods of Analysis In Example 13.1 the initial concentration of analyte is determined by measuring the amount of unreacted analyte at a fixed time. Sometimes it is more convenient to measure the concentration of a reagent reacting with the analyte or the concentration of one of the reaction’s products. The one-point fixed-time integral method can still be applied if the stoichiometry is known between the analyte and the species being monitored. For example, if the concentration of the product in the reaction A+R→P is monitored, then the concentration of the analyte at time t is [A]t = [A]0 – [P]t 13.10

627

since the stoichiometry between the analyte and product is 1:1. Substituting equation 13.10 into equation 13.6 gives ln([A]0 – [P]t) = ln [A]0 – k′t which is simplified by writing in exponential form [A]0 – [P]t = [A]0e–k′t and solving for [A]0. [A]0 = [P]t 1 − e −k ′t 13.12 13.11

EXAMPLE

13.2

The concentration of thiocyanate, SCN–, can be determined from the pseudofirst-order kinetics of its reaction with excess Fe3+ to form a reddish colored complex of Fe(SCN)2+. The reaction’s progress is monitored by measuring the absorbance of Fe(SCN)2+ at a wavelength of 480 nm. When a standard solution of 0.100 M SCN– is used, the concentration of Fe(SCN)2+ after 10.0 s is found to 0.0516 M. The analysis of a sample containing an unknown amount of SCN– results in a concentration of Fe(SCN)2+ of 0.0420 M after 10.0 s. What is the initial concentration of SCN– in the sample? SOLUTION The pseudo-first-order rate constant is determined by solving equation 13.11 for k′ and making appropriate substitutions k′ = ln[A]0 − ln([A]0 − [P]t ) ln(0.1) − ln(0.1 − 0.0516) = = 0.0726 s −1 t 10.0 s

Equation 13.12 then can be used to determine the initial concentration of SCN–. [A]0 = [P]t 0.0420 M = = 0.0814 M −k ′t −(0.0726 s −1 )(10.0 s) 1−e 1−e

The one-point fixed-time integral method has the advantage of simplicity since only a single measurement is needed to determine the analyte’s initial concentration. As with any method relying on a single determination, however, a

628

Modern Analytical Chemistry one-point fixed-time integral method cannot compensate for constant sources of determinate error. Such corrections can be made by making measurements at two points in time and using the difference between the measurements to determine the analyte’s initial concentration. Constant sources of error affect both measurements equally, thus, the difference between the measurements is independent of these errors. For a two-point fixed-time integral method, in which the concentration of analyte for a pseudo-first-order reaction is measured at times t1 and t2 , we can write [A]t1 = [A]0e–k′tl [A]t2 = [A]0e–k′t2 Subtracting the second equation from the first equation and solving for [A]0 gives [A]0 = [A]t 1 − [A]t 2 e −k ′t 1 − e −k ′t 2 13.14 13.13

The rate constant for the reaction can be calculated from equation 13.14 by measuring [A]t1 and [A]t2 for a standard solution of analyte. The analyte’s initial concentration also can be found using a calibration curve consisting of a plot of ([A]t1 – [A]t2) versus [A]0. Fixed-time integral methods are advantageous for systems in which the signal is a linear function of concentration. In this case it is not necessary to determine the concentration of the analyte or product at times t1 or t2 , because the relevant concentration terms can be replaced by the appropriate signal. For example, when a pseudo-first-order reaction is followed spectrophotometrically, when Beer’s law (Abs)t = εb[A]t is valid, equations 13.6 and 13.14 can be rewritten as (Abs)t = [A]0(e–k′t)εb = c[A]0 [A]0 = (Abs)t 1 − (Abs)t 2 1 × = c[(Abs)t 1 − (Abs)t 2 ] −k ′t 1 −k ′t 2 εb e −e

where (Abs)t is the absorbance at time t, and c is a constant. An alternative to a fixed-time method is a variable-time method, in which we measure the time required for a reaction to proceed by a fixed amount. In this case the analyte’s initial concentration is determined by the elapsed time, ∆t, with a higher concentration of analyte producing a smaller ∆t. For this reason variabletime integral methods are appropriate when the relationship between the detector’s response and the concentration of analyte is not linear or is unknown. In the onepoint variable-time integral method, the time needed to cause a desired change in concentration is measured from the start of the reaction. With the two-point variable-time integral method, the time required to effect a change in concentration is measured. One important application of the variable-time integral method is the quantitative analysis of catalysts, which is based on the catalyst’s ability to increase the rate of a reaction. As the initial concentration of catalyst is increased, the time needed to reach the desired extent of reaction decreases. For many catalytic systems the relationship between the elapsed time, ∆t, and the initial concentration of analyte is 1 = Fcat[A]0 + Funcat ∆t

Chapter 13 Kinetic Methods of Analysis where Fcat and Funcat are constants that are functions of the rate constants for the catalyzed and uncatalyzed reactions, and the extent of the reaction during the time span ∆t.8 EXAMPLE

629

13.3
As3+ + 2Ce4+ → As5+ + 2Ce3+

Sandell and Kolthoff9 developed a quantitative method for iodide based on its catalytic effect on the following redox reaction.

Standards were prepared by adding a known amount of KI to fixed amounts of As3+ and Ce4+ and measuring the time for all the Ce4+ to be reduced. The following results were obtained:
Micrograms I–
5.0 2.5 1.0

∆t (min)
0.9 1.8 4.5

How many micrograms of I– are in a sample for which ∆t is found to be 3.2 min? SOLUTION The relationship between the concentration of I– and ∆t is shown by the calibration curve in Figure 13.4, for which 1 = −8.67 × 10 −9 + 0.222(µg I − ) ∆t Substituting 3.2 min for ∆t in the preceding equation gives 1.4 µg as the amount of I– originally present in the sample.
1.2 1.0 0.8 1 Dt 0.6 0.4 0.2 0.0 0 1 2 3 4 µg I – 5 6 ∆t ∆[P ] [P] ∆[P ] ∆t

Figure 13.4
Calibration curve for the variable-time integral determination of I–.

(rate)t =

Direct-Computation Rate Methods Rate methods for analyzing kinetic data are based on the differential form of the rate law. The rate of a reaction at time t, (rate)t, is determined from the slope of a curve showing the change in concentration for a reactant or product as a function of time (Figure 13.5). For a reaction that is firstorder, or pseudo-first-order in analyte, the rate at time t is given as (rate)t = k[A]t

t
Time

Figure 13.5
Determination of reaction rate from a tangent line at time t.

630

Modern Analytical Chemistry Substituting an equation similar to 13.13 into the preceding equation gives the following relationship between the rate at time t and the analyte’s initial concentration. (rate)t = k[A]0e–kt If the rate is measured at a fixed time, then both k and e–kt are constant, and a calibration curve of (rate)t versus [A]0 can be used for the quantitative analysis of the analyte. The use of the initial rate (t = 0) has the advantage that the rate is at its maximum, providing an improvement in sensitivity. Furthermore, the initial rate is measured under pseudo-zero-order conditions, in which the change in concentration with time is effectively linear, making the determination of slope easier. Finally, when using the initial rate, complications due to competing reactions are avoided. One disadvantage of the initial rate method is that there may be insufficient time for a complete mixing of the reactants. This problem is avoided by using a rate measured at an intermediate time (t > 0). EXAMPLE

13.4

The concentration of aluminum in serum can be determined by adding 2-hydroxy-1-naphthaldehyde p-methoxybenzoyl-hydrazone and measuring the initial rate of the resulting complexation reaction under pseudo-first-order conditions.10 The rate of reaction is monitored by the fluorescence of the metal–ligand complex. Initial rates, with units of emission intensity per second, were measured for a set of standard solutions, yielding the following results
[Al3+] (µM) (rate)t = 0 0.300 0.261 0.500 0.599 1.00 1.44 3.00 4.82

A serum sample treated in the same way as the standards has an initial rate of 0.313 emission intensity/s. What is the concentration of aluminum in the serum sample? SOLUTION A calibration curve of emission intensity per second versus the concentration of Al3+ (Figure 13.6) is a straight line, where (rate)t = 0 = 1.69 × [Al3+ (µM)] – 0.246 Substituting the sample’s initial rate into the calibration equation gives an aluminum concentration of 0.331 µM.
5 4 (rate)t = 0 3 2 1 0 0 1 2 [Al 3+] 3 4

Figure 13.6
Result of curve-fitting for the kinetic data in Example 13.4.

Chapter 13 Kinetic Methods of Analysis Curve-Fitting Methods In the direct-computation methods discussed earlier, the analyte’s concentration is determined by solving the appropriate rate equation at one or two discrete times. The relationship between the analyte’s concentration and the measured response is a function of the rate constant, which must be measured in a separate experiment. This may be accomplished using a single external standard (as in Example 13.2) or with a calibration curve (as in Example 13.4). In a curve-fitting method the concentration of a reactant or product is monitored continuously as a function of time, and a regression analysis is used to fit an appropriate differential or integral rate equation to the data. For example, the initial concentration of analyte for a pseudo-first-order reaction, in which the concentration of a product is followed as a function of time, can be determined by fitting a rearranged form of equation 13.12 [P]t = [A]0(1 – e–k′t) to the kinetic data using both [A] 0 and k′ as adjustable parameters. By using data from more than one or two discrete times, curve-fitting methods are capable of producing more reliable results. Although curve-fitting methods are computationally more demanding, the calculations are easily handled by computer.

631

EXAMPLE 13.5 The data shown in the following table were collected for a reaction known to follow pseudo-zero-order kinetics during the time in which the reaction was monitored.
Time (s)
3 4 5 6 7 8 9 10 11 12

[A]t (mM)
0.0731 0.0728 0.0681 0.0582 0.0511 0.0448 0.0404 0.0339 0.0217 0.0143

What are the rate constant and the initial concentration of analyte in the sample? SOLUTION For a pseudo-zero-order reaction a plot of [A] t versus time should be linear with a slope of –k, and a y-intercept of [A]0 (equation 13.8). A plot of the kinetic data is shown in Figure 13.7. Linear regression gives an equation of [A]t = 0.0986 – 0.00677t

632

Modern Analytical Chemistry
0.10 0.08 0.06 [A]t 0.04 0.02 0.00 0 2 4 6 8 10 12 14 Time

Figure 13.7
Result of curve-fitting for the kinetic data in Example 13.5.

Thus, the rate constant is 0.00677 s–1, and the initial concentration of analyte is 0.0986 mM.

Miscellaneous Methods At the beginning of this section we noted that kinetic methods are susceptible to significant errors when experimental variables affecting the reaction’s rate are difficult to control. Many variables, such as temperature, can be controlled with proper instrumentation. Other variables, such as interferents in the sample matrix, are more difficult to control and may lead to significant errors. Although not discussed in this text, direct-computation and curve-fitting methods have been developed that compensate for these sources of error.3 Representative Method Although each chemical kinetic method has its own unique considerations, the determination of creatinine in urine based on the kinetics of its reaction with picrate provides an instructive example of a typical procedure.

Representative Methods

Method 13.1

Determination of Creatinine in Urine11

Description of Method. Creatine is an organic acid found in muscle tissue that supplies energy for muscle contractions. One of its metabolic products is creatinine, which is excreted in urine. Because the concentration of creatinine in urine and serum is an important indication of renal function, rapid methods for its analysis are clinically important. In this method the rate of reaction between creatinine and picrate in an alkaline medium is used to determine the concentration of creatinine in urine. Under the conditions of the analysis, the reaction is first-order in picrate, creatinine, and hydroxide. Rate = k[picrate][creatinine][OH–] The rate of reaction is monitored using a picrate ion-selective electrode.

Procedure. Prepare a set of external standards containing 0.5 g/L to 3.0 g/L creatinine (in 5 mM H2SO4) using a stock solution of 10.00 g/L creatinine in 5 mM H2SO4. In addition, prepare a solution of 1.00 × 10–2 M sodium picrate. Pipet 25.00 mL of 0.20 M NaOH, adjusted to an ionic strength of 1.00 M using Na2SO4, into a thermostated reaction cell at 25 °C. Add 0.500 mL of the 1.00 × 10–2 M picrate solution to the reaction cell. Suspend a picrate ion-selective electrode in the solution, and monitor the potential until it stabilizes. When the potential is stable, add 2.00 mL of a
—Continued

Chapter 13 Kinetic Methods of Analysis

633

creatinine external standard, and record the potential as a function of time. Repeat the procedure using the remaining external standards. Construct a calibration curve of rate (∆E/∆t) versus [creatinine]. Samples of urine (2.00 mL) are analyzed in a similar manner, with the concentration of creatinine determined from the calibration curve.

Questions
1. The analysis is carried out under conditions in which the reaction’s kinetics are pseudo-first-order in picrate. Show that under these conditions, a plot of potential as a function of time will be linear. The response of the picrate ion-selective electrode is E = K − RT ln [picrate] F

We know from equation 13.6 that for a pseudo-first-order reaction, the concentration of picrate at time t is ln [picrate]t = ln [picrate]0 – k′t where k′ is the pseudo-first-order rate constant. Substituting the kinetic expression into the equation for the ion-selective electrode’s potential leaves us with Et = K − RT (ln [picrate]0 − k ′t ) F RT RTk ′ ln [picrate]0 + t F F

Et = K −

Since both K and (RT/F) ln [picrate]0 are constants, a plot of E t versus t will be a straight line whose slope, RTk′/F, is the reaction’s rate (∆E/∆t). 2. As carried out the rate of the reaction is pseudo-first-order in picrate and pseudo-zero-order in creatinine and OH–. Explain why it is possible to prepare a calibration curve of rate versus [creatinine]. Since the reaction is carried out under conditions in which it is pseudo-zeroorder in creatinine and OH–, the rate constant, k′, is k′ = k[creatinine][OH–] where k is the reaction’s true rate constant. The rate, therefore, is RTk[creatinine][OH− ] = c [creatinine] F where c is a constant. Rate = 3. Why is it necessary to use a thermostat in the reaction cell? The rate of a reaction is temperature-dependent. To avoid a determinate error resulting from a systematic change in temperature or to minimize indeterminate errors due to fluctuations in temperature, the reaction cell must have a thermostat to maintain a constant temperature. 4. Why is it necessary to prepare the NaOH solution so that it has an ionic strength of 1.00 M? The potential of the ion-selective electrode actually responds to the activity of picrate in solution. By adjusting the NaOH solution to a high ionic strength, we maintain a constant ionic strength in all standards and samples. Because the relationship between activity and concentration is a function of ionic strength (see Chapter 6), the use of a constant ionic strength allows us to treat the potential as though it were a function of the concentration of picrate.

634

Modern Analytical Chemistry

13A.2 Instrumentation
Quantitative information about a chemical reaction can be made using any of the techniques described in the preceding chapters. For reactions that are kinetically slow, an analysis may be performed without worrying about the possibility that significant changes in concentration occur while measuring the signal. When the reaction’s rate is too fast, which is usually the case, significant errors may be introduced if changes in concentration are ignored. One solution to this problem is to stop, or quench, the reaction by suitably adjusting experimental conditions. For example, many reactions involving enzymes show a strong pH dependency and may be quenched by adding a strong acid or strong base. Once the reaction is stopped, the concentration of the desired species can be determined at the analyst’s convenience. Another approach is to use a visual indicator that changes color after the reaction occurs to a fixed extent. You may recall that this variable-time method is the basis of the so-called “clock reactions” commonly used to demonstrate kinetics in the general chemistry classroom and laboratory. Finally, reactions with fast kinetics may be monitored continuously using the same types of spectroscopic and electrochemical detectors found in chromatographic instrumentation. Two additional problems for chemical kinetic methods of analysis are the need to control the mixing of the sample and reagents in a rapid and reproducible fashion and the need to control the acquisition and analysis of the signal. Many kinetic determinations are made early in the reaction when pseudo-zeroorder or pseudo-first-order conditions are in effect. Depending on the rate of reaction, measurements are typically made within a span of a few milliseconds or seconds. This is both an advantage and a disadvantage. The disadvantage is that transferring the sample and reagent to a reaction vessel and their subsequent mixing must be automated if a reaction with rapid kinetics is to be practical. This usually requires a dedicated instrument, thereby adding an additional expense to the analysis. The advantage is that a rapid, automated analysis allows for a high throughput of samples. For example, an instrument for the automated kinetic analysis of phosphate, based on reaction 13.9, has achieved sampling rates of 3000 determinations per hour.6 A variety of designs have been developed to automate kinetic analyses.6 The stopped-flow apparatus, which is shown schematically in Figure 13.8, has found use in kinetic determinations involving very fast reactions. Sample and reagents are loaded into separate syringes, and precisely measured volumes are dispensed by the action of a syringe drive. The two solutions are rapidly mixed in the mixing chamber before flowing through an observation cell. The flow of sample and reagents is stopped by applying back pressure with the stopping syringe. The back pressure completes the mixing, after which the reaction’s progress is monitored spectrophotometrically. With a stopped-flow apparatus, it is possible to complete the mixing of sample and reagent and initiate the kinetic measurements within approximately 0.5 ms. The stopped-flow apparatus shown in Figure 13.8 can be modified by attaching an automatic sampler to the sample syringe, thereby allowing the sequential analysis of multiple samples. In this way the stopped-flow apparatus can be used for the routine analysis of several hundred samples per hour. Another automated approach to kinetic analyses is the centrifugal analyzer, a partial cross section of which is shown in Figure 13.9. In this technique the sample and reagents are placed in separate wells oriented radially around a circular transfer disk attached to the rotor of a centrifuge. As the centrifuge spins, the

quench To stop a reaction by suddenly changing the reaction conditions.

stopped flow A kinetic method of analysis designed to rapidly mix samples and reagents when using reactions with very fast kinetics.

Syringe drive

Figure 13.8
Schematic diagram of a stopped-flow analyzer.

Reagent syringe

Sample syringe

Mixing chamber Light source

Monochromator

Detector

Observation cell Stopping syringe Syringe drive Detector Cuvette Monochromator Optically transparent windows (b) (c) Rotor Reagents Sample Cross-section Transfer disk

Light source

(a)

Figure 13.9
Schematic diagram of a centrifugal analyzer showing (a) the wells for holding the sample and reagent; (b) mixing of the sample and reagent; and (c) the configuration of the spectrophotometric detector.

635

636

Modern Analytical Chemistry sample and reagents are pulled by the centrifugal force to the cuvette, where mixing occurs. A single optical source and detector, located above and below the transfer disk’s outer edge, allows the absorbance of the reaction mixture to be measured as it passes through the optical beam. The centrifugal analyzer allows a number of samples to be analyzed simultaneously. For example, if a transfer plate contains 30 cuvettes and rotates with a speed of 600 rpm, it is possible to collect 10 data points per sample for each second of rotation. The ability to collect kinetic data for several hundred samples per hour is of little consequence if the analysis of the data must be accomplished manually. Besides time, the manual analysis of kinetic data is limited by noise in the detector’s signal and the accuracy with which the analyst can determine reaction rates from tangents drawn to differential rate curves. Not surprisingly, the development of automated kinetic analyzers was paralleled by the development of analog and digital circuitry, as well as computer software for the smoothing, on-line integration and differentiation, and analysis of kinetic signals.12

13A.3 Quantitative Applications
Chemical kinetic methods of analysis continue to find use for the analysis of a variety of analytes, most notably in clinical laboratories, where automated methods aid in handling a large volume of samples. In this section several general quantitative applications are considered. enzyme A protein that catalyzes biochemical reactions. substrate The specific molecule for which an enzyme serves as a catalyst.

Enzyme-Catalyzed Reactions Enzymes are highly specific catalysts for biochemical reactions, with each enzyme showing a selectivity for a single reactant, or substrate. For example, acetylcholinesterase is an enzyme that catalyzes the decomposition of the neurotransmitter acetylcholine to choline and acetic acid. Many enzyme–substrate reactions follow a simple mechanism consisting of the initial formation of an enzyme–substrate complex, ES, which subsequently decomposes to form product, releasing the enzyme to react again. E+S

t ES kt E + P k
−1 −2

k1

k2

13.15

If measurements are made early in the reaction, the product’s concentration is negligible, and the step described by the rate constant k–2 can be ignored. Under these conditions the rate of the reaction is d[P] = k2 [ES] dt 13.16

steady-state approximation In a kinetic process, the assumption that a compound formed during the reaction reaches a concentration that remains constant until the reaction is nearly complete.

To be analytically useful equation 13.16 needs to be written in terms of the concentrations of enzyme and substrate. This is accomplished by applying the “steady-state” approximation, in which we assume that the concentration of ES is essentially constant. After an initial period in which the enzyme–substrate complex first forms, the rate of formation of ES  d[ES]   = k1[E][S] = k1([E]0 − [ES])[S]  dt  f and its rate of disappearance  −d[ES]   = k−1[ES] + k2 [ES]  dt  d

Chapter 13 Kinetic Methods of Analysis are equal. Combining these equations gives k1([E]0 – [ES])[S] = k–1[ES] + k2[ES] which is solved for the concentration of the enzyme–substrate complex [ES] = [E]0[S] [E]0[S] = {(k−1 + k2 )/k1} + [S] K m + [S] 13.17

637

where Km is called the Michaelis constant. Substituting equation 13.17 into equation 13.16 leads to the final rate equation d[P] k2[E]0[S] = dt K m + [S] 13.18

Michaelis constant A combination of several rate constants affecting the rate of an enzyme-substrate reaction.

A plot of equation 13.18, shown in Figure 13.10, is instructive for defining conditions under which the rate of an enzymatic reaction can be used for the quantitative analysis of enzymes and substrates. For high substrate concentrations, where [S] >> Km, equation 13.18 simplifies to d[P] = k2 [E]0 = Vmax dt 13.19

Maximum rate = Vmax Analytical region for the analysis of enzymes

d [P] dt
Analytical region for the analysis of substrates Concentration of substrate

where Vmax is the maximum rate for the catalyzed reaction. Under these conditions the rate of the reaction is pseudo-zero-order in substrate, and the maximum rate can be used to calculate the enzyme’s concentration. Typically, this determination is made by a variable-time method. At lower substrate concentrations, where [S]