Practical 2 – UV-Visible Spectroscopy
Iron in dietary supplements

Introduction
UV-Visible spectroscopy is an analytical technique involving measurement of the UV-visible light absorbed by substances, and is therefore commonly used for determining the concentration of a substance in a sample.

Many substances absorb energies of particular wavelengths, while transmitting energy of other wavelengths. A UV-visible spectrum is produced when photons that are present in this particular region of the electromagnetic spectrum gain energy; causing electrons to jump from to higher energy levels than the ones they previously existed in. Not all substances have the same energy levels, since they vary from substance to substance. This in turn means that the energy and wavelength of the light required to move electrons from their energy levels also varies between substances. Hence it is possible to use the specific spectrum produced by each substance to identify what it is.
A substance that absorbs light appears coloured, however the observable colour is the complement of the colour(s) absorbed, i.e. the colour not absorbed, since it is what remains to reach our eyes.
UV-Visible spectroscopy can be used to obtain qualitative data, such as through identifying compounds using spectra compounds, or quantitative analysis. However, it is generally used for quantitative analysis, in order to determine the concentration of substance in a sample. In this procedure, the spectrum of the pure substance as well as a wavelength at which the substance absorbs strongly, while other components in the sample do not, are recorded. Thereafter, the absorbance of the sample is measured at its wavelength and the absorbance of a series of standard solutions are collected to make comparisons.

Iron is an essential human nutrient and can be detected using UV-Visible spectroscopy. In order to do so, Fe3+ is reacted with thiocyanate ions, giving an intensely red-coloured product, which is then used as a qualitative test for the presence of Fe3+. To ensure that the hydrolysis reaction of [Fe(H O) ] with water does not interfere with results, strong acid is added to the sample solution so that the hydrolysis reaction is avoided. For the purpose of identifying the amount of iron present in a dietary supplement, a series of solutions with known concentrations of [Fe(OH)(NCS)(H O) ] from a standard Fe3+ are collected and then compared to a solution that has been prepared from an iron supplement. But the dietary supplement contains mostly Fe2+ as opposed to Fe3+, so prior to reacting the iron with thiocyanate, the Fe2+ present must be transformed into Fe3+ through oxidization by reacting it with a strong oxidising agent, such as hydrogen peroxide (H2O2).

Aim
To determine the concentration of iron in an iron supplement, by measuring the amount of light that it absorbs through UV-Visible spectroscopy

Hypothesis
It is predicted that the sample of iron supplement will contain the same mass as stated by the manufacturer - 5g/25mL

Apparatus
Safety glasses
Lab coat
Five 25mL volumetric flasks
Beaker
Autopipette
Pasteur pipettes
Dispensers
Iron supplement
Fe3+ solution
4 M HNO3 solution
10% KNCS solution
10% H2O2
Water
Spectrophotometer
6 cuvettes

Method
A beaker was rinsed with a small amount of the standard solution and then 40mL of the solution was added to the beaker
Five 25mL volumetric flasks were labeled with numbers 1 to 5
2mL of 4 M HNO3 solution was added to volumetric flask from a dispenser, followed by 10% KNCS solution from another dispenser, and then filled with water using a pipette until the bottom of the meniscus formed reached the line present on the flask.
A stopper was placed on the top of the volumetric flask and shaken well to mix contents thoroughly
1mL of Fe3+ solution was added to volumetric flask 2, using an autopipette
Steps 3 and 4 were repeated with volumetric flasks labeled 2, 3, 4 and 5, with the amount of iron solution being added with an autopipette and increasing by 1mL with each flask.
The solutions were transferred to their own cuvettes, using a clean Paseur each time, so that they were approximately ¾ full.
The spectrum of the solution in flask 4 was recorded over a range of approximately 400-799nm using the spectrophotometer
A small beaker was rinsed with some of the sachet contents of the iron supplement
The beaker was then filled with the rest of the sachet contents
An autopipette was used to add 1.0mL of this liquid to a 250mL volumetric flask
1ml of 10% hydrogen peroxide (H2O2) was added to the flask containing the 1.0mL of iron supplement from the sachet
10 mL of 4M HNO3 and 10 mL of 10% KNCS solution were also added to the same volumetric flask
A plastic pipette was used to make up to the mark with distilled water
The sample solution was transferred to a cuvette and its absorbance was measured at the same wavelength of maximum absorbance that was determined earlier (473nm)
The spectrometer was set to the wavelength where the maximum amount of light was absorbed in the sample solution from step 15
The absorbance of each of the solutions prepared earlier were measured and a calibration curve of absorbance against concentration of these solutions was then plotted

Results
Table 1 displaying the concentration of Fe3+ in solutions and their absorbance
|Solution |Fe3+ concentration` |Absorbance |
|1 |0M |0 |
|2 |0.000008M |0.052 |
|3 |0.000016M |0.096 |
|4 |0.000024M |0.160 |
|5 |0.000032M |0.202 |
|Sample |0.000006M |0.040 |

1. What is the iron concentration in the sachet solution (in the 250 mL flask)?
624 x 10-6 or 0.000006M

2. Assuming the sachet contains exactly 25.00 mL of iron supplement, what is the mass of iron in the sachet?
C1V1 = C2V2
C1 x 0.001 = 0.000006 x 0.25
C1 = 0.0015M

n = cv n = 0.0015 x 0.025 = 0.0000375 mol

m = n x mm m = 0.0000375 x 55.8 = 0.002092g

The mass of iron in the sachet is 0.0020g/25mL or 2.0mg/25mL

3. We assume all the iron exists in the sachet as Fe3(PO4)2. Calculate the mass of Fe3(PO4)2 in the sachet mm = 3(55.8) + 2(31) + 8(16) = 357.4

m = n x mm m = 0.0000125 x 357.4 = 0.0044675g

The mass of Fe3(PO4)2 in the sachet is 0.004g

Discussion At which wavelength is the maximum amount of light absorbed?
473nm

Why is 530 nm less suitable for use in the analysis
Because the maximum amount of light that Iron will absorb is at a wavelength 473nm, so if a 530nm is used, the solution will absorb less light than it is capable of, giving incorrect results.

Briefly describe the method used in your analysis. A diagram illustrating the various steps involved might be helpful
The method I used was UV-Visible Spectroscopy. This process firstly involved gathering solutions containing a precisely known concentration of iron and plotting their absorbance against concentration on a calibration curve. Next a solution from an iron supplement sachet was prepared and H2O2 was added to it so that oxidation would take place, causing the Fe2+ to oxidise to Fe3+. Potassium thiocyanate was also added, which turned the solution a very bright red colour before its absorbance of light was measured. Lastly, the calibration curve was used to determine the iron concentration in the sachet.

Briefly describe the principles of operation and the major components of the spectrometer you used. A labeled diagram might be helpful.
The major components in a double beam scanning/simple spectrophotometer include: • The radiation source – provides ultraviolet and visible light of all wavelengths • The monochromator – selects a particular wavelength from those emitted by the source • Rotating mirror – ‘chops’ up the light beams • Slit • Semitransparent mirror • Detector • Chart recorder • Sample and reference cell • Light detector – measures the intensity of the light that passes through the sample

In the process of using the double beam scanning spectrophotometer, the light beam is passed alternately through the sample and the reference cells by the rotating mirror, which rapidly ‘chops’ up the beams. This compensates for changes in the output of radiation source, enabling the absorbance to be accurately measured.

Conversely, in the process of using a simple spectrophotometer, a special cell containing only pure solvent is first paced in the spectrophotometer and a reference reading is taken, which is required to compensate for any reflection, scattering or absorbance of light by the cell and the solvent. The sample solution then takes the place of the reference cell and the light source produces ultraviolet and visible light of all wavelengths that is either absorbed or transmitted through the sample solution. You can't get this range of wavelengths from a single lamp, and so a combination of two is used - a deuterium lamp for the UV part of the spectrum, and a tungsten / halogen lamp for the visible part. The absorbance is determined by comparing its reading with that of the reference cell’s. Through discovering the solution’s absorbance at different wavelengths, a spectrum for the sample can be made.

Was the analytical procedure qualitative or quantitative, or both? Explain fully
The procedure used was quantitative because it involved measuring the amount of light that is absorbed, which is proportional to the iron concentration of the sample. If, however, the experiment involved determining whether iron was present in a substance as opposed to how much was evident, it would be considered qualitiative. Yet it was a process of discovering how much iron the sachet contained and so is regarded as a quantitative procedure.

What is the analyte content (iron or calcium) as stated by the manufacturer?
5mg/25mL

What uncertainties (or errors) were involved in the procedure
The major uncertainty in the procedure was contamination of the materials being used. If the flasks or beakers were not rinsed properly with the solution prior to use there is a chance there was still leftover substance still present from the last time the equipment was used. This may have caused reactions to take place between the iron and other substance/s present, which in turn will have decreased the amount of Fe3+. Another source of contamination also could have included not having used a fresh or clean pipette every time we used a different solution. Furthermore, there is a high chance that the pipettes used were not all made to the exact same scale, making some results not as accurate as possible due to the use of more or less solution, depending on how the pipette was made. It is also possible that we did not shake our solutions well enough to mix them properly, or our measurements were inaccurate, for example during the stage of adding water to the solutions in the volumetric flasks until they reached the line. Lastly, the sample solutions that were prepared and used were said to be "relatively unstable". This means that there is also a chance that the results were obtained from inaccurate amounts of iron, as they were not all placed in the spectrophotometer at the same time, enabling the Fe3+ Concentration to deteriorate and therefore vary between each of them.

Were there any unexpected results?
The results we obtained for the mass of iron present in the sachet were unexpected. The manufacturer stated that the sachet contained 5g/25mL of iron, yet our results showed that only 2g/25mL were actually present.

What are some other applications or uses of the analytical technique that you investigated?
UV-visible spectroscopy can also be used for: • Determining the amount of coloured dye in plastics • Measuring the concentrations of specific substances in body fluids such as blood or urine – determines the haemoglobin content and sugar levels in blood • Quantitative analysis of DNA and proteins in the field of molecular biology

Why is your analyte (calcium or iron) particularly suited to this form of analysis? Do you think it would be detected by other instruments used in this workshop?
Yes. UV-visible spectroscopy can be used for organic substances and metal ions that absorb light strongly in the visible region or the electromagnetic spectrum. Iron is red in colour and absorbs strongly in the 480nm wavelength, which is green. It can also be detected by Atomic absorption spectroscopy.

Conclusion
After the completion of this prac, it was found that the hypothesis was in fact rejected, as the mass of iron was less in the iron supplement was less than expected - 2mn/25mL instead of 5mg/25mL. It was discovered that the iron supplement absorbed 0.040 of light and so contained a concentration of 624 x 10-6 or 0.000006M of iron. Throughout the duration of the prac, some limitations/possible errors were present, such as the contamination of equipment or not properly shaking the solutions prior to using the spectrophotometer. These may have negatively impacted the results, causing inaccuracy due to the solutions containing unwanted other substances or not having completely reacted. If the experiment were to be repeated, it would be necessary to do it more than once, in order to ensure results are as accurate as possible, as well as wear gloves to avoid fingerprints on the cuvettes interrupting the solutions’ absorbance while in the spectrophotometer.