DETERMINATION OF CONCENTRATION OF MONOSACCHARIDES BY POLARIMETRY

Background

Polarimetry is is a very useful method to analyze chiral substances. The magnitude and direction of rotation of the plane of lineraly polarized light by a chiral compound is a specific physical property of the compound that can be used to characterize it. Most biomolecules are chiral and hence rotate polarized light. In this experiment you will study the optical rotation of a series of mixed monosaccharide solutions and determine the relative concentrations of them.

The instrument used to study optical rotation in chiral molecules is called a polarimeter. A polarimeter takes light vibrating in all planes, isolates the light vibrating in a single plane, projects the light through a tube filled with a solution of chiral compound, and measures the amount of rotation. The observed angle of rotation of the plane of polarization by an optically active liquid, solution, or (more rarely) gas or solid is usually denoted by the symbol α. The angle may be either positive (+) or negative (-) depending on whether the rotation is clockwise, that is, to the right (dextro) or counterclockwise, that is, to the left (levo) as seen by an observer towards whom the beam of polarized light travels (this is opposite from the direction of rotation viewed along the light beam). Biot discovered that the observed rotation is proportional to the length l of the cell or tube containing the optically active liquid or solution and the concentration c (or density in the case of a pure liquid): α = [α] × l × c (Biot's law). The value of the proportionality constant [α] depends on the units chosen; in polarimetry it is customary to express l in decimeters, because the cells are usually 0.25, 0.5, 1, or 2 dm in length, and c in g/100 mL. Thus, 100α = [α] × l (dm) × c (g/100 mL). The value of [α], the so-called specific rotation, depends on wavelength and temperature which are usually indicated as subscripts and superscripts, respectively; thus [α]D25 denotes the specific rotation for light of the wavetength of the sodium D-line (589 nm) at 25 °C. In addition, [α] also depends on the solvent and to some extent on the concentration which must thus also be specified. This is usually done by adding such information in parentheses, thus [α]54620 -10.8 ± 0.1 (c 5.77, 95% ethanol) denotes the specific rotation at 20 °C for light of wavelength 546 nm in 95% ethanol solution at a concentration of 5.77 g/100 mL. The dimensions of [α] are deg cm2 g-1 not degrees. It is common to give [α] without the units (understood to be 10-1 deg cm2 g-1) and (in contrast to the observed rotation α) is not given in degrees. The value of [α] varies in a large range in different compound classes, e.g. it is a small value for amino acids, a small-medium one for carbohydrates and it is extremely large for helicenes. The choice of solvent particularly affects the rotation of polar compounds because of its intervention in solvation and association phenomena. While in dilute solutions the specific rotation is independent of concentration, substantial changes of specific rotation with solvent or concentration are not uncommon, e.g. for L-(+)-tartaric acid: [α]D20 = +13.5 ± 0.5 (c = 10 in H2O) and [α]D20 = +12.4 ± 0.4 (c = 20 in H2O). A pH dependence of rotation is also common in the case of acids and bases and reversals are recorded, for example, for (S)-(+)-lactic acid, dextrorotatory in water, whose sodium salt is levorotatory and for L-leucine, which is levorotatory in water but dextrorotatory in aqueous hydrochloric acid. An even more remarkable change in rotation, from positive to negative, is seen in 2-methyl-2-ethylsuccinic acid as its solution in chloroform (containing 0.7% ethanol) is diluted, with a reversal of sign (corresponding to null rotation) occurring at a concentration of 6.3%. The phenomenon (presumably due to association) is confined to solvents of low polarity (CHCl3 or CH2Cl2); no reversal is seen in alcohol solvents, pyridine, diglyme, or acetonitrile.

In the present experiment we model the enzymatic isomerization of dextrorotatory D-(+)-glucose (old name: dextrose) to levorotatory D-(-)-fructose (old name: levulose), following the process by polarimetry. The enzyme D-glucose/D-xylose isomerase (D-xylose ketol isomerase) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium 1:1 mixture of D-glucose and D-fructose, is 1.3 times sweeter than sucrose and 1.7 times sweeter than D-glucose and serves as a sweetener for use by diabetics. The annual world consumption of HFCS is estimated to have reached 10 million tons (dry weight) in 1995. A related process is the acid-catalyzed hydrolysis of dextrorotatory common table, beet or cane sugar [D-(+)-sucrose] into a 1:1 mixture of D-(+)-glucose and D-(-)-fructose. D-(-)-Fructose rotates more to the left than D-(+)-glucose does to the right thus the sign of optical rotation turns from right to left in the process. This process is called sugar inversion and the syrupy product is named inverted sugar. The same process occurs in Nature with the aid of invertase enzyme (β-fructofuranosidase); e.g. this enzyme is used by bees to convert nectar into honey.

Required reagents, tools and instrument

• 2 × 75 cm3 of 5% (m/v) stock solutions of D-(+)-glucose and D-(-)-fructose, respectively, in water-isopropanol (9:1 v/v) mixture (isopropanol is used to prevent bacterial degradation);
• 20 cm3 of unknown solution;
• 6 beakers, each of 50 cm3 volume;
• 2 safety pipettes, each of 10 cm3 volume;
• AA-55 series polarimeter (Optical Activity Ltd., Ramsey, England)

Measurement

1. Connect the electrical cord of the polarimeter to the mains.
2. Switch on the instrument using the button on the right-handed side. Let the instrument warm up for 30 min.
3. Before commencing the measurement, the digital scale must be zeroed with the sample compartment empty (and its lid closed!) by pressing the set-zero button adjacent to the display.
4. Prepare 6 calibration solutions using beakers and safety pipettes from the stock solutions of D-(+)-glucose and D-(-)-fructose using the volumes specified in the following table:

No. vol. of D-(-)-fructose stock solution (cm3) conc. of D-(-)-fructose in the resulting soln. [% (m/v)] vol. of D-(+)-glucose stock solution (cm3) conc. of D-(+)-glucose in the resulting soln. [% (m/v)] α (degrees)
1 0 0 20 5
2 4 1 16 4
3 8 2 12 3
4 12 3 8 2
5 16 4 4 1
6 20 5 0 0 unknown 5. Fill in the polarimeter sample tube with sample no. 1 prepared as described above using the built-in funnels and make sure that no bubbles remained in the tube (this can be checked by looking through the tube by holding it up to bright light). When filling the sample tube with a new solution, the tube must be carefully rinsed twice with a few cm3 of the solution to be measured before undertaking the measurement.
6. Place the sample tube containing the solution on the stainless steel rods in the sample compartment with the funnels in up-right position and close the compartment lid. The sample rotation is displayed in about 8 to 10 seconds when the digital display comes to rest. Sample rotation is given, with the correct sign (+ or -), in degrees to two decimal places. Take a note of this reading.
7. Repeat steps 5 and 6 with all solutions.

Calculation

Draw a graph of optical rotation (α values) vs. concentration of the calibration solutions. Fit a straight line onto the data points. Determine the D-(-)-fructose and D-(+)-glucose concentration [% (m/v)], with two-decimal accuracy, of the unknown solution with the aid of the calibration curve. Alternatively, you may use exact linear regression available in programmable scientific calculators or computer programs (e.g. Excel’s or QuattroPro’s ‘linear regression’ or ‘trend’ function). Determine the concentration of D-(-)-fructose and D-(+)-glucose required to produce an optically inert mixture (α = 0°).

Results to be registered in the lab-report:
• measurement data in table format;
• graph of the measured data and the fitted curve;
• D-(-)-fructose and D-(+)-glucose concentration [% (m/v)] of the unknown solution, with two-decimal places accuracy;
• the concentration of D-(-)-fructose and D-(+)-glucose required to produce an optically inert mixture (α = 0°).