Glucose Concentration by Optical Activity
Glucose is optically active, which means that a solution containing glucose can rotate the plane of polarized light that passes through it. In this assay, cuvettes with various concentrations of glucose are placed between two polarized filters that have planes of polarization set at right angles. The first filter allows light to pass that is vertically polarized, and blocks light in other planes. The second - horizontal - filter efficiently blocks vertically polarized light. If the solution rotates the plane of polarization, the light striking the second polarized filter will be able to pass through and be measured by the light detector.
The goal of this assay is to use rotation of polarized light by an optically active substance, glucose, to measure its concentration. We will not measure the actual angle of rotation. Rather, we will use a standard curve to determine how the light output varies with concentration. Light polarization is a fascinating topic, involving both fundamental physics of electromagnetic waves and the "handedness" of certain molecules. It also has practical applications such as polarized sunglasses and computer screens. Please see the bibliography for sources of background information.
Materials & Equipment
Photometer (see table below for photometer set-up)
Glass cuvettes, 10 (plastic cuvettes are optically active)
Polarized filters, one vertically polarized and the other horizontally polarized.
Glucose solution (Karo syrup, approximately 35% glucose in water)
Pipets, beakers, plastic cups, etc, as needed.
Table 1. Set-up for measuring glucose in solution based on optical activity.
|Experimental set-up for photometer
||Visible light LED, white or colored light
The procedure uses Karo syrup as a source for glucose. (The concentration of glucose in Karo syrup is approximately 35% by weight). This allows you to determine relative concentrations. To obtain precise values, purchase pure glucose from a chemical supply company and make standards with exact concentrations.
Make the standard solutions.
- Label plastic cups as follows, indicating the % Karo syrup: 0, 12.5, 25, 50, 100.
- Pour 100 ml of water into the cup labeled "0". This is the blank.
- Add 100 ml of water and 100 ml of Karo syrup to the cup labeled "50". Mix thoroughly by stirring. The syrup is very viscous. Make sure to rinse the syrup from the sides of the mixing vessels.
- Add 100 ml of water and 100 ml of 50% Karo syrup solution to the the cup labeled "25%", mix thoroughly with a clean spoon.
- Continue 2-fold dilutions to 12.5%.
- Add 100 ml of Karo syrup to the cup labeled 100%.
- Thoroughly clean plastic ware in between uses.
Dilute the unknowns. The procedure below uses a 1:10 dilution. You may need to try other dilutions to get the unknown into the range of the standards.
- Label a cup "1:10, unknown X" for each unknown.
- Add 10 ml of each unknown into the appropriate cup.
- Add 90 ml of water to each cup with 10 ml of unknown.
Transfer to cuvettes and take measurements.
- Transfer 3 ml from the standard solutions (0 to 100% Karo) to cuvettes.
- Transfer 3 ml of the "unknowns" to cuvettes.
- Turn on the light box.
- Make sure polarized filters are in place on either side of cuvette.
- Adjust the brightness of the LED so that you get a reading of approximately 1.0 volts when the blank (cuvette with plain water) is placed in the light box.
- As the concentration of glucose increases, more light passes through. Make sure that the highest signal (pure Karo, in this case) is less than 4.8 volts as that is near the maximum voltage for the instrument.
- Take readings for each standard and sample.
- Take two more readings so that you have data for each point in triplicate.
Analyze the results.
- Average the voltage output readings for the standards and unknowns.
- Subtract the voltage reading for the blank.
- Graph the concentration of the standards on the x-axis and the average voltages from the standards on the y-axis.
Use the graph to estimate the concentration of glucose in the unknown sample(s).
- Locate the concentration on the x-axis that corresponds to the voltage output from the unknown.
- Correct for the dilution factor.
- For example, if the unknown sample has a concentration of 400 micrograms per ml after it was diluted 100 X, the original solution must have been 100 times more concentrated.
- Record the value obtained for the glucose concentration of the unknown sample or samples.
Figure 1. The graph shows raw data for the voltage output of the photometer vs percent Karo syrup. The light signal is in millivolts. For the procedure, the recommended voltages are higher (up to 4.8 V) but the results should be the same.
- How does the color of light affect the polarization (use filters or colored LEDs)
- How does temperature affect polarization?
- Can you modify one of the filters to rotate so that you can measure the angle of polarization?
- Add a trendline to the graph and use the equation for the trendline to calculate concentration based on voltage output.
- Draw the glucose molecule and identify the chiral centers.
Determine concentration of glucose using its optical activity.
Terms and definitions
Optical activity - The turning of the plane of linearly polarized light about the direction of motion as the light travels through certain materials. It occurs in solutions of chiral molecules such as glucose and sucrose (sugar), solids with rotated crystal planes such as quartz, and spin-polarized gases of atoms or molecules. It is used in the sugar industry to measure syrup concentration.
Linearly polarized light - Ordinary white light is made up of waves that fluctuate at all possible angles. Light is considered to be "linearly polarized" when it contains waves that only fluctuate in one specific plane. A polarizer is a material that allows only light with a specific angle of vibration to pass through. The direction of fluctuation passed by the polarizer is called the "easy" axis.
Chiral molecules - A chiral molecule is one that is not superimposable on its mirror image. It has the property of rotating the plane of polarization of plane-polarized light that is passed through it. This phenomenon is called optical activity.