GLPHOTO (2009-2010) DETERMINATION OF GLUCOSE WITH ORTHO-TOLUIDINE Theory of measurement Aldehyde group of aldoses forms a colorful condensation product with aromatic amines in a stoichiometric reaction. The formed colored, water-soluble compound has its absorption maximum at 635 nm wavelength and its solution’s absorbance is proportional to the concentration at low concentrations. Course of measurement Preparation of o-toluidine reagent: Dissolve o-toluidine (60 mL) and thiocarbamide (1.5 g) in glacial (concentrated) acetic acid. (Ready-to-use reagent is provided for students. This is a caustic, volatile and toxic reagent. Use safety pipette for measuring it.) Standard glucose solution for recording the calibration curve: Using a volumetric pipette measure 10.0-10.0 mL of distilled water into 5 test tubes numbered from 1 to 5. Then add 10 mM glucose solution to the test tubes according to the following: 1 mL of glucose solution to test tube 1; 2 mL of glucose solution to test tube 2; 3 mL to test tube 3; 4 mL to test tube 4 and 5 mL of glucose solution to test tube 5. Mix the content of the test tubes by shaking and calculate the concentration of glucose in each of them. Measure 2 mL of the o-toluidine reagent into another set of test tubes, numbered from 0 to 6. (That is, there are 7 test tubes in this set.) Use a labeled, 2 ml-safety pipette for measuring the o-toluidine reagent. Add 2.0 mL of the appropriate standard, diluted glucose solution samples to the test tubes 1 to 5. Use an automatic pipette for measuring the glucose solution. The test tube “0” is the reference (or blank), add 2.0 mL of distilled water to it instead of glucose solution. Finally measure 2.0 mL of the unknown concentration solution to test tube 6. Use automatic pipette for measuring both the reagent and the standard glucose solution. Put all samples into boiling water bath for 10 min and after cooled, measure their absorbance at 680 nm. Register the results in a table. Draw the calibration curve on mm paper (absorbance in function of concentration) and read the concentration of the unknown sample. Provide perspicuous lab note. Measured data should be incorporated in a table. The table should have title, data fixed out with units. The whole notebook has to be clear, that anybody could reproduce both the measurement and calculations (how the dilutions and the measurements were performed). GLPHOTO (2009-2010) Use of WPA CO 7500 photometer Switch on the photometer (colorimeter) with the ON/OFF button. Set the wavelength to 680 nm by the dial at the right bottom of the instrument. Set the instrument to absorbance measurement by the Abs/T button. Fill a cleaned cuvette with sample from the test tube 0 (reference) up to ¾ levels. (Sugar concentration is 0 mM in this sample.) Dry the outside of sample cuvette, put into the photometer and push the „R” button. The instrument sets this sample’s absorbance to zero level. Fill cuvettes with each of the remaining samples, put into the photometer, push the “T” button and read their absorbance.