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LAB EXPERIMENT 1___ _______ Calorimetric Determination of Glucose by the 3,5-dinitrosalicylic acid Method. Principle: Several reagents have been employed which assay sugars by using their reducing properties. This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino-5-nitrosalicylic acid under alkaline conditions, as illustrated in the equation below: The chemistry of the reaction is complicated since standard curves do not always go through the origin and different sugars give different color yields. The method is therefore not suitable for the determination of a complex mixture of reducing sugar. Materials: 1. Standard Glucose Solution: 0.1g anhydrous glucose is dissolved in distilled water and then raised the volume to 100 ml with distilled water. 2. Dinitro salicylic acid reagent: a. Solution "a" is prepared by dissolving 300g of sodium potassium tartarate in about 500 ml distilled water. b. Solution "b" is prepared by dissolving 10 g of 3,5-dinitrosalicylic acid in 200 ml of 2N NaOH solution. c. The dinitrosalycilate reagent is prepared by mixing solutions a & b and raising the final volume to 1 litre with distilled water. Procedure: 1. Pipette in duplicate the following reagents into a series of dry-clean and labelled test tubes and as indicated in the following table, take Section A. SECTION A Tube No. ml. Stand. Glucose. ml. H2 O ml. Dinitrosalicylic reagent SECTION B ml. H2O bbbbbB BB 7.0 1 0.0 1.0 2.0 2 0.2 0.8 2.0 7.0 3 0.4 0.6 2.0 7.0 4 0.6 0.4 2.0 7.0 5 0.8 0.2 2.0 7.0 6 1.0 0.0 2.0 7.0 2. After replacing the above mentioned solutions as in section A in the labelled tubes, shake well and then place them in a boiling water bath for 5 minutes. 3. Cool the tubes thoroughly and then add 7.0 ml of distilled water to each tube as indicated in section B of the previous table, Read the extinction (Optical density) of the colored solutions at 540 nm using the solution in tube 1 as a blank (control). Note: All the tubes must be cooled to room temperature before reading since the extinction is sensitive to temperature change. 4. Record the readings in section B, and plot the relationship between the optical density and the concentration of glucose solution. See whether there is a linear relationship between the concentrations of glucose solutions and their corresponding optical densities. 5. Use the already prepared standard curve for the determination of the unknown concentration of the glucose solution provided and tissue extract form exp.6 or any other unknown reducing sugar sample. Name: No. Experiment 1: Results Sheet The concentration of standard glucose solution : mg/ml - After conducting your test, fill the following table : Tube Concentration Absorbance No. (Mg/ml) (At 540 nm) 1 2 3 4 5 6 7 - Plot the standard curve of the absorbance (y- axis) against the concentration ( x-axis ) - Use this plot to estimate the concentration of your unknown glucose sample. - Express your results in mg/dl , mg% , g/ml and g/l Name: Experiment 1: Results Sheet No.