However, it is suspected that there are many side reactions, and the actual reaction stoichiometry is more complicatedthan that previously described. The type of side reaction depends on the exact nature of the reducing sugars. Differentreducing sugars generally yield different color intensities; thus, it is necessary to calibrate for each sugar. In additionto the oxidation of the carbonyl groups in the sugar, other side reactions such as the decomposition of sugar alsocompetes for the availability of 3,5-dinitrosalicylic acid. As a consequence, carboxymethyl cellulose can affect thecalibration curve by enhancing the intensity of the developed color.Although this is a convenient and relatively inexpensive method, due to the relatively low specificity, one must runblanks diligently if the colorimetric results are to be interpreted correctly and accurately. One can determine thebackground absorption on the original cellulose substrate solution by adding cellulase, immediately stopping thereaction, and measuring the absorbance, i.e. following exactly the same procedures for the actual samples. When theeffects of extraneous compounds are not known, one can effectively include a so-called internal standard by first fullydeveloping the color for the unknown sample; then, a known amount of sugar is added to this sample. The increase inthe absorbance upon the second color development is equivalent to the incremental amount of sugar added.
List of Reagents and Instruments
Dinitrosalicylic Acid Reagent Solution, 1%
Dinitrosalicylic acid: 10 g
Phenol: 2 g (optional, see Note 1)
Sodium sulfite: 0.5 g
Sodium hydroxide: 10 g
Add water to: 1 liter
Potassium sodium tartrate solution, 40%
1. Add 3 ml of DNS reagent to 3 ml of glucose sample in a lightly capped test tube. (To avoid the loss of liquiddue to evaporation, cover the test tube with a piece of paraffin film if a plain test tube is used.)2. Heat the mixture at 90º C for 5-15 minutes to develop the red-brown color.3. Add 1 ml of a 40% potassium sodium tartrate (Rochelle salt) solution to stabilize the color.4. After cooling to room temperature in a cold water bath, record the absorbance with a spectrophotometer at 575nm.
3ml 1ml O.D.
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