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Glucose Assay

Glucose Assay

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Glucose Assay
EXPERIMENT NO. 4AGLUCOSE ASSAYBY DINITROSALICYLIC COLORIMETRIC METHOD
Prepared by
 
Nam Sun Wang
 Department of Chemical & Biomolecular EngineeringUniversity of MarylandCollege Park, MD 20742-2111ENCH485
Table of Contents
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Method
This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves theoxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group infructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkalineconditions:
oxidationaldehyde group ----------> carboxyl groupreduction3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic acid
Because dissolved oxygen can interfere with glucose oxidation, sulfite, which itself is not necessary for the colorreaction, is added in the reagent to absorb the dissolved oxygen.The above reaction scheme shows that one mole of sugar will react with one mole of 3,5-dinitrosalicylic acid.
http://www.eng.umd.edu/~nsw/ench485/lab4a.htm (1 of 4)6/14/2008 11:04:58 AM
 
Glucose Assay
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
A. Equipment
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Test tubes
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Pipets
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Spectrophotometer
B. Reagents
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Dinitrosalicylic Acid Reagent Solution, 1%
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Dinitrosalicylic acid: 10 g
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Phenol: 2 g (optional, see Note 1)
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Sodium sulfite: 0.5 g
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Sodium hydroxide: 10 g
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Add water to: 1 liter
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Potassium sodium tartrate solution, 40%
Procedures
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.
http://www.eng.umd.edu/~nsw/ench485/lab4a.htm (2 of 4)6/14/2008 11:04:58 AM
 
Glucose Assay
reagent --->----+ Rochelle soln --->----++-------> at 575nm| | | | || |+-+--+ +-++-+| | heat | || | --------> | || | | || | | ||____| |____|3ml sample soln
Notes
1. Phenol, up to 2g/l, intensifies the color density. It changes the slope of the calibration curve of absorbanceversus glucose concentration but does not affect the linearity. The above procedure yields an absorbance of 1for 1 g/l of glucose in the original sample in the absence of phenol in the reagent, as opposed to an absorbanceof 2.5 for 1 g/l of glucose in 2 g/l of phenol. This property can be exploited to achieve the maximum sensitivityfor dilute samples.
Questions
1. How much time was needed for the complete color development? Justify your answer with a plot of color intensity as a function of time.2. Obtain an absorption spectrum over wavelengths in the visible range (i,e. 400-700 nm). Justify the useof 575 nm chosen in the Procedure.3. Find the procedures for at least two other methods commonly employed to measure sugarconcentrations. List the advantages and disadvantages of these methods.
References
1. Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar,
 Anal. Chem.,
 
31,
 426, 1959.Return to Prof. Nam Sun Wang'sHome Page Return toBiochemical Engineering Laboratory (ENCH485) 
Glucose Assay
 Forward comments to:
Nam Sun Wang
 Department of Chemical & Biomolecular EngineeringUniversity of Maryland
http://www.eng.umd.edu/~nsw/ench485/lab4a.htm (3 of 4)6/14/2008 11:04:58 AM

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