EXPERIMENT NO. 9D
SUCROSE ASSAY
BY THE DINITROSALICYLIC COLORIMETRIC METHOD
Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
ENCH485
Table of Contents
Method
Unlike other carbohydrates, sucrose is the only non-reducing
common disaccharide. Consequently, most tests for sugar
detection utilizing such reagents as Benedict's solution,
Fehling's solution, and DNS (3,5-dinitrosalicylic acid) solution
result in negative readings for sucrose. (The student should
convince himself of this fact by performing the test with a pure
sucrose solution.) However, these methods can still be applied if
sucrose is first hydrolyzed in an acid solution to yield glucose
and fructose. This method is a straightforward modification of
the original DNS method for glucose analysis.
List of Reagents and Instruments
A. Equipment
- Test tubes
- Pipets
- Spectrophotometer
B. Reagents
- 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%
- HCl, concentrate (37.3%, 11.9 N) solution
- KOH, 5N solution
Procedures
- Add 1 drop, or 20 µl, of concentrate HCl solution to 1
ml of the sucrose solution. Allow the hydrolysis to proceed at
90ºC for 5 minutes.
- Add 3 drops, or 0.05 ml, of the 5 N KOH solution to
neutralize the acid, because the DNS method must be applied in an
alkaline condition to develop the red brown color which
represents the presence of reducing sugars.
- Add the DNS reagent and follow the DNS method henceforth.
- Generate a calibration curve to correlate the absorbance
to the sucrose concentration.
Discussions
The DNS method can be applied twice to measure the individual
concentrations of a mixture of glucose and sucrose. First, a
small part of the original sample is consumed in measuring the
glucose concentration by following the original DNS procedure.
Another part of the sample is hydrolyzed and subsequently
subjected to the same DNS procedure. The difference in the
absorbance between the acid treated sample and the untreated
sample is due to the presence of sucrose. The sucrose
concentration can then be calculated from a calibration curve
based on that difference in the absorbance.
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Sucrose Assay by the Dinitrosalicylic Colorimetric Method
Forward comments to:
- Nam Sun Wang
- Department of Chemical & Biomolecular Engineering
- University of Maryland
- College Park, MD 20742-2111
- 301-405-1910 (voice)
- 301-314-9126 (FAX)
e-mail: nsw@umd.edu