SUCROSE ASSAY

BY THE DINITROSALICYLIC COLORIMETRIC

METHOD

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 %

o Dinitrosalicylic acid: 10 g
o Phenol: 2 g (optional, see Note 1)
o Sodium sulfite: 0.5 g
o Sodium hydroxide: 10 g
o Add water to: 1 liter
 Potassium sodium tartrate solution, 40%
 HCl, concentrate (37.3%, 11.9 N) solution
 KOH, 5N solution
Procedures

1. 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.
2. 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.
3. Add the DNS reagent and follow the DNS method henceforth.
4. 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.