The document describes methods for estimating reducing and non-reducing sugars, including the colorimetric method using dinitrosalicylic acid (DNSA) and the anthrone method for total sugars. It details the principles, reagents, procedures, and calculations for both methods. Non-reducing sugars are determined by subtracting estimated reducing sugars from total sugars.

1. Estimation of reducing and
non-reducing sugar
Jasmine Juliet .R
Biochemistry
Agricultural College
&
Research Institute,
Madurai.

2. Reducing and non-reducing sugar -
Introduction
o Reducing sugar: Sugar that can act as a reducing agent
due to the presence of free aldehyde or ketone groups
o Non-Reducing Sugar: Sugar which does not have
free aldehyde or ketone functional group is the non-
reducing sugar. Also, they do not get oxidized. Sucrose
is their most common source. They give a negative
reaction for Fehling’s as well as Benedict’s test. All
polysaccharides are non-reducing sugars.

3. Sugars

4. https://youtu.be/lZ_0MkUzdbo
Estimation of reducing and non-reducing
sugar

5. Estimation of reducing and non-reducing
sugar – colorimetric method

6. Reducing sugar by DNSA method

7. Estimation of reducing sugar by DNSA method
o Estimation of reducing sugar by dinitro salicylic acid
method is:
 very simple,
 sensitive and
 adoptable during handling of a large number of
samples at a time.

8. Estimation of reducing sugar - Principle
o 3, 5-Dinitrosalicylic acid (DNSA) is used extensively in
biochemistry for the estimation of reducing sugars.
o It detects the presence of free carbonyl group (C=O) of reducing
sugars.
o This involves the oxidation of the aldehyde functional group (in
glucose) and the ketone functional group (in fructose).
o During this reaction DNSA is reduced to 3- amino5-nitrosalicylic
acid (ANSA) which under alkaline conditions is converted to a
reddish brown coloured complex which has an absorbance
maximum of 510 nm.

9. Estimation of reducing and non-reducing
sugar – colorimetric method
Principle: Reducing sugars have the property to reduce 3,5-DNS in
alkaline solution into 3 amino 5 nitro salicylic acid (ANSA).

10. Estimation of reducing sugar – Reagents
required
o Sodium potassium tartrate: Dissolve 40 gms of sodium potassium
tartrate in 60 mL of distilled H2O.
o DNS reagent: Dissolve by stirring 1 g dinitrosalicylic acid, 200 mg
crystalline phenol and 50 mg sodium sulphite in 100 ml 1%NaOH. Store
at 4ºC.
o Standard sugar solution:
(i) Stock standard : 250 mg of glucose in water and make up the
volume to 100 mL.
(ii) Working standard : Take 10 mL from this stock solution and make
up the volume to 100 mL.

11. Estimation of reducing sugar -
Procedure
o Weigh 100 mg of the sample and extract the sugars with
hot 80% ethanol twice ( 5 ml each time).
o Collect the supernatant and evaporate it by keeping it on
a water bath at 80° C.
o Add 10 ml water and dissolve the sugars.
o Pipette out 0.5 to 3 ml of the extract in test tubes and
equalizes the volume to 3 ml with water in all the tubes.

12. Estimation of reducing sugar - Procedure
o Add 3ml of DNS reagent.
o Heat the contents in a boiling water bath for 5 min.
o When the contents of the tubes are still warm, add 1 ml
of 40% Rochelle salt solution.
o Cool and read the intensity of dark red colour at 510
nm.
o Run a series of standards using glucose ( 0-500µg) and
plot a graph.

13. Estimation of reducing sugar -
Calculation
o Calculate the amount of reducing sugars
present in the sample using the standard
graph.

14. https://www.youtube.com/watch?v=NtqsWKRW7N8
Estimation of reducing sugar by DNSA method

15. Estimation of total sugar -
Introduction
 The total saccharides moiety in a sample can be estimated by the
anthrone method which is a simple colorimetric method.
 The first step in total carbohydrates measurement is to hydrolyze
the polysaccharides and to dehydrate the monomers.
 When anthrone it reacts with monomers to give colored
component.
 The amount of total carbohydrates in the sample is then estimated
via reading absorbance of the resulting solution against glucose

16. Estimation of total sugar -
Principle
 The soluble sugars are extracted with 80% ethanol.
 Carbohydrates are first hydrolysed into simple sugars
using strong acid.
 In hot acidic medium glucose is dehydrated to
hydroxymethyl furfural.
 This compound forms with anthrone a gree colored
product with an absorption maximum at 630nm.

17. Estimation of total sugar – Anthrone test

18. Estimation of total sugar -
Material
 80% ethanol
 Anthrone Reagent: Dissolve 200mg anthrone in 100mL of ice cold
95% H2SO4. Prepare fresh before use.
 Standard Glucose: Stock – Dissolve 100mg glucose in 100mL
water.
 Working standard – 10mL of stock diluted to 100mL with distilled
water.

20. Estimation of total sugar -
Extraction
Extraction of sugars:
Weigh 100mg of the sample & extract the sugar
with hot 80% ethanol twice (5 ml each time).
Collect the supernatant and evaporate it by
keeping it on a water bath at 80ºC.
Add 10 ml water and dissolve the sugars

21. Estimation of total sugar - Procedure
 Pipette out known volume of test solutions (0.5 ml) in two different tubes.
 Prepare standards by pipetting out 0.2,0.4,0.6,0.8,1.0 ml of the working standard
in different tubes.
 Make up the volume to 1 ml with distilled water.
 Set up a blank tube add 1ml water.
 Add 4 ml of anthrone reagent to all test tubes.
 Mix well and keep for 8 minutes in a boiling water bath. Cool rapidly under
running tab water and read the colour at 630nm.
 Draw a standard graph and calculate the amount of total sugars present in the
sample.

22. Estimation of total sugar -
Calculation
Calculation:
Amount of carbohydrate present in 100mg of
the sample =(mg of glucose ÷ Volume of test
sample) X 100

23. Estimation of total sugar -
Extraction
• Note:
Cool the contents of all the tubes on ice before
adding ice-cold anthrone reagent.

24. Estimation of total sugar by Anthrone
method

25. Estimation of Non- reducing
sugar
Non-reducing sugar
= (total sugar - reducing sugar)

26. Estimation of total sugar by Anthrone
method
• https://youtu.be/VzYDk4t97Ok