Estimation of reducing and nonreducing 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