Estimation of reducing and non reducing sugar

Estimation of reducing and non reducing sugar

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  Estimation of reducingand non-reducing sugar Jasmine Juliet .R Biochemistry Agricultural College & Research Institute, Madurai.
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  Reducing and non-reducingsugar - 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.
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  Sugars
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  https://youtu.be/lZ_0MkUzdbo Estimation of reducingand non-reducing sugar
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  Estimation of reducingand non-reducing sugar – colorimetric method
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  Reducing sugar byDNSA method
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  Estimation of reducingsugar 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.
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  Estimation of reducingsugar - 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.
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  Estimation of reducingand 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).
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  Estimation of reducingsugar – 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.
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  Estimation of reducingsugar - 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.
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  Estimation of reducingsugar - 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.
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  Estimation of reducingsugar - Calculation o Calculate the amount of reducing sugars present in the sample using the standard graph.
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  https://www.youtube.com/watch?v=NtqsWKRW7N8 Estimation of reducingsugar by DNSA method
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  Estimation of totalsugar - 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
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  Estimation of totalsugar - 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.
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  Estimation of totalsugar – Anthrone test
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  Estimation of totalsugar - 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.
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  Estimation of totalsugar - 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
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  Estimation of totalsugar - 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.
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  Estimation of totalsugar - Calculation Calculation: Amount of carbohydrate present in 100mg of the sample =(mg of glucose ÷ Volume of test sample) X 100
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  Estimation of totalsugar - Extraction • Note: Cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.
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  Estimation of totalsugar by Anthrone method
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  Estimation of Non-reducing sugar Non-reducing sugar = (total sugar - reducing sugar)
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  Estimation of totalsugar by Anthrone method • https://youtu.be/VzYDk4t97Ok