Estimation of total carbohydrate

Total carbohydrate - Introduction, Extraction from plant sample, Estimation, principle, materials required, procedure, observation and calculation

1. Estimation of total Carbohydrate Presented by: Jasmine Juliet .R Biochemistry AC&RI, Madurai.

2. Carbohydrate - Introduction Carbohydrates are the important components of storage and structural materials in the plants. They exist as free sugars and polysaccharides. The basic units of carbohydrates are the monosaccharides which cannot be split by hydrolysis into more simpler sugars . Carbohydrates are sugars – and includes both single sugar units called glucose and chains of sugar units chemically linked together called starch.

3. Total carbohydrate - Introduction Dietary fiber, sugars, and starches make up the Total Carbohydrate. The term " total available carbohydrate" may be defined as including all those carbohydrates which can be used in the plant body as a source of energy or as building material, either directly or indirectly after having been broken down by enzymes.

4. Estimation of total carbohydrate - 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 standard curve.

5. Estimation of total carbohydrate - 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.

6. Estimation of total carbohydrate – Anthrone test

7. Estimation of total carbohydrate - Material 80% ethanol Anthrone Reagent:   Dissolve 200mg anthrone in 100mL of ice cold 95% H 2 SO 4 . Prepare fresh before use. Standard Glucose:   Stock – Dissolve 100mg glucose in 100mL water. Working standard – 10mL of stock diluted to 100mL with distilled water.

8. Estimation of total carbohydrate - 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

9. Estimation of total carbohydrate - 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.

10. Estimation of total carbohydrate - Procedure 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.

11. https://youtu.be/QdufRwbkeKo Estimation of total carbohydrate

12. Estimation of total carbohydrate

13. Estimation of total carbohydrate

14. Estimation of total carbohydrate

15. Estimation of total carbohydrate

16. Estimation of total carbohydrate

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

18. Estimation of total carbohydrate Note: Cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.

19. Estimation of total carbohydrate

20. Estimation of total carbohydrate

21. Estimation of total carbohydrate https://youtu.be/VzYDk4t97Ok