Capsaicin and Sugar estimation in vegetables .pptx

1. Extraction of Capsicum
Babanjeet
L-2019-H-208-M
 Capsaicin is an alkaloid 8-methyl-N-vanillyl-6-nonenamide) produced by chillies and
is used in an almost pure form in police tear gas or as active ingredient in pepper
spray. Pure capsaicin is a hydrophobic, colorless, highly pungent crystalline to waxy
solid compound. It imparts pungency to chillies and bell pepper. It increases the
oleoresin content in the chilies. It provides characteristic colour to the produce.
 Apparatus: Beaker, Filter paper, trays, Glass wool, spectrophotometer, volumetric
flask, Column chromatography unit
 Chemical & Reagent: Acetone, Methanol, AL2O3, Folin- Ciocalteu reagent, Na2CO3,
Sodium Sulphate

2.  Procedure: Take 0.5g of red chilli powder, add 25 ml of Acetone shake well and Keep
it in dark place Using Decant filter extract the solution and repeat this step atleast twice.
Collect the crude extract for further estimation
 By using Column Chromatography (absorption followed by elution). Place glass wool
at the base of column. Add AL2O3 1.5g and add Sodium Sulphate 1 cm layer at the top.
Add 2 ml of sample in the column and Wash the column with 5 ml Acetone (3 times).
 Capsaicin is eluted by using solution of Acetone: Methanol in 75:25 ratio . Make up the
volume to 50 ml. Allow the capsaicin to the elute from the column. After this allow the
solution to evaporate up to 10 ml.
 Add 0.5 ml of FC reagent (1 N) Add 6 ml of distilled water and wait for 3 minutes. Add
1 ml of Na2CO3 Sodium carbonate and make the volume to 10 ml with distilled water.
Keep in dark for 1 hour. The OD is taken at 760nm.
 Multiply the obtained value with 12.5 and we will get the capsaicin percentage of given
sample.
 Optimum range of capsaicin content is 0.6-1.1%

3. Estimation of Sugars in Vegetables
Babanjeet
L-2019-H-208-M
 Sugars play important role in Vegetables. They add texture, taste, and color to processed
products. Estimation of sugars is very important in fruits and vegetables as they are
indicators of:
 Flavor/Acceptance of product
 Sugar/acidity which ratio is an indicator of flavor
 Shelf life of product (Sugar content is inversely proportional to shelf life)
 Sweetness (taste)
 Sugars are indicators of maturity.
 Apparatus: Conical flasks, Funnel, Whatmen number 1 Filter paper, Reflux system with
water bath, Spectrophotometer, Test tubes, Volumetric flask
 Colorimetric tests for reducing sugars and polysaccharides have been known for a
considerable time.

4.  Reagents: Phenol (5%), Concentrated Sulphuric acid, Solvent-Ethanol (70%), Ethanol
(80%), Glucose, Saturated solution of lead acetate, Sodium oxalate.
 Procedure: Take 0.5 g sample and reflux with about 20 ml of 80 % ethanol for 2 hrs,
maintaining the volume of solvent. Filter through Whatman number 1 filter paper.
 Repeat the extraction twice with the residue, using 70% ethanol in subsequent
extractions, 20 minutes each time.
 Pool the filtrates and add 5 ml saturated solution of lead acetated, in order to precipitate
the proteins in the extract. If not removed, proteins will interfere with the estimation.
Keep one hour and filter through Whatman number 1 filter paper.
 Add few crystals of sodium oxalate to the filtrate , keep one hour and again filter as
before. This is done to remove excess of lead acetate. Make volume of the filtrate up to
100 ml with distilled water.
Estimation: Take 0.2 ml of the extract , add 0.8 ml distilled water.
 Add one ml of 5% phenol, mix well.
 Add 4.0 ml of conc. H2SO4, mix and cool the test tubes.
 Read optical density at 490 nm in the spectrophotometer.

5. Preparation of standard curve: For standard curve, make a stock solution of glucose(100
μg/ml).
 Take 12 test tubes and mark them B1, B2,20A, 20 B, 40A, 40B, 60A, 60B, 80A, 80B,
100A, 100B.
 From the stock solution, pipette out 0.2 ml in 20A and 20B, 0.4 ml in 40A and 40B, 0.6
ml in 60A and 60B, 0.8 ml in 80A and 80B, 1.0 ml in 100A and 100B.
 Take 1 ml distilled water in tubes marked B1 and B2.
 Add distilled water in all the tubes so as to make 1.0 ml volume.
 Proceed for color development as for the sample, using phenol-sulphuric acid.
 Calculation: On a graph paper, plot the concentrations(0-100 μg/ml) along X-axis and
OD values along Y-axis.
 The graph will be more or less a straight line, as OD is directly proportional to
concentration.
 Plot the value of sample OD on graph and calculate the sugar concentration.

7. References:
 Dubois M, Giles A, Hamelton JK and Reber PA (1956) Colorimetric method for the
determination of sugar and related substances. Anal Chem 28: 350-6
 Alothman ZA, Rafi M Y, Ahmed A Y, and Usmaan M G (2014) Capsaicin and
dihydrocapsaicin determination in chilli pepper genotypes using ultra fast liquid
chromatography. Mol 19: 6474-88