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  Section: Plant Lab Protocols
 
 
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Methodology for Carbohydrates

 
     
 
Estimation of pectic substances by Gravimetric / Colorimetric method
 
Pectic substances abundantly exist in the middle lamella of the plant cells. There are three types of pectic substances – pectic acids, pectic and protopectin. Pectic acid is an unbranched molecule made up of about 100 units of D-galacturonic acid residues. The monomers are linked through 1-4 linkages. Pectin is an extensively esterified pectic acid. Several carboxyl groups exist as methyl esters. Pectic acid is water soluble where as oectin forms a colloidal solution. Protopectin is a larger moleculethan pectic acid and pectin. During ripening of fruits, conversion of protopectin in pectic acid and pectin takes place. The pectins in fruits vary in their methoxyl content and in jellying powder.
Two methods are described for the estimation of pectin: one gravimetric and the other, colorimetric.


Content
I. Gravimetric Method
II. Colorimetric method
III. Extraction and purification of pectin



I. Gravimetric Method



Principle

Pectin is extracted from plant material and saponified. It is precipitated as calcium pectate by the addition of calcium chloride to an acid solution. After thoroughly washing to eliminate chlorides ions, the precipitate is dried and weighed.
 


Materials
1N Acetic acid (Dilute 30mL of glacial acetic acid to 500mL with water.
1N Calcium chloride solution: Dissolve 27.5g anhydrous CaCl2 in water and dilute to 500mL.
1% Silver nitrate: Dissolve 1g AgNO3 in 100mL water.
0.01N HCl
0.05N HCl
0.3N HCl
 


Procedure
1.
Weigh 50g of blended sample into a 1L beaker and add 300mL 0.01N HCl. Boil for 30min and filter under suction. Wash the residue with hot water and collect the filtrate.
2.
To the residue add 100mL 0.05N HCl, boil for 20min filter, wash and collect the filtrate.
3.
To the residue now add 100mL 0.3N HCl, boil for 10min, filter, wash and collect the filtrate.
4.
Pool the filtrates. Cool and make to volume 500mL.
5.
Pipette out 100 to 200mL aliquots into 1L beakers.
6.
Add 250mL water and neutralize the acid with 1N NaOH using phenolphthalein indicator. Add an excess of 10mL of 1N NaOH with constant stirring and allow it to stand overnight.
7.
Add 50mL 1N acetic acid and after 5min, add 25mL 1N calcium chloride solution with stirring. Allow it to stand for 1h.
8.
Boil for 1 to 2min.
9.
Filter through a pre weighed Whatman No. 1 filter paper (see note 1).
10.
Wash the precipitate with almost boiling water until the filtrate is free from chloride.
11.
Test the filtrate with silver nitrate for chloride.
12.
Transfer the filter paper with the calcium pectate, dry overnight at 100°C in a weighing dish, cool in a desiccators and weigh.
 
 
Calculation
The pectin content is expressed as % calcium pectate
 
% calcium pectate =
Wt. of calcium pectate x 500 x 100
mL of filtrate taken x Wt. of sample for estimation
 
 
Notes
1. The filter paper for set No. 9 should be prepared as described below.
Wet the filter paper in hot water, dry in oven at 102°C for 2h. cool in a desiccators and weigh in a covered dish.
2. The therotical yield of calcium pectate from pure galacturonic anhydride is 110.6%.
 
II. Colorimetric method
 



Principle

Galacturonic acid is reacted with carbazole in the presence of H2SO4 and the color developed is measured at 520nm.
 


Materials
60% ethyl alcohol (Mix 500mL 95% alcohol and 300mL water).
95% Ethyl alcohol
Purified ethyl alcohol (reflux 1L of 95% ethyl alcohol with 4g zinc dust and 2mL conc. H2SO4 for 15h and distill in glass distillation apparatus. Redistill with 4g zinc dust and 4g KOH)
1N and 0.05N Sodium hydroxide.
H2SO4 (Analytical grade).
0.1% Carbazole Reagent: Weigh 100mg recrystallized carbazole, dissolve and dilute to 100mL with purified alcohol.
 

Procedure
1.
Weigh 100mg pectin (see notes section for the preparation of pectin) and dissolve in 100mL of 0.05N NaOH.
2.
Allow it to stand for 30min to deesterify the pectin.
3.
Take 2mL of this solution and make up to 100mL with water.
4.
Pipette out 2mL of deesterified pectin solution and add 1mL carbazole reagent. A white precipitate will be formed.
5.
Add 12ml conc. H2SO4 with constant stirring.
6.
Close the tubes with rubber stopper and allow to stand for 10min to develop the color.
7.
To set a blank add 1mL of purified ethyl alcohol in the place of carbazole reagent.
8.
Read the color at 525nm against blank, exactly 15min after the addition of acid.
 
 
Standard
Weigh 120.5mg galacturonic acid monohydrate (from a sample vacuum dried for 5h at 30°C) and transfer to a 1L volumetric flask. Add 10mL 0.05N NaOH and dilute to volume with water. After mixing, allow it to stand overnight. Dilute 10, 20, 40, 50, 60 and 80mL of this standard solution to 100mL with water. Take 2mL of these solutions for color developing and proceed as in the case of the sample. Draw a standard curve – the absorbance versus concentration.
 



Calculation
Read the concentration of the anhydrogalacturonic acid corresponding to the reading of the sample, and calculate as follows:
 
% anhydrogalacturonic acid =
mg of anhydrogalacturonic acid in the aliquot x dilution x 100
mL taken for estimation x wt of pectin sample x 1,000,000
 
 
Notes
1. Carbazole is recrystallized from toluene
2. An alternate procedure adopted for color development is a follows:
Take 12mL of Conc. H2SO4 in a test tube, cool in an ice-bath, and add 2mL of the deesterified pectin solution and again cool. Heat the contents in a boiling water bath for 10min, cool to 20°C and add 1mL of 0.15% carbazole reagent in purified ethyl alcohol. Allow it to stand for 25 ±5min at room temperature to develop the color. Read the absorbance at 520nm. Standards should also be treated similarly.
 
 

III. Extraction and purification of pectin
1.
Blend the fresh sample. If the material is dry grind.
2.
Transfer 100g macerated sample (10g dry tissue) to a pre-weighed 1L beaker containing 400mL water.
3.
Add 1.2g freshly ground sodium hexametaphosphate and adjust to pH 4.5.
4.
Heat with stirring at 90-95°C for 1h. check the pH every 15min and maintain at pH 4.5 with citric acid or NaOH. Replace water lost by evaporation at intervals. However do not add water at the last 20min.
5.
Add 4g filter aid and 4g ground paper pulp. Filter rapidly through a fast filter papercoated with 3g moistened fast filter aid.
6.
Collect atleast 200mL of the filtrate in a preweighed container. Cool as rapidly as possible. Now, note the weight of the filtrate.
7.
If the filtrate contains less than 0.2% pectin, concentrate the filtrate under vacuum to attain this concentration.
8.
To three volumes of ethanol, isopropanol or acetone containing 0.5N HCl, pour the cooled, weighed filtrate. The slurry should be at pH 0.7-1. Stir for 30min.
9.
Centrifuge or filter. Wash the precipitate with the same solvent containing HCl. Then wash repeatedly with 70% alcohol or acetone until the precipitate is essentially chloride-free or the pH is above 4.
10.
Dehydrate the precipitate further in 400mL acetone.Dry overnight in vacuo with a slow stream of dry air passing through the oven.
11.
Weigh the precipitate and use this pectin for analysis.
12.
The dried pectin should be free from ammonia for which a small sample of the pectin is heated with 1mL of 0.1N NaOH and ammoniacal odour can be noticed or tested with a moistened litmus paper. If ammonium ions are present wash with acidified 6% alcohol, followed by neutral alcohol to remove the acid and dry.
 
 
Reading
1. Ranganna, S (1979) Manual of Analysis of Fruit and Vegetable Products Tata McGraw-Hill Publ Co Ltd New Delhi p 634.

 
     
 
 
     




     
 
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