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.
ContentI. Gravimetric Method
II. Colorimetric method
III. Extraction and purification of pectin
I. Gravimetric Method
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.
» 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
The pectin content is expressed as % calcium pectate
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
Galacturonic acid is reacted with carbazole in the presence of H2SO4 and the color developed is measured at 520nm.
» 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.
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.
Read the concentration of the anhydrogalacturonic acid corresponding to the reading of the sample, and calculate as follows:
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.
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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