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Extraction and Isolation of Pectin From Citrus Peels

Extraction and Isolation of Pectin From Citrus Peels



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 Biochemistry Laboratory
(BCM 362L), Experiment No. 6, © November 22, 20052
Quarter A.Y. 2005-2006
Extraction and Isolation of Pectin from Citrus Peels
Mr. *****
, *****
, *****
, *****, *****
Professor, School of CHE-Chm, Mapua Institute of Technology 
Student, BCM 362L/ C20, School of CHE-Chm, Mapua Institute of Technology 
Experiment number 6 deals with the isolationof pectin by computing the % methoxy contentof the isolated sample. Pectin is basically alinear  polysaccharide containing from a few hundred toabout 1000 saccharide units in a chain-likeconfiguration. It is made up of arabinose, D-galacturonic acid, and galactose.The eliminationreaction of glycosidic linkages occurs morereadily when galacturonic acid units have anesterified carboxyl group. Isolation of pectin wasdone by collecting the filtrate of acidifiedhomogenized citrus peels. The filtrate waswashed with 60 % ethanol to remove Cl ions;heated and washed with 60 % ethanol to removeammonia ions. Some errors done in the procedure like the heating of the homogenized peels was supposed to be for 1 hour but it wasdone in 45 minutes. When the sample was driedovernight, it was difficult to dissolve it the nextday because the sample hardened. Some of itmight have been left on the surface of the mortar and pestle when it was ground. These mistakescan account for the low percentage recovery of  pectin. And since there was low percentagerecovery of pectin, the percent methoxy contentwas also low. But without these errors, it should be accounted that pectins with high ester contentrequire a minimum amount of soluble solids anda pH within a pretty narrow range, around 3.0, inorder to form gels. Degree of esterification of high-ester pectins controls their relative speed of gelation as reflected by the designations ‘slowset’ and ‘rapid set’ high ester pectin. Degree of esterification of low-ester pectins controls their calcium reactivity.
Pectin, pectic acid, glycosidiclinkage, saponification, esterification.
Pectin is an essentially linear polysaccharidecontaining from a few hundred to about 1000saccharide units in a chain-like configuration;this corresponds to average molecular weightsfrom about 50,000 to 150,000.D-galacturonic acid is the principalconstituent of the pectin molecule, but someneutral sugars are also commonly present in pectin. The D-galacturonic acid units are linkedtogether by alpha-1.4 glycosidic linkages.The polygalacturonic acid is partly esterifiedwith methyl groups and the free acid groups may be partly or fully neutralized with sodium, potassium or ammonium ions. The ratio of esterified galacturonic acid groups to totalgalacturonic acid groups - termed the degree of esterification (DE) - has vital influence on the properties of pectin, especially the solubility andthe gel forming characteristics. The highest DEthat can be achieved by extraction of natural rawmaterial is approx. 75%. Pectins with DE from20-70% are produced by controlled de-esterification in the manufacturing process.The DE of 50% divides commercial pectinsinto high ester (HM) and low ester (LM) pectin.These two groups of pectin gel by differentmechanisms.HM-pectin require a minimum amount of soluble solids and a pH within a pretty narrow
range, around 3.0, in order to form gels. LM- pectins require the presence of a controlledamount of calcium or other divalent cations for gelation and do not require sugar and/or acid.Degree of esterification of HM-pectinscontrols their relative speed of gelation asreflected by the designations ‘slow set' and‘rapid set' high ester pectin. Degree of esterification of LM-pectins controls theicalcium reactivity. Some types of LM-pectinsalso contain amide groups, which stronglyaffects the calcium reactivity.
The degree of esterification is determined bythe saponification of pectin, and titration of theliberated carboxylic acid group. This isexpressed as % methoxy content:
%methoxy content=total mass methoxy
mass pectin sample
The reagents used in this experiment werecitric acid, 60% ethanol, 70% ethanol, 95%ethanol, 0.1 N NaOH, 0.25 HCl, 1% NaCl, 0.1 MAgNO
, phenol red, acetone. The equipmentused were pH paper/pH meter, top loading balance, hot plate, thermometer, burette, and blender.Citrus peels were homogenized with 150 mLdistilled water in a blender. The pH should beabout 5 using citric acid or NaOH. It was heatedat about 90-95 C for 1 hour and the pH waschecked every 15 minutes. It was filteredthrough cheesecloth and filtrates were collectedin a 250 mL beaker immersed in an ice bath.95% ethanol was acidified with 1 M HCl whose pH is 0.7 to 1.0 and it was added to the filtrateand stirred for 10 minutes. Precipitate wasfiltered through the cheesecloth and was washedwith 10 mL portions of 60% ethanol repeatedly.This was done so that the precipitate is chloridefree. The precipitate was washed with acetoneand was tested for the presence of NH
. The precipitate was washed with 60% ethanolfollowed by 95% ethanol to remove excess NH
.The precipitate was washed with acetone anddried overnight.The air-dried pectin was weighed and did notexceed 0.25 g and was dissolved to 2.5 mL 95%ethanol. 50 mL of 1% NaCl was added to thesample. 100 mL of CO
free distilled water wasadded and the pH was adjusted to 7.5 by adding0.1 N NaOH. 12.5 mL of 0.25 N NaOH wasadded to the pectin solution and was incubatesfor 30 minutes at room temperature. 12. 5 mL of 0.25 N HCl was added into the incubated sampleand 6 drops of phenol red and was titrated to pH7.5 with 0.1 N NaOH. The methoxy content wasdetermined. 
Calamansi peels were used for the isolation of Pectin. Below are the tabulated results of theexperiment:
Table 1.
A. Isolation of Pectin
Mass of Pectin (g)0.14gPercentage yield of pectin0.56%
B. Determination of Methoxy Content of Pectin
 NaOH volume (mL), V
14.2 mL NaOH volume (mL), V
3.5 mL Net volume NaOH (mL), V17.7 mLPercentage Methoxy Content7.75%

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