Pectin PECTINS from different sources have different compositions resulting in their varying characteristics.

The beet pectin contains acetyl group which inhibits jelly formation. The pectins of fruits vary in their methoxyl content and in jellying power. Methoxyl content of commercial pectins generally varies from 8 to 11.0% and can form high sugar (65.0% and more) gels. Low methoxyl pectins (methoxyl content less than 7.0%), which cannot form high sugar gels, can form gels with. lower concentrations of sugar, and even without sugar in the presence of polyvalent cat ions. While this property has been made use of with advantage to prepare low calorie jellies, degradation of high methoxyl pectins to low methoxyl pectin by enzymes causes loss of stability and/or gelation of citrus concentrates. The method necessary to analyze and characterize the pectin have been standardized by Owens et al. at the Western Regional Research Laboratory, U.S.A. The methods described for analyses and characterizations of pectin are mainly based on these methods and have been included here with their kind permission.

EXTRACTION AND PURIFICATION OF PECTIN Macerate fresh sample in a blender or grind the dried tissue. Weigh 100 g of fresh material after maceration or 10 g of ground dried tissue and transfer to the tared 1000-mi beaker containing 400 ml water. Add 1.2 g of freshly ground sodium hexametaphosphate. Adjust the p11 to 4.5 and heat with stirring at 9095 C for 1 hr. Check the pH at intervals of 15 mm and ensure that it is 45. Adjust pH, if necessary, with citric acid or NaOH. Replace 1wscer bst by evaporation at intervals. Do not add water during the last 20 min of the extraction period. Add .4 g of filter aid and 4 g of ground paper pulp. Check the weight. Filter rapidly through a

st lilter paper coated with 3 g of moistened, fast filter aid. Collect not less than 200 ml of the filtrate. Cool the filtrate as rapidly as possible and note its weight in the bred container. If the concentration of pectin is below 0.2% in the filtrate, concentrate to that level under vacuum before precipitation with alcohol. Pour the cooled, weighed, filtrate into 3 volumes of ethanol, iso-propanol, or acetone containing 0.5 Al Ha (the pH of the slurry should be between 0.7 and 1.0). Stir for 30 mm. Centrifuge, filter, or separate the precipitate on conic mesh nylon. Wash again at the same pH to remove all but traces of ash. Wash repeatedly in 400-mI portions of 70% alcohol or acetone until the precipitate is essentially chloride free or the pH is above 4.0. Dehydrate the precipitate further in 400 ml of acetone. Dry overnight is rams (5 mm of Hg pressure) with a slow stream of dry alt passing through the oven. Weigh the precipitate. Use this pectin for further analysis. Qualitative test for ammonia: The dried pectin should be free horn ammonia which otherwise will interfere with many of the proposed analytical methods. Therefore, test the pectin for ammonia as follows: Add 1 ml of 0.1 N NaOH to a small amount of dried sample. On heating, the presence of ammonia can be detected by its odor or, better, by moistened litmus paper. Wash out ammonium ion, if present, with acidified 60% alcohol, followed by neutral alcohol to remove the acid, and dry.

CHARACTERIZATION OF PECTIN Store the dry pectin samples prepared by the foregoing procedure under dry, cool conditions. Expose the samples to the laboratory atmosphere for 1 or 2 days until they reach an equilibrium

moisture level. Then determine the moisture content and apply correction for it in all other analyses and physical measurements. Results of analyses and physical measurements should be expressed on a moisture and ash-free basis. Commercial pectins are likely to contain reducing sugars which should be removed by washing with 60% alcohol and finally with acetone.

Moisture Weigh 1 g of sample, ground to pass 80-mesh, into a tared metal dish (5 cm in diameter with cover). Dry in vacuo (5 to 20 mm of Hg) for 4 hr at 100 C. Cool in a desiccator over phosphorus pentoxide. Do not use this sample for subsequent measurement as pectin would have been degraded. If the sample is to be used for other measurements, drying should be done at 70 C frr 16 hr. Add 1% to the percent moisture observed to obtain agreement with the Fischer method.

Ash Weigh I to 2 g of pectic substance ground to pass 80-mesh into a tared crucible. Ignite slowly, then heat for 34 hr at 6000 C. Cool the crucible to room temperature in a desiccator and weigh. To determine the alkalinity of the ash, dissolve the ash in 25 ml of 0.1 N HG. Heat it gently to boiling, and then cool. Titrate with 0.1 N NaOH using phenolphthalein as indicator. (The normality of HO and NaOH used should be the same or else carry out a blank titration using 25 ml of the HG used.)

CALCULATION Wt of ash X 100

Ash

Titer x Normality of NaOH x 60 x 100 Wt. of pectin

Alkalinity % as carbonate = of ash X 1000 Carbonate free ash % = Ash % Carbonate %

Equivalent Weight Equivalent weight is used for calculating the anhydrouronic acid content and the degree of esterification. It is determined by titration with sodium hydroxide to pH 7.5 using either phenol red or Hintons indicator. REAGENTS 1. Ethanol. 2. 0.1 N Standard sodium hydroxide. 3a. Phenol red indicator: Grind 0.1 g of the dry powder in a mortar with 28.2 ml of 0.01 M NaOH. Dilute to 250 nil with distilled water; or b. Hintons indicator: Mix together 20 ml of 0.4% bromothymol blue, 60 ml of 0.4% phenol red, 20 ml of 0.4% cresol red and 20 ml of distilled water. Use sodium salts of the indicators for preparing the solutions. 4. Carbon dioxide-free distilled water Boil distilled water for 15 mm. Cool to room temperature. Protect from atmospheric carbon dioxide.

PROCEDURE Weigh 0.5 g of pectic substance (ammonia- and ash-free) into a 250-mi conical flask. Moisten with 5 ml ethanol. Add I g of sodium chloride to sharpen the end point. Add 100 ml of carbon

dioxide-free distilled water and 6 drops of phenol red or Flintons indicator. Make sure that all the pectic substance has dissolved and that no lumps are retained on the sides of the flask. Titrate slowly (to avoid possible deesterification) with 0.1 N NaOH until the color of the indicator changes (pH 7.5); the color change should persist for at least 30 sec. 1-lintons indicator gives a magenta end point. CALCULATION Wt of sample x 1000 Equivalent weight = Methoxyl Content The methoxyl content or degree of esterification is an important factor in controlling the setting time of pectins, the sensitivity to polyvalent canons, and their usefulness in the preparation of low solid gels, films and fibers. It is determined by saponification of the pectin and titration of the liberated carboxyl group. ______ml of alkali x Normality of alkali

REAGENTS 1. 0.25 N and 0.1 N Standard sodium hydroxide. 2. 0.25 N Standard hydrochloric acid.

PROCEDURE

To the neutral solution titrated by equivalent weight, containing 0.5 g of pectic substance, add 25 ml of 0.25 N sodium hydroxide, shake thoroughly, and allow to stand for 30 mm at room

temperature in a stoppered flask. Add 25 nil of 0.25 N HG (or an amount equivalent to the basc added) and titrate with 0.1 N NaOH to the same end point as before. CALCULATION Methoxyl = ml of Alkali x Normality of alkali x 3.1 Wt of sample

Anhydrouronic Acid Pectin, which is a partly esterified polygalacturonide, contains 1O% or more of organic material composed of arabinose, galactose and perhaps sugars. Estimation of anhydrouronic acid content is essential to determine the purity and degree of esterification, and to evaluate the physical properties. PROCEDURE Making use of the equivalent weight, methoxyl content and the alkalinity of the ash data, calculate the anhydrouronic acid from the expression given below. 17jm.e. Alkali for m.e. Alkali for m.e. Titra free acid +saponification + table ash = x100 Wt of sample (mg) where m.e. = milli equivalents

Acetyl Value Sugar-beet pectin contains acetyl group. Perhaps other pectins may also contain this group. If acetyl group is present in pectin, it inhibits jelly format ion. Analysis for this functional group by simple alkaline saponification procedure followed by back titration does not yield satisfactory

results. MocWied-Clarks method as applicable to pectic substances is given here.1 A colon metric method based on hydroxamic acid reactions been described by McComb and McCready. REAGENTS 1. 0.05 N and 0.1 N Standard sodium hydroxide solutions. 2. Magnesium sulfatesulfuric acid solution: Mix 100 g of magnesium sulfate crystals and 1.5 g of HSO and dilute to 180 ml. 3. Phenol red indicator. PROCEDURE Weigh 0.5 g of pectin into a 250-mi conical flask and add 25 ml of 0.1 N NaOH. Stopper the flask and stir the contents until the pectin is dissolved. Set aside for at least 1 hr or preferably overnight. Dilute the contents to 50 ml with water. Pipette 20 ml into the distillation apparatus. Add 20 ml of magnesium sulfatesulphuric acid solution. Steam distil and collect about 100 ml of distillate, keeping the volume in the distillation flask low. Titrate the acet ic acid with 0.05 N NaOH to a phenol red end point. Carry out a blank dist illation using 20 ml water and 20 ml of the magnesium sulfatesulfuric acid solution and titrate the distillate as dcscribcd in thc case of the sample. The titer should be less than 0.1 ml of the standard alkali.

Viscosity The contribution that pectin makes to the viscosity of the food products, to the firmness of texture and to dlly formation is due in part to its molecular weight. The simplest measure of viscosity average molecular weight is the ihtrin.m v1scosity. Ihis is defined as the limiting value for the ratio ,.1IG ii C the concentration, approaches zero; 1, is the viscosity of the solution relative to the solvent. The intrinsic viscosity is indicated as or 7j

Pectin contains carboxyl groups, which, when ionized, contribute to the flow behavior of pectin solutions, because electrostatic repulsion between them causes lengthening of the chain. This effect can be reduced by addition of either salt or acid.

PROCEDURE

Weigh exactly 0.1 g of pectic substance (ash- and moisture-free basis). Dissolve in 50 ml of water. Stir for 2 hours. Add 0.8 g of sodium chloride and 0.2 g of sodium hexametaphosphate or neutral versene in 15 ml of distilled water and stir for another hour. Adjust the pH, if necessary, with dilute acid or alkali to 6 f 0.2. Rinse the detrudes of the pH meter into the solution, transfer to a 100-mL volumetric flask and make to volume. Stir rapidly and thoroughly for 1 min and stopper the flask. If the solution is cloudy or contains dust or fibers, centrifuge in covered tubes or filter using a coarse sintered-glass funnel. Determine the viscosity of the solution within an hour after the pH adjustment, using an Ostwald-Cannon-Fenske No. 50 pipette with 10 ml of solution at 25 0.03 C. Determine the flux time in the same instrument for the solvent (0.8% sodium chloride and 0.2% sodium hexametaphosphate solution). Since the density difference between the solution and the solvent is only 4 parts in 10,000, the relative viscosity is essentially equal to the ratio of the time of reflux for solution to that for solvent. Read the intrinsic viscosity directly from the plot in Fig. 2.1. The plot is based on Martins exponential equation, in which the constant, k, is assumed to be 0.40. Maximum errors in intrinsic viscosity resulting from deviations in k (1) with citrus or apple pectic substances are not more than 0.06 at = 3.5 and 0.20 at = 7.0. If greater accuracy is required, determine the relative viscosity at three concentrations, such as 0.15, 0.10 and 0.05 g

per 100 ml, plot the ratio (i-1)/C against C on a semi-log paper, and extrapolate to zero concentration to get the intrinsic viscosity. Care should be exercised in cleaning the viscometer. When in doubt, use cleaning solution. When determinations are made in series on the same day, rinse thoroughly five times with distilled water followed by a pure 95% ethanol or acetone rinse and dry by suction. The degree of esterification influences the intrinsic viscosity value which decreases with increasing esterification. The viscosity value can be a useful index of jelly grade or molecular weight.