Emulsifying and stabilizing agents

i. Lecithin

Lecithin, food grade widely used as an antioxidant and emulsifier, is permitted under the Prevention of Food Adulteration rules (1955) and the Fruit Product Order (1955).

Purity

Dissolve 5 g of phophatides from previous acetone insoluble matter in 10 ml of petroleum ether and add 25 ml of acetone to the solution. The precipitate portions are transferred to 4 centrifuge tubes with additional amount of acetone, stir again,chill for 15 min in an ice bath, stir again and then centrifuge for 5 min. Decant the acetone, crush the solids with a stirring rod, refilled with acetone and repeat the centrifuge. 5 g of the purified phosphatides are required to saturate about 16 L of acetone.

Add a quantity of purified phosphatides to sufficient acetone, previously cooled to a temperature of about 5°C to form a saturated solution, and maintain the mixture at this temperature for 2 h, shaking it vigorously at 15 min intervals.Transfer 2 g of sample accurately weighed into a 40 ml centrifuge tube, previously tarred with a glass stirring rod. Add 15 ml of phosophatide-acetone solution from a burette. Warm the mixture in a water bath until the lecithin melts, but avoid evaporation of the acetone. Chill for 5 min, add about one-half of the required volume of phosphatide-acetone solution and stir for 15 min. Centrifuge the content at 2000 rpm for 5 min. Decant the supernatant liquid from the centrifuge tube,crush the centrifuged solids with the stirring rod. Repeat the process. Mix the residue again, dry the centrifuge tube and its contents at 105°C for 45 min in oven, cool and weigh.

ii. Sodium Alginate

Sodium alginate widely used as stabilizing and thickening agent, is permitted under PFA rules

Purity

Apparatus : Soda lime column (A), a mercury valve (B) connected through a side tube (C) with a reaction flask (D) by means of a rubber connection, the reaction flask (D) is a 100 ml round bottom long necked boiling flask with 24/40 ground joint attached. The oil bath (E) is maintained at 145°C by means of a thermo-regulator and an immersion heater.

The reaction flask is provided with a 20 cm reflux condenser (F) terminating in a trap (G) containing 25 g of 850 m zinc or tic, which is connected with an absorption flask (H) a 250 ml Erlenmeyer flask equipped with a 24/40 ground joint and a side tube attached a little below the ground joint. Flask (H) is provided with an absorption tower (J) the lower part of which consists of an 18 mm tube fitted with a medium porosity fritted Pyrex, sealed to the inner part of the lower end of a 24/40 ground joint and terminating 1-2 mm above the bottom of the absorption flask when the joint is in place.

Appratus for Deternination of Purity

A trap consisting of a bulb of 100 ml capacity, is blown above the ground portion of the joint and the outer portion of 1 24/40 ground joint is sealed on above this bulb. The absorption tower, from the botto0m of the disc to the top of ground joint is 30 cm in length. The top of the tower is fitted with a hollow ground stopper with a short side-tube attached. The tower assembly may be attached to a soda lime tower connected with a water pump by means of capillary tube regulator (L) which serves to seep 1700 to 2000 ml of carbon dioxide free air per hour through the apparatus during the heating period (Figure 1)

Procedure

Transfer about 250 mg of sample previously dried at 105°C for 4 h and accurately weighed to the reaction flask, add 30 ml of 19% HCL, insert a small boiling tube and connect it to the reflux condenser (F) using syrupy phosphoric acid as lubricant.Draw a carbon dioxide free air for about 10 min and then discontinue it. Disconnect the absorption tower (J) rapidly transfer from a pipette 25 ml of 0.25N NaOH into the absorption flask, add 5 drops of butanol and again attach it to the absorption tower. Raise the oil bath, previously heated to 145°C until the oil level is several mm above the liquid level in the reaction flask. After the initial rapid evolution of carbon dioxide has subsided, resume the passage of air through the apparatus and continue the heating at 145°C for 2 h, then disconnect the absorption flask and rest of the assembly. Remove the absorption tower, washing any adhering NaOH solution into the flask with several small portions of water. To the flask add 10 ml of barium chloride solution (1:10), stopper the flask, shake gently for about 2 min, add phenolphthalein solution and titrate with 0.1N HCl. Perform a blank determination make necessary correction.Each ml of 0.25N NaOH is equivalent to 27.75 mg of sodium alginate (equivalent wt 222.0)

iii. Sodium Carboxymethyl Cellulose (CMC)

Sodium carboxymethyl cellulose, food grade widely used as thickening agent,emulsifier and stabilizer, is permitted under the Prevention of Food Adulteration rules (1955) for certain foods.

Degree of Substitution

Weigh 500 mg of sodium CMC in a beaker. Add 80 ml of glacial acetic acid.Heat the mixture on a boiling water bath for 2 h and cool to room temperature.Set the pH meter on mV mode and deep the electrode in the solution. Take a definite volume of perchloric acid in a graduated burette and add to the solution in large increments until the change on mV becomes noticeable. Then add at the rate of 0.1 ml and note the mV reading each time. Continue addition as above until the variation in the readings passes through a maximum. Plot the amount of perchloric acid against the mV readings and determine the quantity of titrant corresponding to half-way up the steepest gradient.

Degree of substitution = (16.2(A/M))/ 1.0 [8.0(A/M)]

Where,

A = volume in ml of 0.1N perchloric acid required, and

M = mass in mg of the sample taken for the test.

Sodium Chloride

Weigh accurately about 5 g of the sample into a 250 ml beaker. Add 50 ml of water and 5 ml of hydrogen peroxide and heat on a steam bath for 20 min, stirring occasionally to ensure complete dissolution. Cool and add 100 ml of water and 10 ml of nitric acid. Titrate with the silver nitrate to a potentiometric end point,using silver and mercurous sulphate- potassium sulphate electrodes with continuous stirring.

Sodium chloride (as NaCl), % by mass =(584.5 × N × V (100-A))/ M

Where,

V= volume in ml of silver nitrate used in the titration with the material, corrected to blank;

N = normality of standard silver nitrate solution;

A= percent loss on drying, and

M = mass in g of sample taken for the test.

Free Glycolate

Weigh 0.5 g of the sample and transfer to a 100 ml beaker. Moisten the sample thoroughly with 5 ml of glacial acetic acid, followed by 5 ml of water, and stir with a glass rod until the solution is complete; usually about 15 min are required.Slowly add 50 ml of acetone while stirring and the 1 g of NaCl. Continue the stirring for several minutes to ensure complete precipitation of the CMC. Filter through a soft, open-texture paper, previously wetted with a small amount of acetone, and collect the filtrate in a 100 ml volumetric flask. Use 30 ml of acetone to facilitate the transfer of the solids and to wash the filter cake. Make up to volume with acetone and mix.

Prepare a blank solution containing 5 ml of water, 5 ml of glacial acetic acid and acetone in another 100 ml volumetric flask. Pipette 2 ml of the sample solution and 2 ml of blank solution into two 25 ml volumetric flasks. Remove the acetone by heating the uncovered flasks upright in a boiling water bath for exactly 20 min.Cool to room temperature and add 5 ml of naphthalenediol, mix thoroughly, then add 15 ml more of the naphthalenediol and mix. Cover the mouth of the flask with a small piece of aluminium foil and heat upright in the boiling water bath for 20 min. Cool to room temperature and make up to volume with conc sulphuric acid.

Measure the absorbance of sample solution against blank solution at 540 nm using 1 cm cell. Read the corresponding mg of glycolic acid from the calibration curve obtained as follows:

Introduce 0, 1, 2, 3 and 4 ml aliquots of standard glycolic acid solution (1mg/ml) prepared by weighing accurately 0.1 g of glycolic acid, previously dried in a vacuum desiccator for at least 16 h and then dissolving in 100 ml of water; do not keep the solution longer than 30 days. Add water to each flask to a volume of 5 ml, then add 5 ml glacial acetic acid and make up with acetone to mark and mix. Pipette 2 ml of each solution into a series of 25 ml volumetric flasks and proceed in the same manner as described for the test solution.

Sodium glycolate, % by mass = (A×0.129)/ B

Where,

A= mg of glycolic acid read from the calibration curve, and

M = mass in g of dried sample taken for the test.

iv. Guar Gum

Guar gum is obtained from ground endosperms of Cyamopsis tetragonolobus.It consists chiefly of a high molecular weight hydrocolloidal polysaccharide,galactomannan. Use of guar gum as food additive is permitted under the Prevention of Food Adulteration rules (1955).

Identification of Sugars

Boil a mixture of 100 mg of sample and 20 ml of 10% sulphuric acid for 3 h.Allow to cool and add excess barium carbonate (about 10 mg). Mix with a magnetic stirrer until the solution is of pH 7, and filter. Evaporate the filtrate in a rotary evaporator at 30-50°C in vacuum until a crystalline residue is obtained.Dissolve it in 10 ml of 40% methanol.

Prepare a thin layer chromatoplates with a mixture of 15 g cellulose powder and 90 ml water and dry them for 10 min at 100°C.Place 1-10 ml spots of hydrolysate on the starting line of two chromatoplates and spots containing 1-10 mg of the sugars which could be present in the hydrolysate.Use two solvents (a) a mixture of formic acid, methyl ethyl; ketone, tertiary butanl and water (15:30:40:15, v/v) and (b) a mixture of isoprapanol, pyridine, acetic acid and water (40:40:5:20, v/v) to develop the plates.

After development, spray with a solution of 1.23 g anisidine and 1.66 g phthalic acid in 100 ml ethanol and heat the plates at 100°C for 10 min. A greenish-yellow colour is produced with hexoses, a red colour with pentoses and a brown colour with uronic acids. Compare sample spots with those for the solution of galactose and mannose.

Galactomannans

The difference between the sum of the percentages of acid insoluble matter (1% solution in 0.1% sulphuric acid), total ash at 550°C, loss on drying at 105°C for 5 h and protein (N×5.7) and 100 represents the percent of galactomannans.

v. Gum Ghatti

Gum ghatti is a complex polysaccharide composed of l-arabinose, d-galactose, d-mannose and d-glucoronic acid residues. It is used as an emulsifier, stabilizer and thickener in foods and pharmaceuticals. It is a water-soluble gum exuded by the tree Anoqeissus latifolia of the family combretaceae.

Viscosity

Weigh accurately 5 g of sample in a 250 ml beaker. Fix a stirrer and thermometer in it. Add 10 ml iso-propyl alcohol and mix it thoroughly so as to form a slurry.Add 80 ml of boiling water quickly while stirring the solution rapidly. If there are any lumps in the solution, discard and prepare the fresh solution until a clear solution is obtained. Cool and stir the solution till the temperature drops to 80°C.Place the beaker in a water bath maintained at about 85°C and stir frequently for 10 min. Remove the beaker and place it in a water-bath maintained at 27±1°C.Stir the solution and add water so that the final weight of the beaker is 100 g more than the tare weight of the beaker. Adjust the temperature of the solution to 27±1°C and measure its viscosity with Brookefield viscometer at 2o rpm using spindle No. 4.

vi. Tragacanth Gum

Use of tragacanth gum as thickening agent and stabilizer has been permitted in the PFA rules for certain foods. It is dried gummy exudation obtained from Astragalus strobiliferus. Chromatographic identifications of sugars in the gum hydrolysate

Boil a mixture of 100 mg of sample and 20 ml of 10% sulphuric acid for 3 h.Allow to cool and add excess barium carbonate (about 10 mg). Mix with a magnetic stirrer until the solution is of pH 7, and filter. Evaporate the filtrate in a rotary evaporator at 30-50°C in vacuum until a crystalline residue is obtained. Dissolve it in 10 ml of 40% methanol.

Prepare a thin layer chromatoplates with a mixture of 15 g cellulose powder and 90 ml water and dry them for 10 min at 100°C

.Place 1-10 ml spots of hydrolysate on the starting line of two chromatoplates and spots containing 1-10 mg of the sugars which could be present in the hydrolysate.Use two solvents (a) a mixture of formic acid, methyl ethyl; ketone, tertiary butanl and water (15:30:40:15, v/v) and (b) a mixture of isoprapanol, pyridine, acetic acid and water (40:40:5:20, v/v) to develop the plates.

After development, spray with a solution of 1.23 g anisidine and 1.66 g phthalic acid in 100 ml ethanol and heat the plates at 100°C for 10 min. A greenish-yellow colour is produced with hexoses, a red colour with pentoses and a brown colour with uronic acids.

Viscosity

Transfer a 4 g sample, finely powder into the container of a stirring apparatus equipped with blades capable of revolving at 1000 rpm. Add 10 ml of alcohol to the sample, so as to wet the gum uniformly, and then add 390 ml of water,avoiding the promotion of lumps. Immediately stir the mixture for 7 min, pour the resulting dispersion into a 500 ml bottle, insert a stopper, and allow to stand for about 24 h in a water bath at 25°C. Determine the apparent viscosity at a temperature with a Brookefield viscometer using spindle No. 2 at 3 rpm and a factor 10.

Test for Karaya Gum

Boil 1 g of the material with 20 ml of water until mucilage is formed, add 5 ml of HCl and again boil the mixture for 5 min. No permanent pink or red colour shall develop.

vii. Gelatin

Gelatin is a protein produced by partial hydrolysis of collagen derived from skin,tendons, ligaments and bones of animals. Gelatin is widely used in the food processing industry as a stabilizer, gelling agent, emulsifying agent and a crystallization inhibitor. It is permitted as a food additive under the PFA rules (1955).

Gel strength

Weigh accurately about 1 g and place with 99 of water in a 200 ml flask. Allow to stand for 15 min; then place the flask in a water bath at 60°C and swirl occasionally until solution is complete. Transfer 10 ml of the solution to a test tube having an internal I.D. of 12 mm and place the tube in ice bath, making certain that the top of the solution is below the level of the ice and water. Place the bath containing the tube in a refrigerator, and maintain it at about 0°C for 6 h. When the tube is removed from the bath and inverted, no movement of the gel shall be observed.

viii. Sodium Citrate

Sodium citrate, food grade widely used as emulsifying and stabilizing agent, is permitted under the Prevention of Food Adulteration rules (1955) for certain foods.

Purity

Weigh accurately about 2 g of the sample. Heat until carbonized. Cool. Boil the residue with 50 ml of water and 50 ml of the sulphuric acid. Filter and wash the filtrate with water. Titrate the excess of acid in the filtrate and washings with the NaOH using solution of methyl orange as indicator. Each ml of 0.5N sulphuric acid is equivalent to 0.04902 g of C6H5O7Na3.2H2O.

ix. Dicalcium Phosphate

Dicalcium phosphate has been permitted as stabilizer, dough conditioner and as nutrient supplement in certain foods.

Purity

Weigh accurately about 400 mg of the sample, dissolve in 150 ml of water, and add 15 ml of NaOH and 300 mg hydroxynaphthol blue indicator. Titrate with disodium ethylenediamine tetraacetate until the solution is clear. Each ml of 0.05M disodium EDTA is equivalent to mg of CaHPO4.

x. Edible Common Salt (Dairy Salt)

Purity

Accurately weigh about 20 g of the dried sample, dissolve it in 200 ml of water in a beaker and heat to boiling and cool. Filter the solution through a weighed Gooch or sintered glass crucible (G No. 4) and wash the residue till it is free from soluble salts. Collect the filtrate and washings in a 1 litre graduate flask and dilute to mark. Transfer 10 ml of the solution into a conical flask and add 1 ml of potassium chromate indicator solution. Titrate against standard silver nitrate solution till the reddish brown tinge persists after brisk shaking. Carry out a blank determination.

Sodium chloride (as NaCl), % by mass =(584.5× N×V)/ M

 Where,

V= volume in ml of silver nitrate used in the titration with the material, corrected to blank;

N = normality of standard silver nitrate solution; and

M = mass in g of the dried sample in 1000 ml of the solution taken for the test.

xi. Glyceryl Monostearate

Use of glyceryl monostearate is permitted as emulsifier for certain food products in the Prevention of Food Adulteration rules (1955). It shall preferably be made from edible vegetable oils.

Monoglyceride

The method is based upon their quantitative oxidation with periodic acid. Quantitative oxidation is obtained only if excess of periodic acid is used. The monoglyceride  or glycerine is determined by the periodic acid consumed.

Reagents

•  Periodic acid stock solution (Dissolve 12 g of periodic acid in water and dilute to 100 ml. Store in an amber colour bottle)
•  Reagent solution (Dilute 5 ml of stock solution to 100 ml with 95% acetic acid)
•  Starch indicator (1%)
•  Chloroform
•  Acetic acid (Dilute redistilled glacial acetic acid to 95%)
•  Potassium iodide solution (15% in water)
•  Standard sodium thiosulphate solution (0.1N)

 Procedure

Weigh 0.7-0.8 g sample into 100 ml glass stoppered volumetric flask accurately and rapidly. Add 50 ml chloroform. Gently swirl to dissolve the sample. If the sample is not soluble (due to the presence of soap) add 5 drops of acetic acid and shake. Add 25 ml distilled water and stopper the flask. Shake vigorously for 1 min to mix the contents thoroughly. Allow to settle for some time. If the emulsion does not break, add 3-4 drops of glacial acetic acid, but do not shake.The two layers will separate. When the water layer is perfectly clear, pipette out with an aspirator bulb as much water as possible without sucking the chloroform layer and store it in a 100 ml; volumetric flask. Again add 25 ml of water and repeat the procedure 3 times more to extract all the glycerol. Combine all water extracts; make to 100 ml and mix thoroughly. Keep this extract for estimation of glycerol.

Add chloroform to the mark, taking off as much as the residual water as possible.Stopper the flask and mix the contents thoroughly. Allow the flasks to stand for 15 min.Pipette out 25 ml aliquot from chloroform extract into 500 ml glass stoppered flask. Prepare a blank containing 25 ml of chloroform only. Pipette out 25 ml periodic acid reagent solution into each sample and also the blank. Stopper the flasks and mix the contents by gentle swirling. Allow the flasks to stand for 1-1.5 h during which the contents of all the flasks should be frequently shaken to mix the sample with the reagent. At the end 1.5 h, add 15 ml of 15% KI solution; wait for 1 min and add 100 ml water. Shake gently and titrate immediately with 0.1N sodium thiosulphate solution, using starch solution as indicator.

Monoglyceride, % by mass = ((B -S) *N *M* 4)/ W *20

Where,

B= titre of periodic acid reagent in chloroform blank;

S=titre of sample for monoglyceride;

N=normality of sodium thiosulphate solution;

M= molecular mass of monoglyceride; and

W= mass of sample taken for the test.

Glycerol

Pipette out 25 ml aliquot from the combined aqueous extract. Prepare a blank containing 25 ml distilled water only. Pipette out 25 ml of periodic acid reagent solution into each flask. Stopper the flasks and mix the contents by gentle swirling.Allow the flasks to stand for 30 min. At the end of 30 min, add 15 ml of 15% KI solution; wait for 1 min and add 100 ml water, shake gently and titrate immediately with 0.1N sodium thiosulphate solution using starch solution as indicator.

Glycerol, % by mass = ((B−S)× N× 2.302× 4)/M

Where,

B= titre of periodic acid reagent solution in water blank;

S=titre of sample for monoglyceride;

N=normality of sodium thiosulphate solution; and

W= mass of sample taken for the test.

xii. Polyglycerol Esters of Fatty Acids

Polyglycerol esters of fatty acids are the esters of fatty acids of food fats with a mixture of polyglycerols. These are being permitted for use as release agents in bakery industry and in chocolate manufacture.

Test for Fatty Acids

Reflux 1 g of sample with 15 ml of 0.5N ethanolic KOH for 1h. Add 15 ml of water, acidify with about6 ml of dilute HCl. Oil drops or a white to yellowish white solid is produced which is soluble in 5 ml of hexane. Remove the hexane layer. Extract again with 5 ml of hexane and again remove the hexane layer. Use the aqueous layer for detection of acids other than fatty acids.

Detection of acids other than fatty acids

Acetic Acid : Transfer about 5 ml of the aqueous layer into a dish. Add excess calcium carbonate and evaporate until dry. Transfer the major part of the residue into a glass tube. Place a filter paper, moistened with reagent for acetone (a saturated solution of o-nitrobenzaldehyde in sodium hydroxide, freshly prepared) on the top of the tube. Heat and observe the yellow colour changes into greenish blue by reaction of the reagent for acetone, with the calcium acetate formed.

Succinic Acid: Transfer one drop of the aqueous layer and a drop of a 0.5% solution of ammonium chloride and several mg of zinc powder into a micro test tube. The mouth of the tube is covered with a disk of filter paper moistened with a solution, in benzene of 5% p-dimethylaminobenzaldehyde and 20% trichloroacetic acid. The bottom of the test tube is heated vigorously with a micro flame for about 1 min. Depending on the amount of succinic acid or succinimide, red-violet or pink stain appears on the paper.

Fumaric Acid : transfer 1 ml of the aqueous layer with 1 ml of 2N sodium carbonate into a test tube. Add 2 or 3 drops of 0.1N potassium permanganate.The solution is promptly discoloured.

Tartaric Acid : Evaporate about 5 ml of the aqueous layer in a porcelain dish until dry. Add 2 ml of conc sulphuric acid containing 0.5% pyragallol and heat on a steam bath. An intense violet colour is produced.

Citric Acid : To 3 ml of the aqueous layer add a few drops of 1% potassium permanganate and warm until the colour has disappeared. Then add an excess of bromine solution. A white precipitate (pentabromacetone) is formed immediately on cooling. Evaporate 1 ml of the aqueous layer resulting from test in a porcelain dish add 1 ml of a mixture of 1 volume acetic anahydride and 5 volume of pyridine into the warm dish. A violet colour is produced. Tartaric acid produces

a green colour.

Lactic Acid : Transfer 0.2 ml of the aqueous layer add 2 ml of conc sulphuric acid into a test tube and place for 2 min in boiling water. Cool and add 1-2 drops of a 5% guaiacol solution in ethanol. A red colour is immediately produced.

Polyols Content by GC

Polyglycerol esters are saponified with alcoholic KOH solution and the fatty acids removed by extraction. The polyols are converted to trimethyl silyl derivatives and analyzed by GC.

Procedure

Weigh about 0.5 g of sample and reflux with 20 ml of ethanolic 1N KOH solution for 2 h. Reduce the volume of ethanol by evaporation at 45-50°C in a stream of nitrogen. Add 10 ml of water and convert the soaps to free fatty acids by acidifying with conc HCl. Extract the fatty acids from the aqueous phase with successive 20 ml portions of light petroleum extracts with water (20 ml) and combine the wash with the aqueous phase. Adjust the aqueous polyol solution to pH 7.0 with aqueous KOH solution with the aid of a pH meter. Evaporate to a small volume (2-3 ml) under reduced pressure and extract three times with 30 ml of boiling ethanol. Filter off any residue and evaporate the ethanol under reduced pressure to yield a viscous liquid sample of polyols. Dissolve a 0.1 g sample of polyol in 0.5 ml of warm pyridine (previously dried over KOH) in a 10 ml capped phial. Add 0.2 ml hexamethyl disilazane, shake, add 0.2 ml trimethyl chlorosilane and shake again. Stand on a warm plate (about 80°C) for 3-5 min. Check that white fumes are present indicating an excess of reagent.

GLC

GC with a FID and a column (1.5 m×4 mm I.D.) packed with 3% OVI on diatomite CO (100=-200 mesh) or on Gas chrom 0 (100-120 mesh). Operating conditions are as follows:

•  Oven temperature : Programmed from 90-330°C at 4-6°C/min
•  N2 carrier gas flow rate : 86 ml/min
•  Injection temperature : 275°C
•  Detector temperature : 350°C

 Inject a 2 ml sample of TMS derivative of polyols.

Total Fatty Acid Esters

Take 20 g of a homogeneous sample of emulsifier in a 1 litre Erlenmeyer flask.Dissolve in 100 ml chloroform. Add to it 200 ml methanol and mix the blend with a magnetic stirrer. After mixing, add another 100 ml chloroform and continue the mixing. Transfer the solution into 1 litre separating funnel. Wash the Erlenmeyer flask with 30 ml chloroform and transfer to the separating funnel. Then add 180 ml water and mix the contents of the separating funnel. Let the two phases separate from each other. Extract chloroform phase two times with a 10 ml mixture of 9:10-water:methanol. After separating, the water: methanol mix is combined with the original water phase. Wash the combined water phase, once in a 500 ml separating funnel with 50 ml chloroform.

Transfer the chloroform phase which contains the fatty acid ester to a 500 ml round bottomed flask and distil off the solvent in rotary vacuum drier. After the chloroform has been removed, treat the flask further for 20 min. Dry the flask for 20 min in oven at 105°C and after cooling in a desiccator weigh.

Total fatty acid ester, % by mass = X×100/ M

Where,

M = mass, in g, of sample and

X= mass, in g, of residue after drying.

Total Glycerol

Weigh 1 g of sample in a 300 ml Erlenmeyer flask. Add 25 ml of 0.5N alcoholic KOH solution and saponify the sample for 30 min by boiling under reflux. After saponification neutralize the sample with an equivalent amount of 0.5N HCl.Further add o.5 ml of HCL. Distil off the alcohol in the rotating evaporator.

Transfer the contents of the flask to a 500 ml separating funnel with 100 ml hexane and 25 ml distilled water. Shake, after the separation, transfer the water phase to a 200 ml flask fitted with NS 29. Wash the hexane phase two times with 25 ml distilled water each time. Both the water phases are united with the first phase in the flask. Distil the water in the rotating evaporator. Dissolve the remaining glycerol in 2-propanol and filter through a Whatman filter paper No. 41 into a 100 ml flask fitted with NS 29. Wash the flask and filters with 2-propanol in order to transfer the sample quantitatively. After distilling off the 2-propanol in the rotating evaporator on a water bath, apply full vacuum from the water jet pump for 20 min followed by drying for 20 min in the oven and finally cool in the desiccator. Weigh.

Total glycerol, % by mass = B×100/ A

Where,

A= mass, in g, of the sample, and

B=mass, in g, of the dried polyalcohol phase.

Free Glycerol

Take 20 g of a homogeneous sample of emulsifier in a 1 litre Erlenmeyer flask.Dissolve in 100 ml chloroform. Add to it 200 ml methanol and mix the blend with a magnetic stirrer. After mixing, add another 100 ml chloroform and continue the mixing. Transfer the solution into 1 litre separating funnel. Wash the Erlenmeyer flask with 30 ml chloroform and transfer to the separating funnel. Then add 180 ml water and mix the contents of the separating funnel. Let the two phases separate from each other. Extract chloroform phase two times with a 10 ml

mixture of 9:10-water:methanol. After separating, the water: methanol mix is combined with the original water phase. Wash the combined water phase, once in a 500 ml separating funnel with 50 ml chloroform.

Transfer the water phase into a weighed 500 ml round bottom flask and distil off the solvent in a rotating evaporator under vacuum. As soon as the chloroform and methanol has been distilled off, treat the flask further full vacuum in the rotating evaporator. Dry the flask for 20 min in an oven at 105°C and after cooling in a desiccator, weigh.

Free glycerol, % by mass = (M1×100)/ M

Where,

M= mass, in g, of the sample, and

M1=mass, in g, of free glycerol remained in flask after drying.