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INTRODUCTION PHYSICAL PROPERTIES ACID VALUE IDENTIFICATION & QUANTIFICATION OF FA’S SEPERATION OF FATTY ACIDS SAPONIFICATION VALUE ESTER VALUE IODINE VALUE PEROXIDE VALUE ESTIMATION OF OILS ACETYL VALUE HYDROXYL VALUE

FATS
These compounds are so named because the first isolated member was from fat.

The general formula is R-COOH, R being an alkyl group with a short or long chain.

FATTY ACIDS

The general chemical structure of fatty acid is CH3(CH2)n.COOH,where n has a value ranging from 0 to 20. By virtue of being carboxylic acid, fatty acids are capable of forming salts with inorganic bases like NaOH or KOH,MG(OH)2… The mineral salt of fatty acid is called a soap.

TRIGLYCERIDES

Fatty acids can form esters with alcoholic hydroxyl also Those esters are called mono glycerides, di glycerides, depending on the no. of –OH groups that are esterified to fatty acids.

As there are no polar groups triglycerides are called as the neutral fats.

odorless. tasteless substances than water in which they are insoluble but soluble in organic solvents.PHYSICAL PROPERTIES  They are colorless.  . The melting point of fats are higher than their solidification points.

of KOH required to neutralize the free acids present naturally in 1g. of neutral fat. . is called as acid number. This indicates the degree of purity of fat. which is purer with lower values of this number.ACID VALUE / ACID NUMBER The number of mg.

ESTIMATION OF FREE FATTY ACIDS PRINCIPLE:  The free fatty acid in an oil is estimated by titrating it against KOH in the presence of phenolphalein indicator. of sample. However the free fatty acid content is expressed as oleic acid equivalents.  .  The acid number is defined as the mg of KOH required to neutralize the fatty acids present in 1g.

1N KOH ↓ Shake constantly until pink color persists for 15 sec.MATERIALS:  1% phenolphalein in 95% ethanol  0. PROCEDURE: 1 to 10g of oil in 50ml. .1N KOH  Neutral solvent.the neutral solvent in 250ml conical flask ↓ 2drops indicator added ↓ Titrate against 0.

of sample (g) The free fatty acid is calculated as oleic acid using the eq.028g oleic acid .1 wt.CALCULATION: Acid value (mg KOH/g) = titer value× N of KOH×56. N/10 KOH =0. 1ml.

 . The conversion of FA’s in methyl esters is carried out directly by trans esterification.IDENTIFICATION & QUANTIFICATION OF FATTY ACIDS PRINCIPLE:  FA are made volatile by converting them into methyl esters. The esters are identification & quantified by injecting into GLC & comparing with a set of std esters.

pre tested 10% silar 10ºc on GC with 100 to 200 mesh. -280ºc  .MATERIALS: 10% BCl3 or 14% BF3 in methanol  Saturated NaCl solution  Na2SO4(anh)  Hexane  N2 gas  GLC  Column.  Detector – FID  Injector temp.

   Detector temp. ↓ . -250ºc Carrier gas –N2 at 50ml/min Column/oven temp. -165ºc PROCEDURE: 150 to 300mg oil ↓ 3ml of 10% BCl3 added + boiling chips & heat at 83ºc for 6 min ↓ Transfer the contents to seperatory flask & collect the washings which is done 4 times with 1ml portions of hexane.

Na2SO4 ↓ Rinse funnel with 2ml hexane& collect the hexane layer ↓ Filter the hexane extract ↓ Reduce the vol. to 2-3ml by drying with N2 gas steam ↓ .↓ Shake & allow to separate ↓ 4ml sat. NaCl solution added ↓ Shake & collect hexane layer over anh.

of C.atoms . Esters appear in order of increasing no. of Catoms & of increasing unsaturation for the same no.↓ Inject an aliquot to pre conditioned GLC ↓ Inject std methyl esters separately & calculate the retention time. CALCULATION: By identifying the peaks in their relative positions.

DETERMINATION OF THE ACID VALUE OF A FAT PRINCIPLE:  During storage. . O2 & hydrolysis by micro organisms with the liberation of free acids. fats may become rancid as a result of peroxide formation at the double bonds by atm.  The amt of free acid gives an indication of the age & quality of the fat.

butter& margarine .50 METHOD: 10g of test comp.200g  Fat solvent .1Lt  Burettes .200ml  KOH .6Lt  Phenolphthalein .MATERIALS: Olive oil. weighed ↓ Melted fat suspended in 50ml of fat solvent ↓  .

1 mol. of std alkali required & calculate the acid value of fat./lit. of ml. . KOH ↓ Faint pink color persists for 20-30 sec  Note the no.↓ 1ml phenolphthalein ↓ Mix thoroughly ↓ Titrate with 0.

Acid value 0–1 1–4 4 – 15 Sample weight 20 10 2.5 0.5 15 – 74.1 .9 ≥75 0.

Whatmaan no. the S. The M. Ether.SEPERATION OF FATTY ACIDS BY REVERSE PHASE PAPER CHROMATOGRAPHY  In this method.1 & 3 filter paper of size 20×20cm is dipped in the 10% liq. paraffin in pet.P is the organic solvent adsorbed to an inactive supporting material.   .P immiscible with the first is aq.

by identifying the FA content of sample. for detecting the spots with phosphomolybdic acid reagent.  After development the papers are hanged to room temp. palmatic acid. Ether.  Unsaturated FA’s (1-3µg each): linolenic acid.  Develop the papers in the chromatographic chambers saturated with the solvents like acetone-water 80:20 or acetic acid-acetonitrile 1:1v/v saturated with liq.paraffin in pet. myristic acid. . oleic acid. stearic acid.The std FA’s samples are applied to the paper in the followed manner:  Saturated FA’s (2-5µg each): lauric acid.  Heat in hot air oven at 120ºc for 10-15 min.

SAPONIFICATION VALUE PRINCIPLE:  The saponification value is the no. of KOH required to neutralize the FA’s resulting from the complete hydrolysis of 1g.  The saponification value gives an indication of the nature of the FA’s in the fat since longer the C-chains the less acid is liberated per gram of fat hydrolyzed. . Of fat. of mg.

100  .ether) – 1Lt  Alcoholic KOH – 3Lt  Reflux condenser .100  Boiling water bath .100  Phenolphthalein – 50ml  HCl – 3Lt  Burettes – 100  Conical flasks .MATERIALS: Fats – 20g  Fat solvent(95% ethanol .

METHOD: 1g of fat weighed in tarred beaker ↓ Dissolve in 3ml.of 0.of fat solvent ↓ Contents transferred to 250ml conical flasks ↓ Rinsing beaker with further ml of solvent 3 times ↓ 25ml.5mol/Lt alc. KOH added ↓ .

↓ Leave to room temp. ↓ Titrate with 0.5mol/Lt HCl ↓ Phenolphthalein indicator was added .↓ Attached to reflux condenser ↓ Attach another reflux condenser as blank ↓ Heat on boiling water bath for 30min.

mol wt. of fat = 3× 56×1000 s .CALCULATION: Saponification value (s) = 3×56×1000 avg.of fat Avg mol wt.

Of the substance. of KOH required to saponify the esters present in 1g.  Ester value = saponification value-acid value. of mg.DETERMINATION OF ESTER VALUE PRINCIPLE:  The ester value is the no. .

 Higher the iodine value. greater is the proportion of unsaturated FA’s in fat.THE IODINE NUMBER OF A FAT PRINCIPLE:  The number of grams of iodine absorbed by 100g of fat by virtue of its unsaturation is termed as iodine value. .  Halogens add across the double bonds of unsaturated FA’s to form additional compounds.

5Lt  Sod.thio sulfate(0.2mol/Lt) – 3Lt  KI(100g/Lt) – 1.1g/Lt) – 4Lt  Starch indicator(10g/Lt) -250ml  Stoppered bottles(250ml) – 200  Burettes (25ml) – 100  Chloroform – 1Lt  .MATERIALS: Fats – 1Lt  Iodine chloride –(0.

METHOD: Pipette out 10ml of fat solution ↓ 25ml of iodine chloride solution added ↓ Leave to stand in dark for 1hr. necks with 50ml water ↓ 10ml. Shaking thoroughly ↓ Rinse the stoppers.KI solution added ↓ .

CALCULATION: Iodine number = (B-T) × 6.When the solution is pale straw color add 1ml. Of starch solution until blue color disappears ↓ Bottles must be shaken thoroughly to ensure that all iodine is removed from chloroform layer.35g per 100g of fat .

000 0.400 0.200 101 – 150 151 – 200 0.100 .000 1.Iodine value Weight in grams <5 5 – 20 21 – 50 51 – 100 3.130 0.

SIGNIFICANCE:  The iodine value is a measure of the unsaturation of an oil. The higher the iodine value the more double bonds are present. . which consequently reflects the reactivity of the oil.

.DETERMINATION OF PEROXIDE VALUE PRINCIPLE:  Peroxide value is a measure of peroxides contained in the oil. The peroxides present are determined by the titration against thio sulfate in the presence of KI.   Starch is used as indicator.

of glacial acetic acid with 1 vol.MATERIALS:  Solvent mixture – mix 2 vol. of chloroform 5% KI solution 1% starch solution N/500 sod. Thio sulfate solution    .

↓ Transfer the contents to conical flask containing 20ml of 5% KI solution ↓ Wash the tube with 25ml water twice ↓ .PROCEDURE: 1g of oil/fat are weighed in dry boiling tube ↓ 1g of powdered KI & 20ml of solvent mixture ↓ Place the tube in boiling water for 30sec.

CALCULATION: Peroxide value = S × N × 1000 sample (g) .5ml starch added ↓ Shake vigorously till blue color just disappears.↓ Titrate against N/500 sod. Thio sulfate solution until yellow color disappears ↓ 0.

. MATERIALS:  Pet.  It is then distilled off completely & dried. Ether(40-160°c)  Whatmaan no.ESTIMATION OF OIL IN OIL SEEDS PRINCIPLE:  Oil from known quantity of the seed is extracted with pet. Ether.2 filter paper  Absorbent cotton  Soxhlet apparatus.

PROCEDURE: Fold a piece of filter paper in such a way hold the seed meal. Ether for 6hrs without heating ↓ Cool & dismantle the apparatus ↓ . ↓ Cotton wool is placed at the top to distribute evenly ↓ Place the sample packet in butt tubes of soxhlet apparatus ↓ Extract with pet.

↓ Evaporate the ether on steam or water bath until no odour remains ↓ Cool to room temp. ↓ Carefully remove the dirt outside the flask & weigh ↓ Repeat heating until constant wt. . is obtained.

basis = % oil in ground sample 100% moisture in whole seed.CALCULATION: Oil in ground sample % = wt. . of oil (g) ×100 wt. of sample (g) Oil in dry wt.

which express in mg the amount of KOH required to neutralize the acetic acid liberated by the hydrolysis of 1g of the acetylated substance.DETERMINATION OF ACETYL VALUE PRINCIPLE:  The acetyl value is the no. METHOD: 10g with 20ml acetic anhydride in 200ml RB flask ↓ Support the flask on sheet of heat resistant material with diameter of 4cm ↓ Boil gently for 2 hrs & cool ↓ .

↓ Shake with 20ml warm water ↓ .2g of pumice powder added ↓ Boil for 30min & cool ↓ Transfer to a separator & discard the lower layer ↓ Wash the acetylated product with each 50ml of warmed sat.↓ Pour the contents into 600ml water containing beaker ↓ 0. solution of NaCl.

into small dish ↓ 1g of powdered anh. Sulfate added ↓ Stir thoroughly & filter. Layer completely ↓ Pour the acetylated sub. CALCULATION: Acetyl value = 1335 (b-a) / (1335-a) .↓ Remove the aq. Sod.

of mg of KOH required to neutralize the acid combined by acetylation in 1g of the substance.DETERMINATION OF HYDROXYL VALUE PRINCIPLE:  The hydroxyl value is the no. METHOD: Accurately weigh the substance in 150ml acetylation flask ↓ Pyridine acetic anhydride reagent added ↓ .

.5M ethanolic KOH using dilute phenolphthalein as indicator.↓ Boil for 1hr on water bath & maintain it to 3cm above the level of liquid in flask ↓ 5ml water added from top of the condenser ↓ If cloudiness is observed add pyridine to produce clear liquid ↓ Shake & replace the flask for 10min & cool ↓ Rinse the walls & condenser with 5ml ethanol ↓ Titrate with 0.

0 0. of reagent (ml) 5.75 0.60/1.00 5.00 5.00 15.5 1.00 .00 15.Presumed hydroxyl value 10 – 100 101 – 150 151 – 200 201 – 250 251 – 300 Qty of sub (g) 2.0 0.00 301 – 350 351 – 700 701 .5 10.950 1.75 0.00 5.0 1.00 5.00/10.20 Vol.

CALCULATION: Hydroxyl value = acid value + 28. .05 v/w V = difference in ml during titration W = wt. in grams of substance.

1N alkali with phenolphthalein indicator to find out the amount of volatile FA’s. .ESTIMATION OF VOLATILE ACIDS PRINCIPLE:  Steam distillation after acidifying the sample with 1+1 H2SO4 results in the evaporation of volatile acids which on condensation yield liquefied volatile acids.  These acids could be titrated against 0.

H2SO4 with 1 vol. titrant 0. . Water Diatomaceous silica filter aid Magnesium sulfate Std NaOH.5g FeCl3.6H2O in 1Lt dist. Ferric chloride solution – dissolve 82. of water.MATERIALS:  Dilute H2SO4 1+1 – mix 1 vol. of conc.1N – dissolve 4g NaOH in 1Lt distilled water & std against any std acid      Phenolphthalein indicator.

PROCEDURE: Adjust the sample containing volatile FA’s to Ph 3.5 with 1+1 H2SO4 ↓ 6ml of FeCl3 solution/Lt is added ↓ 50g of filter aid /Lt is added & mixed well ↓ Filter the contents ↓ Wash the residue with water 3 -4 times ↓ Adjust the Ph to 11 with NaOH solution ↓ .

↓ Evaporate to 150ml & cool in refrigerator ↓ Adjust cooled titrate to Ph4 with dil. H2SO4 ↓ MgSO4 is added for saturation ↓ Heat the contents till volatile acids evaporates ↓ Steam distillation is used so that 200ml of distillate can be collected in 25 min ↓ .

CALCULATION: µg/Lt volatile acids as acetic acid =ML 0.1N NaOH using phenolphthalein indicator.↓ Increase the rate of distillation till 600ml is collected ↓ Titrate the distillate against 0.1N NaOH ×6000 ML sample .

absorb iodine & visible as brown spots on yellow background These are not stable. however iodine in chloroform solution(1%conc) can be sprayed on.  The phospholipids on exposure to iodine vapors.  .THIN LAYER CHROMATOGRAPHY OF PHOSPHOLIPIDS  The preferred solvent for phospholipids separation is chloroform-methanol-acetic acid-water(25:15:4:2).

H2SO4 added with stirring ↓ Mixture shld be colorless but if blue color appears discard the reagent ↓ Std solution of cholesterol (100µg/ml)prepared in chloroform ↓ Std solution pipetted ranging from 50-500µg/tube & upto 1ml with chloroform ↓ .ESTIMATION OF CHOLESTEROL 40ml of acetic anhydride kept in ice bucket ↓ 2ml conc.

↓ Absorbance-640nm. .↓ 5ml reagent added to each tube & mixed well ↓ If rosy red to blue or greenish blue color is noticed tubes covered with dark cloth for 15min.

D. They impart special flavor & texture to our food which increases palatability.SIGNIFICANCE OF FATS     They are the concentrated source of energy as 1g of fat contributes 9 kilo calories of energy.E. They are used by the body to make prostaglandins involved in vital physiological functions. . They are good source of vitamin A.K.

D.INDIRA M.PHARM (ANALYSIS) .GLORY HEPSIBAH P.SRAVANTHI M.J.

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