CONTENTS

INTRODUCTION PHYSICAL PROPERTIES ACID VALUE IDENTIFICATION & QUANTIFICATION OF FAS 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.

PHYSICAL PROPERTIES

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

ACID VALUE / ACID NUMBER

The

number of mg. 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.

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.

The acid number is defined as the mg of KOH required to neutralize the fatty acids present in 1g. of sample. However the free fatty acid content is expressed as oleic acid equivalents.

MATERIALS: 1% phenolphalein in 95% ethanol 0.1N KOH Neutral solvent. PROCEDURE: 1 to 10g of oil in 50ml.the neutral solvent in 250ml conical flask 2drops indicator added Titrate against 0.1N KOH Shake constantly until pink color persists for 15 sec.

CALCULATION: Acid value (mg KOH/g) = titer value N of KOH56.1 wt. of sample (g)

The free fatty acid is calculated as oleic acid using the eq. 1ml. N/10 KOH =0.028g oleic acid

IDENTIFICATION & QUANTIFICATION OF FATTY ACIDS

PRINCIPLE:

FA are made volatile by converting them into methyl esters. The conversion of FAs in methyl esters is carried out directly by trans esterification. The esters are identification & quantified by injecting into GLC & comparing with a set of std esters.

MATERIALS:

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

Detector temp. -250c Carrier gas N2 at 50ml/min Column/oven temp. -165c

PROCEDURE: 150 to 300mg oil 3ml of 10% BCl3 added + boiling chips & heat at 83c for 6 min Transfer the contents to seperatory flask & collect the washings which is done 4 times with 1ml portions of hexane.

 Shake & allow to separate 4ml sat. NaCl solution added Shake & collect hexane layer over anh.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

 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, Esters appear in order of increasing no. of Catoms & of increasing unsaturation for the same no. of C- atoms

DETERMINATION OF THE ACID VALUE OF A FAT PRINCIPLE: During storage, fats may become rancid as a result of peroxide formation at the double bonds by atm. O2 & hydrolysis by micro organisms with the liberation of free acids.

The amt of free acid gives an indication of the age & quality of the fat.

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

 1ml phenolphthalein Mix thoroughly Titrate with 0.1 mol./lit. KOH Faint pink color persists for 20-30 sec
Note

the no. of ml. of std alkali required & calculate the acid value of fat.

Acid value
01 14 4 15

Sample weight
20 10 2.5

15 74.9
75

0.5
0.1

SEPERATION OF FATTY ACIDS BY REVERSE PHASE PAPER CHROMATOGRAPHY

In this method, the S.P is the organic solvent adsorbed to an inactive supporting material. The M.P immiscible with the first is aq. Whatmaan no.1 & 3 filter paper of size 2020cm is dipped in the 10% liq. paraffin in pet. Ether.

The std FAs samples are applied to the paper in the followed manner: Saturated FAs (2-5g each): lauric acid, myristic acid, palmatic acid, stearic acid. Unsaturated FAs (1-3g each): linolenic acid, oleic 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. Ether. After development the papers are hanged to room temp. for detecting the spots with phosphomolybdic acid reagent. Heat in hot air oven at 120c for 10-15 min. by identifying the FA content of sample.

SAPONIFICATION VALUE
PRINCIPLE: The saponification value is the no. of mg. of KOH required to neutralize the FAs resulting from the complete hydrolysis of 1g. Of fat.

The saponification value gives an indication of the nature of the FAs in the fat since longer the C-chains the less acid is liberated per gram of fat hydrolyzed.

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

METHOD:
1g of fat weighed in tarred beaker Dissolve in 3ml.of fat solvent Contents transferred to 250ml conical flasks Rinsing beaker with further ml of solvent 3 times 25ml.of 0.5mol/Lt alc. KOH added

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

CALCULATION: Saponification value (s) = 3561000 avg.mol wt.of fat Avg mol wt. of fat = 3 561000 s

DETERMINATION OF ESTER VALUE

PRINCIPLE: The ester value is the no. of mg. of KOH required to saponify the esters present in 1g. Of the substance.

Ester value = saponification value-acid value.

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. Higher the iodine value, greater is the proportion of unsaturated FAs in fat. Halogens add across the double bonds of unsaturated FAs to form additional compounds.

MATERIALS:
Fats 1Lt Iodine chloride (0.2mol/Lt) 3Lt KI(100g/Lt) 1.5Lt Sod.thio sulfate(0.1g/Lt) 4Lt Starch indicator(10g/Lt) -250ml Stoppered bottles(250ml) 200 Burettes (25ml) 100 Chloroform 1Lt

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

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.

CALCULATION:
Iodine number = (B-T) 6.35g per 100g of fat

Iodine value

Weight in grams

<5 5 20
21 50 51 100

3.000 1.000
0.400 0.200

101 150 151 200

0.130 0.100

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.

MATERIALS:

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

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. Transfer the contents to conical flask containing 20ml of 5% KI solution Wash the tube with 25ml water twice

 Titrate against N/500 sod. Thio sulfate solution until yellow color disappears 0.5ml starch added Shake vigorously till blue color just disappears. CALCULATION: Peroxide value = S N 1000 sample (g)

ESTIMATION OF OIL IN OIL SEEDS

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

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

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.

CALCULATION:

Oil in ground sample % = wt. of oil (g) 100 wt. of sample (g)

Oil in dry wt. basis = % oil in ground sample 100% moisture in whole seed.

DETERMINATION OF ACETYL VALUE

PRINCIPLE:

The acetyl value is the no. which express in mg the amount of KOH required to neutralize the acetic acid liberated by the hydrolysis of 1g of the acetylated substance.

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

 Pour the contents into 600ml water containing beaker 0.2g 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. solution of NaCl. Shake with 20ml warm water

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

DETERMINATION OF HYDROXYL VALUE

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

 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 0.5M ethanolic KOH using dilute phenolphthalein as indicator.

Presumed hydroxyl value 10 100 101 150 151 200 201 250 251 300

Qty of sub (g) 2.0 1.5 1.0 0.75 0.60/1.20

Vol. of reagent (ml) 5.00 5.00 5.00 5.00 5.00/10.00

301 350
351 700 701 - 950

1.0
0.75 0.5

10.00
15.00 15.00

CALCULATION: Hydroxyl value = acid value + 28.05 v/w

V = difference in ml during titration W = wt. in grams of substance.

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 0.1N alkali with phenolphthalein indicator to find out the amount of volatile FAs.

MATERIALS:

Dilute H2SO4 1+1 mix 1 vol. of conc. H2SO4 with 1 vol. of water. Ferric chloride solution dissolve 82.5g FeCl3.6H2O in 1Lt dist. Water Diatomaceous silica filter aid
Magnesium sulfate Std NaOH, titrant 0.1N dissolve 4g NaOH in 1Lt distilled water & std against any std acid

Phenolphthalein indicator.

PROCEDURE:

Adjust the sample containing volatile FAs 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

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

THIN LAYER CHROMATOGRAPHY OF PHOSPHOLIPIDS

The preferred solvent for phospholipids separation is chloroform-methanol-acetic acid-water(25:15:4:2).

The phospholipids on exposure to iodine vapors, absorb iodine & visible as brown spots on yellow background
These are not stable, however iodine in chloroform solution(1%conc) can be sprayed on.

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


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. Absorbance-640nm.

SIGNIFICANCE OF FATS

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

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