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FATS. CONTENTS. 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.

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contents
CONTENTS
  • 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
slide3

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
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
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
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

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
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.
slide9
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.

slide10
CALCULATION:

Acid value (mg KOH/g) =

titer value× N of KOH×56.1

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
IDENTIFICATION & QUANTIFICATION OF FATTY ACIDS

PRINCIPLE:

  • FA are made volatile by converting them into methyl esters. The conversion of FA’s 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
MATERIALS:
  • 10% BCl3 or 14% BF3 in methanol
  • Saturated NaCl solution
  • Na2SO4(anh)
  • Hexane
  • N2 gas
  • GLC
  • Column- pre tested 10% silar 10ºc on GC with

100 to 200 mesh.

  • Detector – FID
  • Injector temp. -280ºc
slide13
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.

slide14

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

slide15

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 C- atoms & of increasing unsaturation for the same no. of C- atoms

determination of the acid value of a fat
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.
materials17
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

slide18

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.
seperation of fatty acids by reverse phase paper chromatography
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 20×20cm is dipped in the 10% liq. paraffin in pet. Ether.
slide21
The std FA’s samples are applied to the paper in the followed manner:
  • Saturated FA’s (2-5µg each): lauric acid, myristic acid, palmatic acid, stearic acid.
  • Unsaturated FA’s (1-3µg 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 120ºc for 10-15 min. by identifying the FA content of sample.
saponification value
SAPONIFICATION VALUE

PRINCIPLE:

  • The saponification value is the no. of mg. of KOH required to neutralize the FA’s resulting from the complete hydrolysis of 1g. Of fat.
  • 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.
materials23
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
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

slide25

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

slide26
CALCULATION:

Saponification value (s) = 3×56×1000

avg.mol wt.of fat

Avg mol wt. of fat = 3× 56×1000

s

determination of ester value
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
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 FA’s in fat.
  • Halogens add across the double bonds of unsaturated FA’s to form additional compounds.
materials29
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
method30
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

slide31
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

slide33
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
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.
materials35
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
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

slide37

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
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(40-160°c)
  • Whatmaan no.2 filter paper
  • Absorbent cotton
  • Soxhlet apparatus.
procedure39
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

slide40

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.

slide41
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
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

slide43

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

slide44

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
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

slide46

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.

slide48
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
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 FA’s.
materials50
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.
procedure51
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

slide52

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

slide53

Increase the rate of distillation till 600ml is collected

Titrate the distillate against 0.1N NaOH using phenolphthalein indicator.

CALCULATION:

µg/Lt volatile acids as acetic acid

=ML 0.1N NaOH ×6000

ML sample

thin layer chromatography of phospholipids
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
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 (100µg/ml)prepared in chloroform

Std solution pipetted ranging from 50-500µg/tube & upto 1ml with chloroform

slide56

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
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.
slide58
D.J.SRAVANTHI

M.GLORY HEPSIBAH

P.INDIRA

M.PHARM (ANALYSIS)

THANK U