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1 Extraction, Separation and Estimation of Lipids from Oil Seed

1 Extraction, Separation and Estimation of Lipids from Oil Seed

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Estimation of Lipids

a mixture of ethanol and diethyl ether or a mixture of chloroform and methanol.

Inclusion of methanol or ethanol in the extraction medium helps in breaking the

bonds between the lipids and proteins.

Materials and Reagents

Iodometric flasks

Separatory funnel

Oil seeds (sunflower, peanuts or soybean)

Anhydrous sodium sulphate

Chloroform: methanol mixture (2:1)

1% sodium chloride


1. Take 1 g of the oil seed and grind it in the presence of 5 g of anhydrous sodium

sulphate in a pestle and mortar. A small amount of acid washed sand may be

used as an abrasive if the seed material is tough.

2. Add 20 mL of chloroform–methanol mixture to it and transfer it to an air tight

glass stoppered iodometric flask. Shake the content of the flask on a mechanical

shaker for 1 h and then filter it through a glass-sintered funnel. Repeat the

extraction of the residue twice and pool the filtrates.

3. Remove the solvent from the residue by distilling under vacuum. Since the

residue left after drying contains crude lipids, extract it again with 10 mL of

chloroform–methanol mixture containing 1 mL of 1% sodium chloride.

4. Take the pooled fractions in a separatory funnel, shake it thoroughly and allow it

to stand for 5 min. The lipids will be recovered in the lower chloroform layer

while soap, glycerol and other water insoluble impurities move into the upper


5. Drain out the lower layer and treat the upper layer again 3–4 times with 5–10 mL

of chloroform–methanol mixture to extract any residual lipid from it.

6. Collect the lipid containing fractions in a pre-weighed beaker.

7. Evaporate the solvent by keeping the beaker in warm water bath (50 C) with a

constant blowing of a slow stream of nitrogen gas over the surface.

8. Record the weight of the beaker and determine the amount of crude lipids in the

sample by subtracting the weight of empty beaker.

9. Express the results in terms of % crude lipid in the given sample of the oil seed.

• The sample should not be exposed to high temperature or light as some lipids

get polymerized or decomposed on exposure to light, heat and oxygen.

6.2 Determination of Saponification Value of Fats and Oils



Determination of Saponification Value of Fats and Oils


Hydrolysis of fat with an alkali results in the formation of salts of fatty acids (also

called soap) and glycerol. This process is called saponification. From the amount of

potassium hydroxide utilized during hydrolysis, the saponification value of a given

fat sample can be calculated. The saponifiaction value is defined as mg of KOH

required to saponify 1 g of the given fat (Fig. 6.1).

It may be recalled that three molecules of KOH are consumed for saponification

of each molecule of tracylglycerol irrespective of chain length of fatty acid. Each

gram of a triacylglycerol with shorter chain fatty acids will contain larger number

of molecules of the triacylglycerol and will thus require much more KOH.

The saponification value is therefore an indication of average molecular weight

of the fatty acids in an acylglyceride.

The procedure involves refluxing of known amount of fat or oil with a fixed but

an excess of alcoholic KOH. The amount of KOH remaining after hydrolysis is

determined by back titrating with standardized 0.5 N HCl and the amount of KOH

utilized for saponification can therefore be calculated.

Materials and Reagents







Reflux condenser

Boiling water bath


Test compounds (tristearin, coconut oil, butter)

Fat solvent: A mixture of 95% ethanol and ether (1:1 u/v)

0.5 N alcoholic KOH: Prepare 0.5 N solution of KOH by dissolving 28.05 g of

KOH pellets in 20 mL water and make the volume to 1 L with 95% ethanol

7. 1% Phenolphthalein solution in 95% alcohol

8. 0.5 N HCl


Fig. 6.1 Saponification

reaction of triacylglycerol










Potassium Glycerol

salt of fatty acid



Estimation of Lipids


1. Weigh accurately 1 g of the fat sample in a conical flask and dissolve it in about

3 mL of the fat solvent (Reagent No. 5).

2. Add 25 mL of 0.5 N alcoholic KOH, attach a reflux condenser to it and reflux the

contents on boiling water bath for 30 min.

3. Cool to room temperature and add a few drops of phenolphthanlein into the


4. Titrate the contents of the flask with 0.5 N HCl till the pink colour disappears.

5. Similarly, run a blank by refluxing 25 mL of 0.5 N alcoholic KOH without any

fat sample.


0.5 N KOH in blank ¼ x mL

0.5 N KOH in test sample ¼ y mL

Titre value for sample ¼ (xÀy) mL

Saponification value ¼

28:05 Â titre value


Weight of sample ðgÞ

The multiplication factor of 28.05 in the above equation is included since 1 mL

of 0.5 N KOH contains 28.05 mg of KOH.


1. As alcohol is highly inflammable therefore precaution is required during heating.

2. During refluxing, effective cooling of condenser is required so that alcohol does

not get evaporated during saponification.


Determination of Acid Value of Fats and Oils


Different fat samples may contain varying amount of fatty acids. In addition, the

fats often become rancid during storage and this rancidity is caused by chemical

or enzymatic hydrolysis of fats into free acids and glycerol. The amount of free fatty

acids can be determined volumetrically by titrating the sample with potassium

hydroxide. The acidity of fats and oils is expressed as its acid value or number

which is defined as mg KOH required for neutralizing the free fatty acids present in

1 g of fat and oil. The amount of free fatty acids present or acid value of fat is a

useful parameter which gives an indication of the age and extent of its deterioration.

6.4 Determination of Iodine Number of Fat


Materials and Reagents







Conical flasks.

Test compounds (olive oil, butter, margarine, etc.).

1% phenolphthalein solution 95% alcohol.

0.1 N Potassium hydroxide: Weigh 5.6 g of KOH and dissolve it in distilled

water and make the final volume to 1 L. Standardize this solution by titrating

known volume of 0.1 N oxalic acid (prepared by taking 630 mg oxalic acid in

100 mL water) using phenolphthalein as an indicator till a permanent pink colour

appears. Calculate the actual normality (N2) of KOH solution from equation

N1V1 ¼ N2V2 where N1 and V1 are normality and volume of oxalic acid taken for

titration and V2 is the volume of KOH solution used.

6. Fat solvent (95% ethanol: ether 1:1, u/v).


1. Take 5 g of fat sample in a conical flask and add 25 mL of fat solvent (Reagent

No. 6) to it. Shake well and add a few drops of phenolphthalein solution and

again mix the contents thoroughly.

2. Titrate the above solution with 0.1 N KOH until a faint pink colour persists for

20–30 s.

3. Note the volume of KOH used.

4. Repeat the steps 1–3 with a blank which does not contain any fat sample.


0.1 N KOH solution used for blank ¼ x mL

0.1 N KOH solution used for sample ¼ y mL

Titre value for sample ¼ (yÀx) mL

Acid value (mg KOH/g fat) ¼

Titre value  Normality of KOH  56:1


Weight of sample (g)

1 mL of 1 N KOH contains 56.1 mg of KOH. Hence, factor of 56.1 is incorporated

in the numerator in the above equation to obtain weight of KOH from the volume of

0.1 N KOH solution used during this titration.


Determination of Iodine Number of Fat


The most important analytical determination of an oil/fat is the measurement of its

unsaturation. The generally accepted parameter for expressing the degree of carbon

to carbon unsaturation of fat, oil or their derivatives is iodine value. Iodine value or


Fig. 6.2 Reaction for the

estimation of Iodine number

of fatty acid


Estimation of Lipids


Oleic acid





I2 + 2Na2SO3



KCI + I2

2NaI + Na2S4SO6

iodine number is defined as grams of iodine absorbed by 100 g of fat. It is a useful

parameter in studying oxidative rancidity of triacylglycerols since, higher the

unsaturated, greater is the possibility of rancidity.

Estimation of iodine number is based on the treatment of a known weight of fat

or oil with a known volume of standard solution of iodine monochloride, and then

determining the amount of unused iodine monochloride from iodine liberated, on

addition of excess of KI. The released iodine is titrated against 0.1 N sodium

thiosulphate solution using starch as an indicator (Fig. 6.2).

Materials and Reagents





Stoppered bottles.

Burette (25 mL).

Test compounds: 2% solution of corn oil, olive oil and butter in chloroform.

Wij’s solution: Dissolve 8.5 g of iodine and 7.8 g of iodine trichloride separately in

450 mL of acetic acid each. Mix both the solution and make the volume upto 1 L.

5. 0.1 N sodium thiosulphate: Dissolve 24.82 g of Na2S2O3. 5H2O in 1 L of water.

To check its normality, take 20 mL of 0.1 N potassium dichromater, add 10 mL

of 15% KI and then 5 mL of HCl. Dilute to 100 mL with water and titrate

with thiosulphate solution till the yellow colour appears. Now add a few drops

of starch solution (Reagent No. 7) and continue the titration till the blue colour

disappears. Note the volume of thiosulphate solution and calculate its exact

normality (N1V1 ¼ N2V2, where N1 is the normality, V1 is the volume of dichromate solution taken for titration and V2 is the volume of thiosulphate solution used).

6. 10% Potassium iodide solution: Dissolve 10 g of KI crystals in water and make

up the volume to 100 mL.

7. 1% Starch indicator: Take 1 g starch and dissolve it in 100 mL water, boil for a

min, cool and centrifuge to get a clear solution.


1. Take 10 mL of fat solution into stoppered bottles and add 25 mL of Wij’s

solution. Shake thoroughly and allow it to stand in dark for 1 h.

2. Similarly, prepare a blank in which fat solution is replaced by chloroform.

3. After the reaction time of 1 h in dark, rinse the stopper and neck of the bottle with

50 mL of water and add 10 mL of potassium iodide solution.

4. Titrate the liberated iodine with standard sodium thiosulphate solution till the

content of the flask becomes pale yellow in colour.

6.5 Solubility Test for Lipids


5. Add a few drops of starch solution and continue to titrate it further with sodium

thiosulphate solution till the blue colour disappears.


The difference between the blank and test readings gives the amount of 0.1 N

sodium thiosulphate required to react with an equivalent volume of iodine. One litre

of 0.1 N iodine solution contains 12.7 g of iodine. The iodine number can thus be

calculated as follow:

Volume of 0.1 N sodium thiosulfate used for blank ¼ x mL

Volume of 0.1 N sodium thiosulfate used for sample ẳ y mL

Iodine number ẳ

x yị 12:7




1; 000

wt. of sample ðgÞ


1. The bottles must be shaken thoroughly throughout the titration to ensure that all

the iodine is expelled from the chloroform layer.


Solubility Test for Lipids

Triaclyglycerols with small chain fatty acids are somewhat soluble in water but

those containing non-polar long chain fatty acids are insoluble and they form

emulsions in water. All triacylglycerols are soluble in diethyl either, chloroform

and benzene. They are slightly soluble in cold methanol, ethanol and acetone but

their solubility increases on warming. Understanding of their solubility characteristics is helpful in developing efficient procedures for extraction of various lipids

from the biological materials.

Materials and Reagents

1. Fatty acids (butyric, palmitic and oleic acids)

2. Fats and oils (butter, olive oil, cod liver oil, phospholipids, etc.)

3. Solvents (water, acetone, ethyl alcohol, chloroform, diethyl either, etc.)


1. Take small amount of different lipids in various test tubes and add water. Shake

well and check their solubility.

2. Note the change, if any, in solubility on warning the above tubes in water bath at

50 C for 5 min.

3. Repeat the solubility test using different solvents.

4. Record the observation regarding solubility of these lipids and conclude about

their solubility characteristics.



Fig. 6.3 Reaction for

Acrolein test

Estimation of Lipids










+ 2H2O



Acrolein Test for Glycerol


When glycerol, either in free form or as an ester of fatty acids, is heated with

potassium hydrogen sulphate till it gets dehydrated to an unsaturated aldehyde

called acrolein. Acrolein can be identified by its characteristic pungent smell

(Fig. 6.3).

Materials and Reagents

1. Lipid samples (butter, olive oil, stearic acid, glycerol, etc.)

2. Anhydrous potassium hydrogen sulphate


1. Take approximately 1.5 g of potassium hydrogen sulphate in a test tube, and add

five drops of the liquid test sample or an approximately equivalent weight of the

test sample if it is solid. Cover the test sample completely by adding more of

solid potassium hydrogen sulphate on top of it.

2. Heat the test tube slowly on burner and note the odour of the fumes evolved from

the tube.


Qualitative Test for the Presence of Fatty Acids

by Titrimetric Method


The presence of non-esterified fatty acids in a given sample can be determined by

titrating it with an alkali using phenolphthalein as an indicator.

Materials and Reagents





Lipid samples (butter, olive oil, stearic acid dissolved in 50% alcohol)

Phenolphathalein: Prepare 1% solution in alcohol

0.1 N NaOH

Erlenmeyer flasks

6.8 Test for Unsaturation of Fatty Acids in Lipid Sample



1. Take 10 mL of 0.1 N NaOH in an Erlenmeyer flask and add a drop of phenolphthalein solution, which will give permanent pink colour.

2. To this, with pipette add the test solution drop by drop with constant shaking of

the flask.

3. Disappearance of pink colour indicates the presence of free fatty acids in the

given test compound.

4. Repeat this with other lipid samples.


Test for Unsaturation of Fatty Acids in Lipid Sample


The fatty acids present in animal fats are usually fully saturated, whereas

those occurring in vegetable oils contain one or more double bonds in their hydrocarbon chain. A semiquantitative estimate about the degree of unsaturation of lipid

samples can be deduced since halogens are readily added across the double bonds

and this reaction results in decolorization of bromine water or iodine solution

(Fig. 6.4).

Materials and Reagents

1. Test solutions (olive oil, corn oil, coconut oil, oleic acid, etc.).

2. Bromine water: Add 5 mL of bromine to 100 mL of water. Shake the mixture

and keep it in dark bottle.


1. Take approximately 5 mL of test solution in a test tube and slowly add bromine

water dropwise and shake the tube after each addition.

2. Keep on adding bromine water till it fails to get decolorized and retains its


3. Note the amount of bromine water added.

4. Repeat the experiment with other test solutions.

CH3(CH2)7CH=CH(CH2)7COOH + Br2

Oleic acid





Dibromostearic acid

Fig. 6.4 Reaction for estimation of unsaturation in oleic acid




Estimation of Lipids

Separation of Different Lipid Fractions

by Thin Layer Chromatography (TLC)

The qualitative and quantitative analysis of non-polar and polar lipids can be

effectively done after separating the components in these fractions by thin layer

chromatography (TLC). It is a chromatographic technique in which different

components in a sample get separated during their passage along a very thin

layer, usually of 0.20–0.25 mm thickness, of a suitable chromatographic media

which is spread as a uniform layer on a glass plate (see Sect. 9.4 for details of TLC).

The chromatographic material used for this experiment is Silica Gel-G. Different

lipids are adsorbed onto activated Silica Gel-G with varying degrees of strength.

Those components, which are not adsorbed or adsorbed with lesser strength, tend to

move faster along with the mobile phase (solvent system), whereas those which are

held more firmly by the adsorbent (stationary phase) travel slowly. This differential

distribution of various components between the mobile and stationary phases

determines the rate of migration of different compounds in a mixture and results

in their separation from each other.

Materials and Reagents

Activated TLC plates

Glass plates (20 Â 20 cm) onto which a 0.2-mm thick layer of silica

gel-G has been layered with the help of a spreader. The prepared

thin layer plates are dried at room temperature and then activated

at 110 C for 45 min before use

Thin layer chromatographic


Developing mixtures

Chloroform:methanol:water (65:25:4) for separation of

phospholipids and galactolipids from polar lipids

Toluene:ethyl acetate:ethanol (2:1:1) for separation of galactolipids

from phospholipids

Hexane:diethylether:acetic acid (80:20:1) for separation of various

neutral lipids

Spraying reagents

Sulphuric acid: 50% H2SO4 in water of spots on TLC plates (u/v)

for location

Perchloric acid: 20% perchloric acid in water (u/v)

Ferric chloride spray: dissolve 50 mg FeCl3. 6H2O in 90 mL water

and add 5 mL each of glacial acetic acid and conc. sulphuric acid

Nihydrin spray: 0.2 g ninhydrin dissolved in 100 mL ethanol

Anthrone spray: dissolve 0.2 g anthrone in 100 mL conc. H2SO4

Molybdate spray: prepare a solution containing 16 g ammonium

molybdate in 120 mL water. To 80 mL of this solution add

40 mL conc. HCl and 10 mL mercury. Shake it for 30 min. To

this solution add 200 mL conc. H2SO4 and remaining 40 mL of

ammonium molybdate, cool the mixture and make the volume to

1 L with water

Iodine spray: 1% iodine in chloroform

Various lipid fractions prepared as discussed previously

Standard lipids: make 0.2% solution of cholesterol, tristearin, phosphatidyl ethanolamine, palmitic

acid, stearic acid, lecithin, galactosyldiacylglycerol


Separation and Identification of Lipids by Column Chromatography



1. Apply 10–20 mL fraction of the lipid sample in the form of a spot at a distance of

2 cm starting from the left bottom edge of the activated TLC plate.

2. Develop the plates in the appropriate developing mixture (depending upon the

types of lipids to be separated) in an air tight chromatographic glass tank till the

solvent front moves up to 4 cm below the top edge of the glass plate.

3. Take out the plates, dry them for 5 min in air and spray the plates with the

required detection reagent. The specificity and use of different spraying reagents

is given below.

4. Locate the position of the lipid spots on the glass plate and measure the distance

travelled by the individual lipid component. Calculate their Rf values and

compare them with those of standards.

Spraying Reagents

1. Sulphuric acid spray: This reagent forms charred black spots with all the lipids.

Spray the plates with H2SO4 and keep the plates in an oven at 120 C for 30 min.

Mark the spots on the plate with pencil.

2. Iodine vapours: This is also a general detection reagent for all lipids. On keeping

the plates in a jar or development tank containing a trough filed with iodine

crystals, yellowish or brown spots appear and then fade away after some time on

exposure of plates to air.

3. Perchloric acid spray: Heat the developed chromatogram at 100 C for 15 min and

spray with perchloric acid. Note the brown spots which are formed by the lipids.

4. Ferric chloride spray: Spray the developed plates with ferric chloride and heat

them at 100 C for 2–3 min in an oven. Lipid samples of cholesterol and

cholesteryl esters will produce red or violet spots.

5. Ninhydrin spray: For detection of lipids containing amino groups, spray the

developed plates with ninhydrin reagent and heat them in oven at 100 C for

5 min. Violet spots will indicate their presence and location on the plates.

6. Anthrone spray: Spray the chromatogram with anthrone reagent and heat the

plate in oven at 100 C for 10 min. Formation of green spots will be due to its

reaction with glycolipids and violet spots due to sulpholipids.

7. Molybdate spray: Phospholipids are identified as violet spots on spraying the

developed plates with molybdates reagent or iodine solution. Heat the plates in

an oven at 100 C for 5 min after spraying them with molybdate reagent.


Separation and Identification of Lipids

by Column Chromatography


The extracted lipids are fractionated into neutral, non-polar and polar

lipids by adsorbing them on solid adsorbent in non-polar solvents. They are then



Estimation of Lipids

eluted stepwise with solvents of increasing polarity. The individual components

in neutral and polar fractions obtained by this method may further be separated

by TLC.

Materials and Reagents

1. For column chromatography








Lipid preparation (as discussed)

Silicic acid




Glass column (2.5 cm diameter  15 cm length)

Glass wool

2. For estimation of galactose

(a) Anthrone reagent: Dissolve 0.2 g anthrone in 100 mL of conc. H2SO4.

(b) Standard galactose solution: Dissolve 25 mg galactose in water and make

the volume to 250 mL. This solution contains 100 mg galactose/mL.

3. For estimation of phosphorus

(a) Digestion mixture: Sulphuric acid and 72% Perchloric acid (9:1, u/v)

(b) Aminonaphthol suphonic acid (ANSA) reagent: Dissolves 3.43 g of sodium

metabisulphite, 0.063 g of 1-amino-2-naphthol-4-sulfonic acid and 0.125 g

of sodium, filter through Whatman No. 1 filter paper and make the volume to

25 mL with water.

(c) 0.26% ammonium heptomolybdate.

(d) ANSA-molybdate reagent: Mix 22 mL of ANSA reagent (Reagent No 11)

and 500 mL ammonium heptamolybdate just before use. This reagent is

stable for 1 h only.

(e) Standard phosphorus: Dissolve 34 mg of KH2PO4 in water and make the

volume to 250 mL. This solution contains 1 mmol of phosphorus/mL.


1. Wash silicic acid thrice with double distilled water to remove fine particles and

activate it at 125 C for 14 h.

2. Suspend the activated silicic acid in chloroform and carefully pour the slurry

into the glass column having glass wool plug at its bottom. Allow the adsorbent

to settle and add more slurry till a column with a bed height of 9 cm is obtained.

3. Wash the packed column with three column volumes of chloroform.

4. Apply lipid preparation (dissolved in chloroform) to the silicic acid column and

elute it with 200 mL of chloroform at constant flow rate of 2 mL/min. The

neutral lipids, i.e. triacylglycerols, diacylglycerols, sterols and sterol esters get

eluted during this step. The different lipid components in these fractions may

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