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11 Estimation of Fatty Acids by Gas-Liquid Chromatography (Morrison and Smith, 1964)

11 Estimation of Fatty Acids by Gas-Liquid Chromatography (Morrison and Smith, 1964)

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Fig. 6.5 Reaction showing

formation of methyl ester of

fatty acid

Estimation of Lipids

CH2O. OC(CH2)14CH3



CH2. OC(CH2)14 CH3


CH3(CH2)14. COONa

Sodium palmitate




( Palmitic acid)

Methyl palmitate

(Methyl ester)

GLC conditions


10% DEGS (diethylene glycol succinate) on chromosorb P or W

(60–80 mesh)


Flame ionization detector (FID)

Carrier gas

Nitrogen/argon with a flow rate of 40–50 mL/min. Convenient

flow rates of hydrogen (0.5 kg/cm2) and air (0.8 kg/cm2) are

also used with the detector as a fuel

Column/oven temperature 170–200 C

Detector temperature

230 C

Injector port temperature

230 C

Recorder speed

1 cm/min

Generally the injector and detector temperatures are kept 50 C above column temperature


1. Preparation of methyl esters

(a) Grind 0.1 g of oilseed with 4 mL of 0.5 N sodium methoxide in instalment of

2 mL each in a pestle-mortar. Transfer the contents to an air tight screw

capped 15 mL vial. Wash the pestle-mortar twice with 1 mL of 0.5 N Sodium

methoxide and transfer into the vial.


Take 20 mg oil (one or two drops of oil) along with 5 mL of 0.5 N sodium

methoxide in a screw capped vial.

(b) Keep the vial in a boiling waterbath for 10–15 min.

(c) Cool to room temperature, add 1–2 drops BF3-methanol reagent and again

heat for 5 min.

(d) Cool and add 1–2 mL of hexane, shake and wait till hexane layer separates out.


Estimation of Fatty Acids by Gas–Liquid Chromatography


2. Fatty acid analysis

(a) Take appropriate aliquot (2–3 mL) of the hexane layer containing methyl

esters and inject into pre-conditioned gas chromatograph.

(b) Measure the retention time and identify individual fatty acids by comparing

with the reaction time of standard methyl esters. Esters appear in the order of

increasing number of carbon atoms and of increasing unsaturation for the

same number of carbon atoms. The area of each peak should be calculated

by measuring peak height and width at half height. After computing total

peak area for the sample, calculate per cent area under peak that would give

per cent of respective fatty acid.

Peak area ¼ Peak height  width at half height:


1. Do not switch on the gas chromatograph without the flow of carrier gas.

2. Solvents used should be of high purity.

3. Add some anhydrous sodium sulphate to the solvents to remove moisture


4. BF3 is a carcinogenic compound. Do not allow it to come in contact with skin

or hand. Do not pipette it out by mouth.

5. In step (a) 4, sometimes the hexane layer does not separate out. Add 2+ or 3

drops of water and tap the vial to separate the layer. Pipette out the upper layer

and remove moisture by adding a pinch of anhydrous sodium sulphate.

6. Do not load more than 2–3 mL of methyl ester in the column at a time.

7. Any unesterified liquid component will spoil the column in most cases and

hence esterification must be checked before injecting into GLC.

8. If proper resolution of the peaks is not there, wait for sometime and find out

the reasons for it. May be flame has extinguished or carrier gas, hydrogen or air

has exhausted. Then load the sample again.

9. Sodium methoxide is generally in powder form. If it has lumps then keep it in a

Petri dish in an oven for 1–2 h at 110 C to remove moisture.

10. In case of power failure, continue the flow of carrier gas and after resumption of

power, allow the instrument to run for about 1 h to remove the sample already

loaded in the column.

11. While switching off the instrument, first switch off injector, detector and oven

but continue the flow of carrier gas till the oven temperature is brought down

to 50 C.

12. To activate the column, allow it to condition at 200–210 C for 4–5 h (In case of

DEGS bleeding temperature is 225 C) to get good resolution.

13. Strictly avoid introduction of moisture in the column through sample, or

solvent or wet glassware.

Chapter 7

Qualitative and Quantitative Estimations

of Amino Acids and Proteins

Proteins are present in the living organisms. They form the structural and functional

basis of the cell, which is the smallest unit of life. The estimation of proteins and

sometimes their constitution of amino acids become necessary for various biochemical and molecular experimentations. Different methods are available for the

estimation of proteins and amino acids. Some of the simple colour reactions of

proteins which could be used as identification tests are given below.


Qualitative Tests for Proteins

See Table 7.1.


Nitrogen Estimation by Micro-Kjeldahl

Method (FAO 1970)

Nitrogen is the major element next to carbon, hydrogen and oxygen found in living

things. Nitrogen occurs in amino acids, purine and pyrimidine bases, vitamins,

aminosugars, alkaloids, compound lipids, etc.; however, the major nitrogen source

is proteins. In most proteins, nitrogen constitutes 16% of the total make-up and

hence, the total nitrogen content of a sample is multiplied by 6.25 to arrive at the

value of the crude protein. By and large micro-kjeldahl technique is adopted to

estimate the total nitrogen content in a variety of samples ranging from microbila

cells to meat. The procedure described here is highly suitable for food samples such

as cereals and pulses flour. In addition, procedures for non-protein nitrogen, protein

nitrogen and amino nitrogen are also presented.

R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,

DOI 10.1007/978-1-4419-9785-2_7, # Springer Science+Business Media, LLC 2011




Qualitative and Quantitative Estimations of Amino Acids and Proteins

Table 7.1 Qualitative test for proteins


Colour of the reaction product

Ninhydrin test

To 4 mL of the solution which Violet or purple colour

should be at neutral pH add

1 mL of 0.1% freshly

prepared ninhydrin

solution. Mix the contents

and boil for a couple of

minutes. Allow to cool

Biuret test

To 2 mL of the test solution and Violet or pink colour

2 mL of 10% NaOH add

two drops of 0.1% CuSO4


Xanthoproteic test

To 5 mL of the solution add

1 mL of conc. HNO3. Boil

the contents. After cooling

add excess 40% NaOH


The ninhydrin test is answered

by amino acids and

proteins. The formation of a

complex called

Rheumann’s purple due to

the condensation of two

molecules of ninhydrin

with one molecular of

ammonia acid is

responsible for the violet

colour. The a-amino group

is the reactive group

Compounds with two or more

peptide bonds give a violet

colour with alkaline copper

sulphate solution

On adding acid, yellow colour The yellow colour is due to the

with be noticed, the when

nitro derivatives of the

NaOH is added deep orange

aromatic amino acids

colour will develop

present in the protein. The

sodium salts of nitro

derivatives are orange in


Sulphur test

To 2 mL of the solution add

Black precipitate

2 mL of 40% NaOH and ten

drops of 2% lead acetate

solution. Boil for a minute

and cool

Glyoxylic test/Hopkins-Cole test for tryptophan

Add 2 mL of glacial acetic acid Violet ring is formed at the


to 2 mL of the test solution.

Then add about 2 mL of

conc. H2SO4 carefully

down the sides of the test

tube. Observe the colour

change at the junction of the

two liquids

Sakaguchi test

To 5 mL of the solution on ice Intense red colour

add 1 mL of 10% NaOH

solution and 1 mL of 0.02%

a-naphthol solution. After

few minutes add ten drops

of alkaline hypobromide


The sulphur in sulphur

containing amino acids of

the proteins in presence of

NaOH, is changed into

Na2S which forms black

lead sulphide when reacted

with lead acetate

The indole group of tryptophan

reacts with the glyoxylic

acid released by the action

of conc. H2SO4 on acetic

acid to give a purple colour

The guanidine group of

arginine reacts with

a-naphthol to form a bright

red colour complex


7.2 Nitrogen Estimation by Micro-Kjeldahl Method

Table 7.1 (continued)


Colour of the reaction product

Modified Millon’s test

To 1 mL of solution add 1 mL Yellow precipitate

of 10% mercuric sulphate in

10% sulphuric acid. Boil

gently for half a minute

Cool under a tap and add a drop Red colour develops

of 1% NaNO2 solution and

warm gently



The yellow precipitate is due to

the precipitation of protein.

Mercury combines with

tyrosine of the protein

The red colour is due to

reaction of the precipitate

with the nitrous acid


The nitrogen in protein or any other organic material is converted to ammonium

sulphate by H2SO4 during digestion. This salt, on steam-distillation, liberates

ammonia which is collected in boric acid solution and titrated against standard

acid. Since 1 mL of 0.1 N acid is equivalent to 1.40 1 mg N, calculation is made to

arrive at the nitrogen content of the sample.


Kjeldahl flasks: 30 mL hard glass flasks

Digestion rack: Commercial heating apparatus

Distillation apparatus: Glass distillation apparatus assembly

Sulphuric acid specific gravity (Sp gr) 1.84

Mercuric oxide

Potassium sulphate

Sodium hydroxide–Sodium thiosulphate solution: Dissolve 600 g of NaOH and

50 g of Na2S2O3Á5H2O in distilled water and make to 1 L

Indicator solution: Methyl red 0.2 g/100 mL ethanol, methylene blue

0.2 g/100 mL ethanol. For mixed indicator, two parts of methyl red solution

are added to one part of methylene blue solution

Boric acid 4% solution

Standard HCl or H2SO4, 0.02 N

Boiling chips


1. Weigh 100 mg of the sample (containing 1–3 mg nitrogen) and transfer to a

30-mL digestion flask.

2. Add 1.9 Ỉ 0.1 g potassium sulphate and 80 Ỉ 10 mg mercuric oxide and 2 mL

conc. H2SO4 to the digestion flask. If sample size is larger than 20 mg dry

weight, 0.1 mL H2SO4 should be added for each 10 mg dry material.



Qualitative and Quantitative Estimations of Amino Acids and Proteins

3. Add boiling chips and digest the sample till the solution becomes colourless.

The time of digestion will vary with regard to the size of the sample, temperature

and the mode of digestion.

4. After cooling the digest, dilute it with a small quantity of distilled ammonia-free

water and transfer the distillation apparatus. When the nitrogen content of the

sample is high, the digest can be made up to a known volume and an aliquot may

be transferred to the distillation flask. The Kjeldahl flask should be rinsed with

successive small quantities of water.

5. Place a 100-mL conical flask containing 5 mL of boric acid solution with a few

drops of mixed indicator with the tip of the condenser dipping below the surface

of the solution.

6. Add 10 mL of sodium hydroxide–sodium thiosulphate solution to the test

solution in the apparatus.

7. Distill and collect the ammonia on boric acid (at least 15–20 mL of distillate

should be collected).

8. Rinse the tip to the condenser, and titrate the solution against the standard acid

until the first appearance of violet colour, the end point.

9. Run a reagent blank with an equal volume of distilled water and subtract the

titration volume from that of sample titre volume.


The nitrogen content of the sample can be calculated based on any one of the

following formulae as the case may be

Ng=kg ¼

ðmL HCl mL blankị normality 14:01


Weight gị

Ng=kg ẳ

mL HCl À mL blankÞ Â normality  14:01  final volume


Weight ðgÞ Â aliquot volume

Non-protein nitrogen

1. Extract a known quantity of powdered material (100 mg) with ice cold 10% TCA

(10 mL). (Proteins are precipitated while non-protein nitrogen gets extracted).

2. Centrifuge, wash the precipitate with TCA, pool all the supernatants and make

up to a known volume (25 or 50 mL).

3. Take an aliquot and distill as described earlier.

4. Titrate against the standard acid, and calculate the nitrogen content. This gives

the percentage of non-protein nitrogen.

Protein nitrogen

Multiplying total nitrogen value with 6.25 will give the crude protein content,

which also includes non-protein nitrogen. To get true protein content, deduct the

non-protein nitrogen from the total nitrogen and then multiply with the factor.

7.2 Nitrogen Estimation by Micro-Kjeldahl Method


Amino nitrogen

1. Estimate the total free amino acid content as per the procedure given elsewhere

in this book.

2. Multiply the percentage equivalent of leucine with 14/131 to get the percentage

of amino nitrogen. If any amino acid other than leucine is used as standard,

introduce the molecular weight of that amino acid in the denominator.


• Care must be taken so as to get a representative and homogeneous sample.

• When greater quantity of sample (500 mg) is used and the digest is diluted before

an aliquot is transferred to the distillation set, care should be taken so that the

actual quantity of sulphuric acid so transferred does not exceed the capacity of

the 10 mL of NaOH–Na2S2O3 solution. The solution being distilled out should

always be strongly alkaline.

• If the test solution is still yellow-coloured even after prolonged digestion,

addition of a few drops of perchloric acid or hydrogen peroxide will ensure

complete oxidation and to get a colourless solution.

• Appropriate factors should be calculated for the acid normalities prepared in the

laboratory based on the following

1 mL 0:1 N acid ¼ 1:401 mg N

• A known concentration of ammonium sulphate solution can be distilled as a

standard check.

• In general, the nitrogen content is multiplied by the factor 6.25 to arrive at the

percentage of crude protein which is based on the assumption that nitrogen

constitutes 16% of a protein. However, the nitrogen percent varies with the

amino acid composition of the proteins. For more refined expression of protein

percentage in samples, different factors are used. These factors were arrived at

by the amino acid composition. Some such factors are given below.

• The method is meant for the conventionally operated distillation apparatus and

does not attempt to give the methodology for automatic distillation sets that are

commercially available. In such cases, the research worker has to follow the

supplier’s manual for specific procedures (Table 7.2).

Table 7.2 Conversion

factors for estimation

of crude protein


Wheat (whole)

Wheat flour

Wheat bran


Rye, barley and oats



Sesame, safflower and sunflower

Milk and cheese

Other foods

Conversion factor














Qualitative and Quantitative Estimations of Amino Acids and Proteins

Sample Preparation for Amino Acid Estimation

Amino acids may be determined colorimetrically, or by chromatography techniques

or in an amino acid analyzer. For estimating the amino acid composition of

foodstuff, feed or any protein, it has to be first hydrolyzed for further estimations.

The free amino acids may be extracted from the tissues or foods in ethanol. If the

extract sample contains higher amounts of salts and/or sugars they have to be

removed before estimation of amino acids.

1. Free amino acids


1. Ethanol

2. 0.01 N HCl


1. Weigh accurately sufficient quantity of the sample, which should have

2–6 mmole of each amino acid.

2. Extract with warm (60 C) 70% ethanol or 0.01 M phosphate buffer, pH 7.0,

3–6 times. Use extractant 5 times the weight of the sample for each


3. Pool the extracts after filtration or centrifugation and evaporate in a rotary

vacuum evaporator to dryness.

4. Take the residue in 1–10 mL of 0.01 N HCl or in a suitable sample diluting


2. Hydrolysis of proteins


1. 6 N HCl

2. 0.01 N HCl


1. Weigh accurately sufficient quantity of the sample containing about 10 mg

protein in a thick-walled heavy glass tube having a constriction at 7.0 cm

from top.

2. Add 5 mL of 6 N HCl and place the tube in liquid nitrogen.

3. Evacuate the frozen sample to 0.01 mmHg.

4. Thaw the sample by shaking and slight warming to allow any dissolved air to

bubble out.

5. Freeze the contents and evacuate to 0.005 mmHg again.

6. Seal the tube at the constriction using a flame till the tube is under evacuation.

7. Place the tube in a hot air oven at 110 Ỉ 1 C for 22 h to hydrolyze proteins.

8. Break open the seal and evaporate the contents in a rotary evaporator to

remove hydrochloric acid.

7.3 Sample Preparation for Amino Acid Estimation


9. Add water and repeat evaporation 2 times.

10. Take the residue in 1–10 mL 0.01 N HCl or 50 mm citrate buffer (pH 2.2).

However, some amino acids may be degraded partly or completely during

acid hydrolysis. Glutamine and asparagines are converted to their respective

acids. Cysteine may be converted to cysteic acid. Serine and threonine are very

rapidly hydrolysed to form the protein and are lost to some extent. Tryptophan is

also partially destroyed during acid hydrolysis.

3. Deproteinization






1% Picric acid

Dowex 1 Â 8 ClÀ Resin

0.02 N HCl

0.01 N HCl


1. Add 50 mL of 1% picric acid to 10 mL of the sample.

2. Centrifuge at 3,000 rpm for 10 min and reserve the supernatant.

3. Place the supernatant in a glass column (2 Â 10 cm) containing Dowex

1 Â 8 ClÀ resin to a height of 3 cm.

4. Immediately after the sample sinks into the resin wash the sides of the column

with 3 mL of 0.02 N HCl.

5. Repeat washing 5 times.

6. Collect all the eluate and evaporate it to dryness.

7. Take the residue in 0.01 N HCl.

Centrifugation of the sample for 30 min at 2,000 g is a better method for

deproteinization. The ultrafiltrate may be used for analysis. Picric acid does

remove some amino acids.

4. Desalting


1. Dower 5 Â 8 (H+) or Amberlite CG-120 (Na+)

2. 2 N NH4OH

3. 0.01 N HCl


1. Load 10–20 mL of sample over the column of resin (2 Â 10 cm) and allow it

to be absorbed by the resin.

2. Elute the amino acids with ammonium hydroxide.

3. Collect the eluate (about 100 mL) and evaporate to remove ammonia.

4. Wash the residue twice with water and repeat evaporation.

5. Take the amino acid residue in 0.01 N HCl.




Qualitative and Quantitative Estimations of Amino Acids and Proteins

Estimation of Total Free Amino Acids

(Moore and Stein 1948; Misra et al. 1975)

Amino acids are the basic building blocks of proteins. Apart from being bound

as proteins, amino acids also exist in the free form in many tissues and are known as

free amino acids. They are mostly water soluble in nature. Very often in plants

during disease conditions, the free amino acid composition exhibits a change

and hence, the measurement of the total free amino acids gives the physiological

and health status of the plants.


Ninhydrin, a powerful oxidizing agent, decarboxylates the alpha-amino acids and

yields an intensely coloured bluish purple product which is colourmetrically

measured at 570 nm.

Ninhydrin ỵ alpha-amino acid ! Hydrindantin ỵ Decarboxylated amino acid

ỵ Carbon dioxide ỵ Ammonia

Hydrindantin þ Ninhydrin þ Ammonia ! Purple coloured product þ water


1. Ninhydrin: Dissolve 0.8 g stannous chloride (SnCl2Á2H2O) in 500 mL of 0.2 M

citrate buffer (pH 5.0). Add this solution to 20 g of ninhydrin in 500 mL of


2. 0.2 M Citrate buffer pH 5.0.

3. Diluent solvent: mix equal volumes of water and n-propanol.


Extraction of amino acid

Weigh 500 mg of the plant sample and grind it in a pestle and mortar with a small

quantity of acid-washed sand. To this homogenate, add 5–10 mL of 80% ethanol.

Filter or centrifuge. Save the filtrate or the supernatant. Repeat the extraction twice

with the residue and pool all the supernatants. Reduce the volume if needed by

evaporation and use the extract of the quantitative estimation of total free amino

acids. If the tissue is tough, use boiling 80% ethanol for extraction.






To 0.1 mL of extract, add 1 mL of ninhydrin solution.

Make up the volume to 2 mL with distilled water.

Heat the tube in a boiling water bath for 20 min.

Add 5 mL of the diluent and mix the contents.

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