Qualitative and Quantitative Tests for Lipids

Read this article to learn about the qualitative and quantitative tests for lipids.

A large number of heterogenous compounds are referred to as lipids including fats, steroids, waxes, and related compounds, which are related more by their physical than their chemical prop­erties.

They have the common property of being:

(1) Relatively insoluble in water and

(2) Soluble in polar solvents such as ether and chloroform.

Fatty acids are aliphatic carboxylic acids. If the aliphatic chain contains no double bond then it is called saturated and if it contains one or more double bond it is called unsaturated. Most naturally occurring unsaturated fatty acids have cis-double bonds. Some of the most common fatty acids are palmitic acid and stearic acid. Palmitic has 16 carbon atoms and stearic has 18 carbon atoms.

As it is clear from the formulae, both are saturated fatty acids. Some fatty acids like oleic acid may be unsaturated. Naturally occurring animal fats consist largely of mixed glyceride of oleic, palmitic and stearic acids. They are usually mixture of individual fats. Fats have more saturated fatty acids whereas oils have more of unsaturated ones.

Lipids are simple, complex or derived. Simple lipids are esters of fatty acids with various alcohols, e.g., fats (esters of fatty acids with glycerol) and waxes (esters of fatty acids with higher molecular weight of monohydric alcohols). Complex lipids are esters of fatty acids containing groups in addition to an alcohol and a fatty acid, e.g., phospholipids or glycolipids etc. Derived lipids include fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, and ketone bodies, lipid soluble vitamins, and hormones.

Phospholipids yield in addition to alcohol and fatty acids, phosphate and a nitrogenous base like choline, ethanolamine, etc. Lecithin’s and cephalous are representatives of the phospholipids. Similarly glycolipids contain carbohydrates, and sulpholipids contain sulphate. Lipoproteins are com­binations of lipids with proteins.

Now we will consider some qualitative and quantitative tests for lipids.

Qualitative Tests:

I. Physical Test:

1. Grease spot test:

Take a small amount of oil on a piece of paper, a greasy spot penetrating the paper will be formed. This happens because lipid does not wet paper unlike water.

2. Test for free fatty acids:

Take a few drops of phenolphthalein solution in a test tube and add to it one or two drops of very dilute alkali solution, just sufficient to give the solution a pink colour. Now add a few drops of the oil and shake. The colour will disappear as the alkali is neutralized by the free fatty acids present in the oil.

3. Emulsification:

Oil or liquid fat becomes finely divided and is dispersed in water when shaken with water to form emulsification. Emulsification is permanent and complete in the presence of emulsifying agent. The important emulsifying agents are bile salts, proteins, soaps, mono- and diglycerides. Emulsification is important in the processes of fat digestion in the intestine. Emulsifying agents lower surface tension of the liquid.

Take 2 clean and dry test tubes, in one test tube added 2 ml water and in other 2ml dilute bile salt solution. Now to each tube added 2 drops of mustard oil and shaken vigorously for about one minute. Allow the tubes to stands for two minutes and note that the water, oil is broken in small pieces and floats on the surface; where as in the bile salt solution, the oil can be seen in minute droplets suspended in the liquid (permanent emulsification).

4. Saponification test:

Esters can be hydrolysed by alkali to yield the parent alcohol and salt. When the fatty acid possesses a long chain the salt formed is a soap which we commonly use. This process is called saponification. Oils and fats usually contain long chain fatty acids and are, therefore, the starting materials for the preparation of soap.

Take 1 ml of the oil in a test tube and add an equal amount of alcoholic KOH solution, mix them thoroughly and keep the mixture during the course of warming and shake up gently with a little distilled water. Appearance of some oil drops will indicate the incomplete saponi­fication. After complete saponification no oil drops will appear.

5. Tests for unsaturation of fatty acids:

Unsaturated fatty acids like oleic acid can react with halogens like bromine and iodine due to presence of double bonds as shown below.

CH₃ (CH₂)₇CH = CH (CH₂)₇COOH + Br₂ → CH₃ (CH₂)₇CHBr-CHBr (CH₂)₇COOH

The amount of Br₂ or I₂ taken up will indicate the amount of unsaturation present in a particu­lar acid. Approximate idea about the unsaturation in a different oils and fats can be obtained by the following test. Set up four clean and dry test tubes each containing 5 ml of CCl₄.

To the first, add one drop of shark liver oil, to the second, one drop of coconut oil, to the third, a drop of vegetable ghee and add nothing to the fourth tube. Now test for the unsaturation of the added oil by adding bromine water drop by drop to each tube followed by shaking.

Record the number of drops required to obtain a permanent yellowish red colour in each tube and infer the relative unsaturation in the three samples used. It may be mentioned here, vegetable ghee is prepared by hydrogenating vegetable oil. Hydrogenation means saturation of unsaturated fatty acid by hydrogen.

6. Isolation of free fatty acids from soap:

Take a few ml of 20% H₂SO₄ in a test tube and gradually add 5 ml of some soap solution. The fatty acids will separate out in a distinct layer due to the hydrolysis of the soap.

RCOONa + H₂O → RCOOH + NaOH

Cool the solution which will become hot and skim off the surface layer and wash it several times with water till free from H₂SO₄. Then dissolve it in some water and add alkaline phenolphthalein solution and shake. The pink colour will be discharged indicating the presence of free fatty acids.

Calcium soap formation:

To a small amount of the soap solution in a test tube add CaCl₂ solution. A white precipitate will be formed. The white precipitate is due to insoluble calcium salt of fatty acid. This is referred to as calcium soap.

Lead soap formation:

To a small amount of the soap solution in a test tube add lead acetate solution, a white precipitate will appear. The white ppt is due to insoluble lead salt of fatty acids. This is referred to as lead soap.

7. Tests for Glycerol:

I. Acrolein test:

Take pure glycerol in a dry test tube; add to it a few crystals of potassium hydrogen sulphate. Warm gently to mix and then heat strongly. A very pungent odour of acrolein is produced. Acrolein is formed due to removal of water from glycerol by potassium hydrogen sulphate.

II. Dichromate Test:

Take in a dry test tube 3 or 4 ml of glycerol solution, to it add a few drops of 5% potassium dichromate solution and 5 ml of conc. HNO₃, mix well and note that the brown colour is changed to blue. This test is given by the substances containing primary and secondary alcohol groups. The chromic ions oxidize the glycerol and in this process they are reduced to chromous ions which give the blue colour. This test is also given by reducing sugars, so before confirming glycerol be sure that the reducing sugars are not present.

Quantitative Tests:

1. Determination of Iodine Number:

The iodine number of a fat is the amount in gm. of iodine taken up by 100 gm. of fat. Not only iodine but also equivalent amounts of other halogens will add at double bonds; so bromine is often used instead of iodine because it is more reactive. The halogenating reagent used in this method is pyridine sulphate di-bromide. This reagent can be prepared by adding carefully 8.1 ml pyridine in 20 ml glacial acetic acid and making the volume up to 1 litre with glacial acetic acid.

Weigh the bottle containing sample of oil plus a medicine dropper and then transfer about 0.1 to 0.3 gm. of oil to a flask. Reweigh the bottle containing oil and dropper to find out the exact quantity of the sample transferred. Add 10 ml of chloroform and then 25 ml of the pyridine sulphate di-bromide reagent.

Shake thoroughly; allow standing for 5 minutes and then determining the residual bromine. To do this, add 10 ml of 10% KI and titrate the equivalent amount of iodine liberated by the residual bromine with the help of 0.1 (N) Na₂S₂O₃ (sodium thiosulphate). The titration can be done by adding sodium thiosulphate solution through a burette to the flask.

When the colour of the solution in flask becomes light yellow add 1 ml of starch solution. It will become blue. Slowly add the thiosulphate solution again till it becomes colourless. Note the total volume of thiosulphate used.

The total amount of bromine originally added is found by titrating 25 ml of the pyridine sulphate di-bromide reagent with thiosulphate after adding KI as in the previous case. The amount of bromine taken up by the fat sample can be determined by the difference between the two titers and then the iodine number can be calculated.

Suppose with a sample of 0.2 gm. oil the data obtained are as follows:

0.1 (N) Na₂S₂O₃ used for titration of blank = 47.0 ml

0.1 (N) Na₂S₂O₃ used for titration of sample = 27.0 ml

0.1 (N) Na₂S₂O₃ equivalent to iodine absorbed by the sample = 20.0 ml

As 1 ml 0.1 (N) Na₂S₂O₃ = 1.0 ml of 0.1 (N) Bromine = 1 ml of 0.1 (N) Iodine

Hence, 20 ml of 0.1 (N) Na₂S₂O₃ = 20 ml of 0.1 (N) Iodine = 20×12.7/1000 gm Iodine = 0.254 gm Iodine.

Thus 0.2 gm of oil can take up 0.254 gm of iodine.

Therefore, iodine number of oil used = 127.

Qualitative Test of Cholesterol:

Cholesterol is a lipid with a structure quite different from that of phospholipids. It is a steroid, built from four linked hydrocarbon rings. A hydrocarbon tail is linked to the steroid at one end, and a hydroxyl group is attached at the other end. In membranes, the molecule is oriented parallel to the fatty acid chains of the phospholipids, and the hydroxyl group interacts with the nearby phospholi­pid head groups.

Cholesterol is absent from prokaryotes but is found to varying degrees in virtually all animal membranes. It constitutes almost 25% of the membrane lipids in certain nerve cells but is essentially absent from some intracellular membranes.

It is found in bile and a common constituent of gall stones.

The main test for cholesterol is known as Liberman-Burchard test. This is carried in the follow­ing way. In a dry test tube take a small amount of solution of cholesterol in chloroform. Add 1 ml of acetic anhydride and 1 drop of conc. H₂SO₄. Mix and observe that a purple colour is formed which soon changes to green. It may take 15-30 min for full development and it is advisable to put the tube in dark during this time.

Quantitative Estimation of Cholesterol:

The above mentioned qualitative test has been developed quantitatively for the estimation of cholesterol.

Follow the following protocol for the purpose:

Shake the tubes well and keep them at room temperature for 30 minutes. Blue colour will develop in all the tubes except blank tube. Measure the absorbencies at 625 m|a. against the blank tube and plot these against the amount of cholesterol.

Note: Acetic anhydride-sulphuric acid reagent.

This reagent has to be freshly prepared before use. Acetic anhydride (20 ml) is taken in a glass stoppered flask which is then chilled in ice water. When cold, add 1 ml of conc. H₂SO₄ to it drop by drop. The contents are mixed and cooled during the addition. After completion of the addition the flask is stoppered and shaken vigorously for a few minutes. The solution has to be kept cold in ice and should be used within an hour.

Enzymatic Methods:

Assays have been developed in which cholesterol oxidase obtained from the bacterium Nocardia erythropolis is used to convert cholesterol into cholest-4-en-3-one with the formation of Hydrogen peroxide. The cholest-4en-3-one formed has been measured by reading at 240 nm after extracting into isopropanol. Alternatively, hydrogen peroxide has been quantified by formation of chelate complex with quadrivalent titanium and xylenol orange.

Other Tests for Cholesterol:

1. Salkowski’s Test (H₂SO₄ Test):

Dissolve cholesterol in 2 ml of chloroform in dry test tube. Add equal amount of con. H₂SO₄. Shake gently. The upper layer turns red and the sulphuric acid layer shows a yellow colour with a green fluorescence.

2. Formaldehyde-H₂SO₄ Test:

Add 2 ml of formaldehyde-sulphuric acid solution (1 part of 40% formaldehyde to 50 parts of the acid) to 2 ml of chloroform solution in a dry test tube. The cherry colour is developed in the chloroform. Pour off the chloroform in another test tube and add 2-3 drops of acid anhydride. The blue colour develops.