Qualitative Analysis of Lipids

The qualitative analysis of Lipid is used to detect the presence or absence of lipid on the basis of colour change. Lipids are the organic biomolecules which are soluble in non-polar solvents like chloroform, ether, acetone etc. and insoluble in water.

In nature, there are different varieties of lipids which shows structural diversity among each other. Some common lipids are fatty acids, soaps, fats, oils, waxes, phospholipids etc.

Content: Qualitative Analysis of Lipids

  1. Definition of Qualitative Analysis of Lipids
  2. Methods for Qualitative analysis of Lipids

Definition of Qualitative Analysis of Lipids

The qualitative analysis of lipid involves some preliminary tests and specific tests to detect the presence or absence of lipids and to classify the different groups of lipids based on their chemical reactivity with the chemical reagent. The presence of lipids in the qualitative analysis is measured by the colour change.

Methods for the Qualitative Analysis of Lipids

There are several methods which are used for the qualitative analysis of lipids and its components.

Solubility Test

Solubility test is the preliminary test which detects the presence of all lipids. This test detects the solubility of lipid in various solvents to check whether it is miscible or immiscible in polar or non-polar solvents.

Principle: Solubility test is based on the property of lipid to dissolve in different solvents. Lipids are readily miscible in non-polar solvents like chloroform, partially soluble in a polar solvent like ethanol and immiscible in a polar solvent like water.

Method:

  • Take the sample of lipid in three different test tubes by labelling it as A, B and C.
  • Then add different solvents like water, ethanol and chloroform in three different test tubes.
  • Shake the tubes and allow it to stand for 1 minute.

Observation: Check the solution for whether lipid is soluble or insoluble.

Interpretation of result:

solubility test

Positive result: Lipids are soluble in non-polar solvent i.e. chloroform and partially soluble in ethanol which can solubilize upon heating.

Negative result: Lipids are insoluble in a polar solvent i.e. water.

Translucent Spot Test

A translucent spot test is also a preliminary test for the lipids which can be detected by the appearance of a translucent and greasy spot.

Principle: The lipid will not wet the filter paper, unlike water. The lipid will form a greasy spot as they are having a greasy texture that will penetrate into the filter paper. In contrast to lipid, the spot of water will disappear from the paper whereas the spot of lipid appears as the “Translucent spot”.

Method:

  • Take a filter paper.
  • Add one drop of water on one end and a drop of oil or lipid on the other end.

Observation: Observe the appearance of a translucent spot on the filter paper.

Interpretation of result:

translucent test

Positive result: Translucent spot will appear on the filter paper.

Negative result: Translucent spot will not appear on the filter paper.

Emulsification Test

Emulsification test is used to detect the presence of lipids.

Principle: Emulsification is the process which stabilizes the water and oil emulsion, by the help of emulsifying agents. The lipid or oil in water appears on the top of the water because of the high surface tension of water which gets together to form a separate layer. On the addition of emulsifying agents like bile salts, soap etc.

Emulsifying agents emulsify the lipid by which the lipid appears as the tiny droplets suspended in the solution.

Method:

  • Take two test tubes and label it as test tube A and test tube B.
  • Add oil to each of the test tubes.
  • Shake the test tube and allow it to stand for about two minutes.

Observation: Observe the test tube for the appearance of tiny droplets in the suspension of liquid.

Interpretation of result:

emulsification test

Positive result: It gives a permanent or stable emulsion of lipid and water.

Negative result: Oil in water emulsion will form at the top, due to the high surface tension of water.

Saponification Test

Principle: It is based on the “Saponification reaction”, where the triglycerides of lipid react with an alkali NaOH and produce soap and glycerol in the presence of ethanol. This reaction also refers to as “Alkaline hydrolysis of esters”.

Method:

  • Take a sample of lipid in a test tube.
  • Then add strong alkali NaOH.
  • Then boil the solution in a water bath for 5 minutes.
  • At last, add ethanol.

Observation: Observe the test tube for the appearance of froth.

Interpretation of result:

saponification test

Positive result: Froth appears in the test tube.

Negative result: Froth does not appear in the test tube.

Sudan IV Test

Sudan IV test is used to detect the presence of lipid in a solution.

Principle: This test is based upon the principle of binding and solubility of lipid to non-polar compounds. As Sudan IV is a non-polar stain, therefore the lipid will bind with it and retain the colour of the stain and gives a red-orange colour. Sudan IV does not stain or binds to the polar compounds.

Method:

  • Take 1ml of the lipid sample in a test tube.
  • Then add 1-2 drops of Sudan IV to the solution.

Observation: Observe the tube for the appearance of red-orange colour to the solution.

Interpretation of result:

sudan IV test

Positive result: Gives red-orange colour to the solution.

Negative result: The solution to the colour will remain unchanged.

Acrolein Test

Acrolein test is used to detect the presence of glycerol and fat.

Principle: This test is based on the “Dehydration reaction”, where the water molecules removed from the glycerol by the addition of reagent potassium hydrogen sulphate. The reaction between glycerol and potassium hydrogen sulphate results in the formation of “Acrolein” that is characterized physically by the release of the pungent smell.

Method:

  • Take 1ml of the lipid sample in a test tube.
  • Add crystals of potassium hydrogen sulphate.
  • Heat the solution for a few minutes.

Observation: Observe the test tube for the pungent smell.

Interpretation of result:

Acrolein test

Positive result: If glycerol present in the sample it will give a pungent smell.

Negative result: If glycerol is absent in a sample, then it will not produce a pungent smell.

Dichromate Test

Dichromate test is also used to detect the presence of glycerol.

Principle: It is based on the principle of “oxidation reaction”. In this, glycerol and dichromate ions react to give a brown colour to the solution. Then the chromic ions oxidize the glycerol and are reduced to chromous ions in the presence of nitric acid giving a blue colour to the solution.

Method:

  • Take 2-3ml of a sample in a test tube.
  • Then add a few drops of 5% potassium dichromate solution.
  • After that, add 5 ml of concentrated nitric acid.

Observation: Observe the test tube for the appearance of a blue colour.

Interpretation of result:

Dichromate test

Positive result: If the colour of the solution changes from brown to blue, then it indicates the presence of glycerol.

Negative result: In this, the brown colour will not change into blue.

Tests for the Free Fatty Acids

Principle: This is based on the “Neutralization reaction” where the alkali is neutralized by the addition of free fatty acids in the lipids.

Method:

  • Add phenolphthalein solution in a test tube.
  • Then, add dilute alkali to the above solution which will give pink colour to the solution.
  • At last, add a lipid sample.

Observation: Observe the tube for the disappearance of the pink colour by the addition of lipid.

Interpretation of result:

free fatty acid test

Positive result: If the pink colour disappears by the addition of sample, then it indicates the presence of free fatty acids in the sample.

Negative test: The pink colour will not disappear.

Unsaturation Test

Unsaturation test is used to detect the presence of unsaturated fatty acids or the amount of double bond in a lipid sample.

Principle: All the neutral fat contains glycerides of fatty acids. Double bond found in the structure of unsaturated fatty acids which becomes saturated by taking up either bromine or iodine. If the lipid contains more unsaturated fatty acids or more double bonds that means, it will take more iodine.

Method:

  • Take 5ml of chloroform and 5ml of Huble’s iodine reagent in a beaker which will give pink colour to the solution.
  • Add lipid sample drop by drop and shake vigorously, until the pink colour disappears.

Observation: Count the number of drops added to the solution of chloroform and Huble’s iodine solution till the pink colour disappears. The number of drops determines the taking up of iodine by the unsaturated fatty acid of lipids.

Interpretation of result:

Unsaturation test

Positive result: Pink colour will disappear by the addition of unsaturated fatty acids.

Negative result: Pink colour will not disappear.

Burchard Test

Burchard test was first given by the scientist “Liebmann” to detect the presence of cholesterol.

Principle: Cholesterol reacts with the strong concentrated acid i.e. sulphuric acid and acetic anhydride. Sulphuric acid and acetic anhydride act as a dehydrating and oxidizing agent.

Method:

  • Take crystals of cholesterol in a test tube.
  • Then add 2ml of chloroform to dissolve the cholesterol.
  • Add 10 drops of acetic anhydride in a solution and then 2-3 drops of concentrated sulphuric acid.

Observation: Observe the test tube for the appearance of a bluish green colour.

Interpretation of result:

Burchard test

Positive result: It indicates the presence of cholesterol in a sample by giving bluish-green colour to the solution.

Negative result: It is indicated by no colour change.

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