Biochemistry and Metabolism

Experiment 5

Introduction

Qualitative tests of lipids Lipids Lipids are a heterogeneous group of compounds, including fats, oils, steroids, waxes, and related compounds, which are related more by their physical than by their chemical properties. Lipids are a class of compounds distinguished by their insolubility in water and solubility in non polar solvents. Lipids are important in biological systems because they form the cell membrane, a mechanical barrier that divides a cell from the external environment. Non polar lipids, such as triglycerides, are used for energy storage and fuel. Polar lipids, which can form a barrier with an external water environment, are used in membranes. Polar lipids include glycerophospholipids and sphingolipids. Fatty acids are important components of all of these lipids. Lipids have the common property of being relatively insoluble in water and soluble in nonpolar solvents such as ether and chloroform. They are important dietary constituents not only because of their high energy value but also because of the fat-soluble vitamins and the essential fatty acids contained in the fat of natural foods.

Saturated fat

Saturated fats contain only single bonds within the carbon chain. Saturated fats are of animal origin and are usually present in solid form. It increases the blood cholesterol level. Some examples are meat fat, butter etc. Coconut oil and palm oil also contain saturated fat.

Unsaturated fat

Unsaturated fats contain double bonds within the carbon chain. Unsaturated fat is found in fish like salmon and tuna, nuts, seeds etc.

Here we discussed about 4 testes.

1.Solubility test

2. Saponification test

3. Translucent Spot test

4. Acrolein test

1.Solubility test

Principle

This test is used to know the solubility of lipids in some solvents, according to polarity feature lipids are insoluble in polar solvents because lipids are non polar compounds, thus lipids are soluble in non polar solvents like chloroform, benzene and iso-propyl alcohol.

Materials required

Refind oil, Ghee, Distilled water, test tubes, pipette and tips, isopropyl alcohol

Interpretation

1. When given samples were mixed with Iso-propyl alcohol and found soluble in Iso-propyl alcohol, that indicates presence of fat in samples.

2. When given samples were mixed with distilled water and found immiscible in d.w. that indicates presence of fat in samples.

2. Saponification test

Principle

This test is used to form soap by saponification process which includes the hydrolysis of fat or oil with alkaline to yield glycerol and salt of fatty acid (soap).

Triglyceride + alkaline KOH--------------> Glycerol +Soap

Materials required

Refind oil, Desi Ghee, Distilled water, test tubes, pipette and tips, 20% alcoholic potassium hydroxide , Boiling water bath, Dropper

Procedure

1. Take two test tubes and respectively add 2 ml of ghee and olive oil in different test tubes.

2. Add 4 ml of 20% alcoholic potassium hydroxide.

3. Shake well and boil the mixture in boiling water bath for 5 minutes.

4. Make sure after the five minutes that the soap is formed, all of the fat is converted to soap.

5. Now add 5 ml of distilled water and shake the tube to observe soap formation.

6. The solid material formed is soap.

Interpretation

The solid material formed is soap and that indicates presence of fat samples.

3. Translucent Spot test

Principle

Fats and oils have higher boiling points so at room temperature they cannot absorb enough heat to evaporate. When fat or oil is place on a sheet of paper, it diffracts light. The diffracted light can pass from one side of the paper to another side and produces a translucent spot.

Materials required

Refind oil, Desi Ghee, Distilled water, test tubes, pipette and tips, Whatmann filet paper , Dropper

Procedure

1.Place a little of the substance (i.e. oil, ghee and distilled water) on a filter paper and press it on the folds of the filter paper and unfold the filter paper.

observation

Interpretation

On unfolding the filter paper, the appearance of translucent spot on the filter paper indicates the presence of fats in given samples .

4. Acrolein test

Principle

Acrolein test is used to detect the presence of glycerol or fat. When fat is treated strongly in the presence of a dehydrating agent like potassium bisulphate (KHSO4), the glycerol portion of the molecule is dehydrated to form an unsaturated aldehyde, acrolein that has a pungent irritating odour.

Materials required

Refind oil, Desi Ghee, Distilled water, test tubes, pipette and tips, potassium bisulphite , Dropper, Burner

Procedure

1. Take 3 clean glass test tubes.

2. Add samples with spatula in respective labelled test tubes.

3. Heat a little of the sample of refined oil, desi ghee and vansapati ghee with some crystals of potassium bisulphate in a test tube.

4. Observe for pungent odour of acrolein .

Observation

Interpretation

A pungent irritating odour of acrolein indicates presence of fats in oil and ghee.

Reference Material

1. Qualitative test by amrita lab

2. Qualitative tests of lipids

Questions

1. List the organic solvents used for solubility test?

2. The solubility test gives us an idea about ---------- of sample with its examples.

3. Enlist other qualitative tests for fats and lipids.