PROCEDURE RESULT PHOTO DOCUMENTATION DISCUSSION CONCLUSION

A. LIEBERMANN-BURCHARD TEST

1. In one test tube containing a small pinch of cholesterol, add chloroform drop by drop until it is dissolved.

2. Add 15 drops of acetic anhydride, mix and cool.

3. Add 5 drops of concentrated sulfuric acid, mix carefully and note the color changes.

B. SALKOWSKI TEST

1. In the other test tube containing a small pinch of cholesterol, add chloroform drop by

The solution has a white cloudy color.

The solution turned to a deep blue-green color.

After:

The Liebermann-Burchard or acetic anhydride test is used for the determination of cholesterol, and in a clinical setting, it s used to determine the serum cholesterol.

When the chloroform solution of cholesterol was mixed with acetic anhydride and sulfuric acid, a characteristic green or deep-blue green was produced. The intensity of the color is proportional to the amount of steroid present. The color is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents and increasing the conjugation of the unsaturation in the adjacent fused ring.http://www.uobabylon.edu.iq/uobColeges/ad_downloads/5_29708_805.pdf

(MN Chatterjea, 2012: Textbook of Medical Biochemistry)

Based on the diagram, cholesterol is first attacked by strongly acid reagent. Such reagents first remove a molecule of water, then oxidize the intermediate to produce -3,5 cholestadiene (two double bonds) or its cations. The cholestadiene reacts further to form cholestapolyne carbonium ion. The stabilities of these cations are dependent on the sulfuric acid concentration In the Liebermann-Burchard reaction, the polyenes are the main chromopheres. In the presence of more concentrated sulfuric acid and ferric ion , the polyene cat ion are the main chromophores . Therefore , depending on the relative concentration of the sulfuric acid, and the presence or absence of ferric ion , one obtains either a green colour (Liebermann -Burchard) due to cholestapolyene sulfonic acid, or a red colour due to the formation of a

Liebermann-Burchard and Salkowski reactions are based from dehydrating, oxidizing and sulphonating properties of the reagents, sulfuric acid and acetic anhydride, to cholesterol which has a structural formula of,

The hydroxyl group in cholesterol is responsible for the result in Liebermann-Burchard test while the double bond present in one cholesterol ring is responsible for the positive result for Salkowski test. These tests are used for qualitative and quantitative determination of cholesterol in serum.

drop until it is dissolved.

2. Incline the tube and add slowly down the side of the test tube 5 drops of concentrated sulfuric acid.

3. Let stand slowly and observe the color produced at the juncture of the two liquids.

A red to crimson color was produced at the lower layer and the upper chloroform layer.

Before: cholestapolyene carbonium ion (iron - salt - acid reaction) .http://www.uobabylon.edu.iq/uobColeges/ad_downloads/5_29708_805.pdf

Salkowski test is a test for cholesterol. A positive reaction for this test is the development of red to crimson color at the upper and lower layers. This test indicates the presence of double bond in one cholesterol ring is responsible for its ability to form colored products in the presence of inorganic acids and it should yield a layering of colors.

Cholesterol is dehydrated to form cholesta-3,5-diene. Later, it is oxidized and two molecules are linked to form bischolesta-3,5-diene. It is later sulphonated by sulfuric acid to form mono and di-sulphonic acids, which are colored products. If excess of sulphuric acid is added and reaction mixture contains ferric ions, disulphonic acids are predominantly formed and give red color.Reference: Ashok Kumar J, Textbook of Biochemistry for Nurses