Lab Activity 7 Separation of blood serum lipids by thin-layer chromatography IUG, 2016-2017 Dr. Tarek Zaida

Chromatography Definition A physical method of separation in which the components to be separated are distributed between two phases: A stationary phase and a mobile phase that moves in a definite direction.

Types of Chromatography Based on stationary phase Column Planar Based on mobile phase Liquid Gas

Planar Chromatography A separation technique in which the stationary phase serves as a plane. The plane can be either a paper (paper chromatography) or a layer of solid particles spread on a support such as a glass or a plastic plate (thin layer chromatography).

Thin Layer Chromatography (TLC) Principle Different compounds in sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.

Measuring Rf

Separation of Blood Serum Lipids by Thin-Layer Chromatography(TLC)

Lipids Lipids are organic compounds formed mainly from alcohol and fatty acids combined together by ester linkage. They are nearly insoluble in water, but soluble in organic solvents such as ether, benzene, chloroform, etc.

Lipids Thy are carried in the body by lipoproteins Subdivided into 4 main groups based on their chemical structure Fatty acids Cholesterol Triglycerides Phospholipids

Lipids Triglyceride

Principle The method is based on different migration rates of blood serum lipid fractions: Phospholipids Free fatty acids Cholesterol Glycerides In a thin layer of adsorbent as an organic solvent moves through it.

Principle The rate of fractions separated is dependent upon their relative polarities. A iodine vapor is then used to identify the separated fractions.

Reagents & materials Ethanol/diethyl ether mixture (3:1) Chloroform N-hexane/diethyl ether/acetic acid mixture (73:25:2) serves as a chromatography solvent Metallic iodine Thin layer chromatography plates silufol UV254 A chromatography chamber A water bath A development chamber A chromatogram viewing cabinet Blood serum sample

Procedure Mix 1 ml of blood serum with 10 ml of ethanol/ether mixture in a standard flask of 25 ml. Place the flask for 30 sec in a boiling water bath, then cool it under a stream of running tap water and make up to the mark with ethanol ether mixture. Filter the lipid extract obtained through a filter paper into a wide-mouth test tube, evaporte the contents to dryness in a boiling water bath, dissolve the dry residue in 0.2 ml chloroform.

4. Pour 2-3 ml of solvent into the chromato-graphy chamber. 5. Apply 0 4. Pour 2-3 ml of solvent into the chromato-graphy chamber. 5. Apply 0.01 & 0.02 ml of chloroformic extract to a chromatograghy plate (leaving 1 cm space between the 2 spots. 6. Cover the chromatography chamber with the lid to keep the vapour concentration at a constant level.

7. Allow the chromatography to proceed at RT until solvent front has reached 1cm from the top edge. 8. Dry the chromatography plate in the air. 9. Place some crystalline metallic iodine on the bottom of the development chamber and transfer the chromatography plate to the development chamber.

10. Cover the development chamber with the lid and heat the chamber in a water bath at 60 c to allow the sublimed iodine vapor to react with the chromatographically separated lipid fractions. 11. Keep the development reaction to run for 1 min. 12. Examine the chromatogram in UV light in the viewing cabinet.

13. Note the separated lipid fractions to appear as brown spots in the developed chromatogram. 14. The separated fractions are positioned from the start line in the order: Phospholipids, nonidentified compounds, cholesterol, monoglycerides, diglycerides, free fatty acids, triglycerides, and cholesterol esters.