Thin layer chromatography (TLC) is a simple, inexpensive technique used to separate and identify components in organic compounds. In TLC, compounds are distributed between a stationary phase, like silica gel, and a mobile phase, like a solvent or solvent mixture. As the mobile phase travels up the plate, different components travel at different rates depending on their solubility, allowing separation. Common adsorbents and solvent systems used for lipid separation are described. Various detection reagents can be used to visualize separated components under UV light or by producing colored spots. Examples are given of separations achieved for different lipid classes and subclasses using TLC.

1. THIN LAYER CHROMATOGRAPHY
 ORDER OF POLARITY( in general)
 HYDROCARBONS <
 STEROL ESTERS/WAXES/FATTY ACIDMETHYLESTERS <
 TRIGLYCERIDES/TOCOPHEROLS <
 FREE FATTY ACIDS /ALCOHOLS <
 CHOLESTEROL/DIGLYCERIDES/MONOGLYCERIDES<
 PHOSPHOLIPIDS

2. THIN LAYER CHROMATOGRAPHY
 TLC IS A SIMPLE,INEXPENSIVE TECHNIQUE USEFUL FOR
IDENTIFICATION OF VARIOUS COMPONENT PRESENT IN AN
GIVEN ORGANIC COMPUND.
 IN TLC COMPOUND TO BE SEPERATED ARE DISTRIBUTED
BETWEEN A STATIONARY PHASE AND AMOBILE
PHASE.DIFFERENT DISTRIBUTION GIVE RISE TO
SEPERATION.
 STATIONARY PHASE IS A SOLID SUBSTANCE LIKE SILICA
GELOR ALUMINA THAT ABSORBS MOLECULE OF THE
COMPOUND TO BE SEPERATED.
 MOBILE PHASE IS A SOLVENT OR MIXTURE OF A SOLVENTS
THAT FLOW PAST STATIONARY PHASE THUS AFFECTING
PARTITION OF THE MOLECULES OF THE COMPOUND
BETWEEN STATIONARY PHASE AND MOBILE PHASE.

3. THIN LAYER CHROMATOGRAPHY
AS THE SOLVENT SLOWLY TRAVELS
UP THE PLATE ,DIFFERENT
COMPONENTS PRESENT IN THE
MIXTURE TRAVEL AT A DIFFERENT
RATES DEPENDING ON THEIR
DIFFERENCE IN SOLUBILITY OF THE
COMPONENTS IN MOBILE PHASE.

4. ADSORBENTS
 THERE IS A BROAD SPECTRUM OF TESTED
ABSORBENTS FOR TLC
 COMMON ADSORBENTS USED FOR SEPERATION OF
LIPIDS IS A SILICA GELCONTAINING 15% CaSO4 AS A
BINDER.
 SILICA GEL CONTAINING ACID,BASES AND SALTS ARE
USEDE FOR SPECIAL PURPOSE.
 DIATOMACEOUS EARTH(KIESULGUHR) IS USED FOR
SEPERATION OF POLAR LIPIDS.
 ALUMINA IS SUPERIOR FOR SEPERATION OF
VITAMINS,HYDROCARBONS,BUT IS RARELY USED
BECAUSE IT CAUSES HYDROLYSIS OF ESTER LINKAGE
AND ISOMERIZATION OF DOUBLE BONDS.

5. ADSORBENTS
 SILICA GEL IMPREGNATED WITH SILVER
NITRATE/BORON IS COMMONLY USED FOR SEPERATION
OF UNSATURATED COMPOUNDS.
 SEPERATION IS BASICALLY ON THE ABILITY OF
COMPONENTS OF COMPOUND TO FORM COMPLEX WITH
Ag+ IONS.
 REVERSED PHASE TLC IS USED IN SEPERATION OF
COMPOUNDS ACCORDING TO NO OF C ATOMS.

6. SOLVENT SYSTEM
 THE CHOICE OF SOLVENTS DEPEND UPON THE TYPE OF
SEPERATION DESIRED.
 SOLVENT MIXTURES,RATHER THAN INDIVIDUAL
SOLVENTS,ARE USUALLY APPLIED,BUT THE MIXTURE
SHOULD BE KEPT AS SIMPLE AS POSSIBLE FOR BETTER
REPRODUCIBILTY.

7. SOLVENT SYSTEM
SAMPLE SOLVENTS
TG,WAXES,FATTY ACIDS HEXANE-ETHYL
ETHER;95:5,90:10,80:20,50
:50
PHOSPHOLIPIDS,SULPHOLIP CHLOROFORM-METHANOL-
IDS AND GLYCOLIPIDS H2O; 70:22:3,65:30:5,
65:25:4,60:35:8
STEROLS BENZENE-ETHYL ACETATE;
9:1,2:1

8. VISUALIZATION AND IDENTIFICATION
S REAGENT COLOUR OF SUBSTANCE
NO SPOT VISUALIZED
1 DAY OR UV LIGHT VARIOUS COLORED
COLOURS COMPOUNDS
2 IODINE VAPOURS BROWNISH- UNSATURATED
YELLOW LIPIDS,SATURATE
BACKGROUND D NITROGENOUS
LIPIDS
3 DICHLORO-FLOUROSCEIN GREEN IN UV NON POLAR LIPID
4 RHODAMINE PURPLE ALL LIPIDS
5 NINHYDRIN RED0 PURPLE AT AMINOPHOSPHATI
105 C DES
6 DRAGENDORFF REAGENT ORANGE CHOLINE
7 DIPHENYL AMINE BLUE-GREY GLYCOLIPID

9. SEPERATION OF NEUTRAL LIPID
1.PHOSPHOLIPIDS
2.FATTY ACIDS
3.CHOLESTEROL
4.TRIGLYCERIDES
5. FAME
6.SQUALENE

10. ARGENTATION TLC
1.Hexaenes
2.Pentaenes
3.Tetraenes
4.Trienes
5.Dienes
6.Monoenes
7.saturates

11. REVERSED PHASE TLC
1.LAURIC ACID
2.MYRISTIC ACID
3.PALMITIC ACID
4.STEARIC ACID
5.MIX 1-4
6.MIX 7-9
7.OLEIC ACID
8.LINOLEIC ACID
9.LINOLENIC ACID

12. BORIC ACID TLC
1.1-MG
2.2-MG
3.FATTY ACID
4.1,2-DG
5.1,3-DG
6.TG

13. TLC OF PHOSHOLIPID
1.PHOSPHATIDIC ACID
2.PHOSPHATIDYL ETHANOL
AMINE
3.PHOSPHATIDYL INOSITOL
4.PHOSPHATIDYL CHOLINE