thin layer chromatography

THIN LAYER CHROMATOGRAPHY

ORDER OF POLARITY( in general)

HYDROCARBONS <

STEROL ESTERS/WAXES/FATTY ACIDMETHYLESTERS <

TRIGLYCERIDES/TOCOPHEROLS <

FREE FATTY ACIDS /ALCOHOLS <

CHOLESTEROL/DIGLYCERIDES/MONOGLYCERIDES<

PHOSPHOLIPIDS


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.


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.

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.

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.

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.

SOLVENT SYSTEM

SAMPLE SOLVENTS

TG,WAXES,FATTY ACIDS HEXANE-ETHYL ETHER;95:5,90:10,80:20,50:50

PHOSPHOLIPIDS,SULPHOLIPIDS AND GLYCOLIPIDS

CHLOROFORM-METHANOL-H2O; 70:22:3,65:30:5, 65:25:4,60:35:8

STEROLS BENZENE-ETHYL ACETATE; 9:1,2:1

VISUALIZATION AND IDENTIFICATIONS NO

REAGENT COLOUR OF SPOT

SUBSTANCE VISUALIZED

1 DAY OR UV LIGHT VARIOUS COLOURS COLORED COMPOUNDS

2 IODINE VAPOURS BROWNISH- YELLOW BACKGROUND

UNSATURATED LIPIDS,SATURATED NITROGENOUS LIPIDS

3 DICHLORO-FLOUROSCEIN GREEN IN UV NON POLAR LIPID

4 RHODAMINE PURPLE ALL LIPIDS

5 NINHYDRIN RED PURPLE AT

1050 C

AMINOPHOSPHATIDES

6 DRAGENDORFF REAGENT ORANGE CHOLINE

7 DIPHENYL AMINE BLUE-GREY GLYCOLIPID

SEPERATION OF NEUTRAL LIPID

1.PHOSPHOLIPIDS2.FATTY ACIDS3.CHOLESTEROL4.TRIGLYCERIDES5. FAME6.SQUALENE

ARGENTATION TLC

1.Hexaenes2.Pentaenes3.Tetraenes4.Trienes5.Dienes6.Monoenes7.saturates

REVERSED PHASE TLC

1.LAURIC ACID2.MYRISTIC ACID3.PALMITIC ACID4.STEARIC ACID5.MIX 1-46.MIX 7-97.OLEIC ACID8.LINOLEIC ACID9.LINOLENIC ACID

BORIC ACID TLC

1.1-MG2.2-MG3.FATTY ACID4.1,2-DG5.1,3-DG6.TG

TLC OF PHOSHOLIPID

1.PHOSPHATIDIC ACID

2.PHOSPHATIDYL ETHANOL AMINE3.PHOSPHATIDYL INOSITOL

4.PHOSPHATIDYL CHOLINE

thin layer chromatography

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