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.
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
