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THIN LAYER CHROMATOGRAPHY

 ORDER OF POLARITY( in general)


 HYDROCARBONS <
 STEROL ESTERS/WAXES/FATTY ACIDMETHYLESTERS <
 TRIGLYCERIDES/TOCOPHEROLS <
 FREE FATTY ACIDS /ALCOHOLS <
 CHOLESTEROL/DIGLYCERIDES/MONOGLYCERIDES<
 PHOSPHOLIPIDS
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.
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.
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,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
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
SEPERATION OF NEUTRAL LIPID



                 1.PHOSPHOLIPIDS
                 2.FATTY ACIDS
                 3.CHOLESTEROL
                 4.TRIGLYCERIDES
                 5. FAME
                 6.SQUALENE
ARGENTATION TLC

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


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

      1.PHOSPHATIDIC ACID

      2.PHOSPHATIDYL ETHANOL
      AMINE
      3.PHOSPHATIDYL INOSITOL

      4.PHOSPHATIDYL CHOLINE

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Thin layer chromatography

  • 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