TLC, thin layer chromatography

 THIN LAYER  CHROMATOGRAPHY  (TLC) by

Mr. Shaise Jacob Faculty, Nirmala College of Pharmacy Muvattupuzha Kerala, India

Chromatography  There are two basic types of  chromatography  ²  Gas  ²  Liquid

Liquid includes TLC and high  performance liquid chromatography (HPLC)

Introduction TLC TLC is is a form form of of liqui liquid d chr chrom omat atog ogra raph phy  y  consisting of:  ² A mobile mobile phase phase (developi (developing ng solvent) solvent) and and  ² A stationary stationary phase (a (a plate or or strip coated coated with with a form of silica gel)  ² Analysis Analysis is performed performed on a flat flat surface surface under under atmospheric pressure and room temperature

Michael Tswett is credited as being the father of liquid chromatography. c hromatography. Tswett developed his ideas in the early 1900·s.

 TLC The The two most most commo common n clas classes ses of TLC are: are:  ² Norm Normal al pha phase se  ² Revers Reversed ed ph phase ase

Normal Phase Norma Normall phas phasee is the termin terminolo ology gy used used when when the the stationary phase is polar; polar; for example silica gel, and the mobile phase is an organic solvent or a mixture of organic solvents which is less polar than the stationary phase.

Reversed Phase Revers Reversed ed ph phase ase is is the the termi termino nolo logy gy used used when when the stationary phase is a silica bonded with an organic substrate such as a long chain aliphatic acid like C-18 and the mobile phase is a mixture of water and organic solvent which is more polar than the stationary phase.

 THIN LAYER  CHROMATOGRAPHY  Chromatography is used to separate mixtures of substances into their components. Similar to P.C, except that a thin layer of  some inert material, i.e. Aluminium oxide, mag.oxid. , sili.oxide sili.oxide is used instead of   paper.  A layer of any one of these oxide is made from a slurry of power in a suitable inert solvent. Slurry is spread over a flat surface ( glass, metal or rigid plastic ) & dried

PRINCIPLE  ADSOR P TION  The

component with more affinity towards the S.P travels slower  The component with lesser affinity towards the S.P travels faster ADVANTAGES OF TLC

simple mtd. & cost of the equipment is low  rapid technique & not time consuming like C.C separation of µg of the substances can be achieved any type of compound can be analyzed corrosive spray reagents can be used without damaging the plate & needs less solvent

Steps in TLC Analysis The The foll follow owing ing are are the import important ant compo compone nents nts of  a typical TLC system:  ² Apparatus Apparatus (developing (developing chamber) chamber)  ² Stationary Stationary phase layer layer and mobile mobile phase phase  ² Appli Applicat cation ion of samp sample le  ² Develo Developme pment nt of the the plate plate  ² Dete Detect ctio ion n of anal analyte yte

General Procedure (1)  ² Decide if you are going going to do Normal Normal or Reversed phase chromatography   ²  Prepare a plate or select a plate with the proper sorbent material  ²  Prepare the mobile phase  ² Mark Mark the the pla plate te  ² Ap Appl plyy the the sam sampl plee  ² Deve Develo lop p the the pla plate te  ² Dete Detect ct the the ana analy lyte tess

PRACTICAL REQUIREME REQUIREMENTS NTS 1.

STATIONARY  PH PH ASE

 Adsorbents mixed with water or other solvents slurry  slurry  Silica gel H ( Silica gel with out binder ) Silica gel G ( Silica gel + CaSO4 ) Silica GF (Silica gel + binder + fluorescent indicator)  Alumina, Cellulose Cellulose powder, Kieselguhr G( Diatomaceous earth + binder)

Coater, Coater, hand operated

2.

GLASS PLATE

Specific dimensions20cm 20 cm  20 20cm cm,, 20c 20cm m  10 10cm cm,, 20c 20cm m  5c 5cm m Microscopic slides can also be used Plates should be of good quality & withstand high temperatures

3. PREP ARATION & ACTIVATION OF TLC PLATES  Pouring  ouring (( simplest methods )  Di Dipp ppiing  ng (used (used for small plates ) Spraying (( difficult to get uniform layers ) Spraying   Sp Spre read adin ing  g (( best technique ) TLC ) TLC Spreader

 Activation of Plates  After spreadi spreading ng  Air dry (5 to to 10 minutes) minutes)  Activ Activated ated by heatin heatingg at about about 100ÜC 100ÜC for 30 min. min.  Then plates may be kept in desiccators 4. APPLICATION OF SAMPLE » Using capillary tube or micropipette » Spotting area should not be immersed in the mobile phase 5. DEVELOPMENT TANK   Better to develop develop in in glass beakers, beakers, jars jars to avoid more more  wastage of solvents  When standard standard method is is used, use twin twin trough trough tanks  Do chamber chamber satu saturatio ration n to avoid ´edge effectµ

6.

MOBILE PH ASE

M.P used depends upon various factors  Nature of the substance  Nature of the S.P Mode of Chromatograph Chromatography  y  Separation to be achieved, Analytical/Preparative e.g.  pyridine, pet. ether, carbon tetrachloride, acetone, water, glycerol, ethanol, benzene« benzene«..

7.

DEVELOPMENT TECHNIQUE

1. One di dime men nsi sio onal de deve velo lop pme men nt 2. Two dimensional development 3. Horizontal developmen developmentt 4. Multiple development 8. DETECTING OR VISUALISING AGENTS a) Non sp specific methods

Iodine chamber method Sulphuric acid spray reagent UV chamber for fluorescent compounds Using fluorescent stationary phase

Specific methods Spray reagents or Detecting agents or Visualizing  agents  Same as P.C QUALITATIVE ANALYSIS 1. Rx value ² The   ratio of distance traveled by the sample & the distance traveled by the standard. 2. R f f  value QUANTITATIVE ANALYSIS > Direct & Indirect method

 APPLICATIONS  APPLICATION S OF TLC

»Purity of sample »Examination of reaction »Identification of compounds »Biochemical analysis »In pharmaceutical industry  »Separation of multicomponent pharmaceutical formulations »In food and cosmetic industry 

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