THIN LAYER CHROMATOGRAPHY (TLC) by Mr. Shaise Jacob Faculty, Nirmala College of Pharmacy Muvattupuzha Kerala, India

Chromatography There are two basic types of chromatography Gas Liquid Liquid includes TLC and high performance liquid chromatography (HPLC)

Introduction TLC is a form of liquid chromatography consisting of: A mobile phase (developing solvent) and A stationary phase (a plate or strip coated with a form of silica gel) Analysis is performed on a flat surface under atmospheric pressure and room temperature

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

TLC The two most common classes of TLC are: Normal phase Reversed phase

Normal Phase Normal phase is the terminology used when the stationary phase is 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 Reversed phase is the terminology used 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 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 ADSORPTION 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 following are the important components of a typical TLC system: Apparatus (developing chamber) Stationary phase layer and mobile phase Application of sample Development of the plate Detection of analyte

General Procedure (1) Decide if you are going to do Normal or Reversed phase chromatography Prepare a plate or select a plate with the proper sorbent material Prepare the mobile phase Mark the plate Apply the sample Develop the plate Detect the analytes

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

Coater, hand operated

3. PREPARATION & ACTIVATION OF TLC PLATES 2. GLASS PLATE Specific dimensions- 20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm Microscopic slides can also be used Plates should be of good quality & withstand high temperatures 3. PREPARATION & ACTIVATION OF TLC PLATES ♦ Pouring ( simplest methods ) ♦ Dipping (used for small plates ) ♦Spraying ( difficult to get uniform layers ) ♦ Spreading ( best technique ) TLC Spreader

Activation of Plates ○ After spreading → Air dry (5 to 10 minutes) ○ Activated by heating at about 100˚C for 30 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 in glass beakers, jars to avoid more wastage of solvents ▫ When standard method is used, use twin trough tanks ▫ Do chamber saturation to avoid “edge effect”

M.P used depends upon various factors 6. MOBILE PHASE M.P used depends upon various factors ► Nature of the substance ► Nature of the S.P ►Mode of Chromatography ►Separation to be achieved, Analytical/Preparative e.g. → pyridine, pet. ether, carbon tetrachloride, acetone, water, glycerol, ethanol, benzene….

7. DEVELOPMENT TECHNIQUE One dimensional development Two dimensional development Horizontal development Multiple development 8. DETECTING OR VISUALISING AGENTS Non specific methods Iodine chamber method Sulphuric acid spray reagent UV chamber for fluorescent compounds Using fluorescent stationary phase

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

APPLICATIONS 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

Thank you