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TLC, thin layer chromatography

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TLC, thin layer chromatography

  1. 1. <ul><li>THIN LAYER CHROMATOGRAPHY </li></ul><ul><li>(TLC) </li></ul><ul><li>by Mr. Shaise Jacob Faculty, Nirmala College of Pharmacy Muvattupuzha Kerala, India </li></ul>
  2. 2. Chromatography <ul><li>There are two basic types of chromatography </li></ul><ul><ul><li>Gas </li></ul></ul><ul><ul><li>Liquid </li></ul></ul><ul><li>Liquid includes TLC and high performance liquid chromatography (HPLC) </li></ul>
  3. 3. Introduction <ul><li>TLC is a form of liquid chromatography consisting of: </li></ul><ul><ul><li>A mobile phase (developing solvent) and </li></ul></ul><ul><ul><li>A stationary phase (a plate or strip coated with a form of silica gel) </li></ul></ul><ul><ul><li>Analysis is performed on a flat surface under atmospheric pressure and room temperature </li></ul></ul>
  4. 4. <ul><li>Michael Tswett is credited as being the father of liquid chromatography. Tswett developed his ideas in the early 1900’s. </li></ul>
  5. 5. TLC <ul><li>The two most common classes of TLC are: </li></ul><ul><ul><li>Normal phase </li></ul></ul><ul><ul><li>Reversed phase </li></ul></ul>
  6. 6. Normal Phase <ul><li>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. </li></ul>
  7. 7. Reversed Phase <ul><li>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. </li></ul>
  8. 8. THIN LAYER CHROMATOGRAPHY <ul><li>Chromatography is used to separate mixtures of substances into their components . </li></ul><ul><li>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. </li></ul><ul><li>A layer of any one of these oxide is made from a slurry of power in a suitable inert solvent. </li></ul><ul><li>Slurry is spread over a flat surface ( glass, metal or rigid plastic ) & dried </li></ul>
  9. 9. PRINCIPLE <ul><li>ADSORPTION </li></ul><ul><li>The component with more affinity towards the S.P travels slower </li></ul><ul><li>The component with lesser affinity towards the S.P travels faster </li></ul><ul><li>ADVANTAGES OF TLC </li></ul><ul><li>simple mtd. & cost of the equipment is low </li></ul><ul><li>rapid technique & not time consuming like C.C </li></ul><ul><li>separation of µg of the substances can be achieved </li></ul><ul><li>any type of compound can be analyzed </li></ul><ul><li>corrosive spray reagents can be used without damaging the plate & needs less solvent </li></ul>
  10. 10. Steps in TLC Analysis <ul><li>The following are the important components of a typical TLC system: </li></ul><ul><ul><li>Apparatus (developing chamber) </li></ul></ul><ul><ul><li>Stationary phase layer and mobile phase </li></ul></ul><ul><ul><li>Application of sample </li></ul></ul><ul><ul><li>Development of the plate </li></ul></ul><ul><ul><li>Detection of analyte </li></ul></ul>
  11. 11. General Procedure (1) <ul><ul><li>Decide if you are going to do Normal or Reversed phase chromatography </li></ul></ul><ul><ul><li>Prepare a plate or select a plate with the proper sorbent material </li></ul></ul><ul><ul><li>Prepare the mobile phase </li></ul></ul><ul><ul><li>Mark the plate </li></ul></ul><ul><ul><li>Apply the sample </li></ul></ul><ul><ul><li>Develop the plate </li></ul></ul><ul><ul><li>Detect the analytes </li></ul></ul>
  12. 12. PRACTICAL REQUIREMENTS <ul><li>STATIONARY PHASE </li></ul><ul><li>Adsorbents mixed with water or other solvents-> slurry </li></ul><ul><li>Silica gel H ( Silica gel with out binder ) </li></ul><ul><li>Silica gel G ( Silica gel + CaSO4 ) </li></ul><ul><li>Silica GF (Silica gel + binder + fluorescent indicator) </li></ul><ul><li>Alumina, Cellulose powder, Kieselguhr G( Diatomaceous earth + binder) </li></ul>
  13. 13. Coater, hand operated
  14. 14. 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
  15. 18. 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”
  16. 19. 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….
  17. 20. 7. DEVELOPMENT TECHNIQUE <ul><li>One dimensional development </li></ul><ul><li>Two dimensional development </li></ul><ul><li>Horizontal development </li></ul><ul><li>Multiple development </li></ul><ul><li>8. DETECTING OR VISUALISING AGENTS </li></ul><ul><li>Non specific methods </li></ul><ul><li>Iodine chamber method </li></ul><ul><li>Sulphuric acid spray reagent </li></ul><ul><li>UV chamber for fluorescent compounds </li></ul><ul><li>Using fluorescent stationary phase </li></ul>
  18. 21. Specific methods <ul><li>Spray reagents or Detecting agents or Visualizing agents </li></ul><ul><li>Same as P.C </li></ul><ul><li>QUALITATIVE ANALYSIS </li></ul><ul><li>Rx value – The ratio of distance traveled by the sample & the distance traveled by the standard. </li></ul><ul><li>R f value - </li></ul><ul><li>QUANTITATIVE ANALYSIS </li></ul><ul><li>> Direct & Indirect method </li></ul>
  19. 22. 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
  20. 23. T h a n k y o u