Thin layer chromatography (tlc)

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  • 1. THIN LAYER CHROMATOGRAPHY (TLC) AND COLUMN CHROMATOGRAPHY M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.
  • 2.  You will have to separate three components of paprika.  The three components can be easily identified because they are colored (absorb visible light).  They have different polarities.  They can be separated using column chromatography.  You can monitor the separation using thin layer chromatography. What is chromatography….
  • 3. Chromatography • Very useful technique in organic chemistry based on differential adsorption. • Used to separate components in a mixture (solid or liquid). • It depends on the polarity of the ingredients involved --- intermolecular forces!! • Thin layer chromatography (TLC) is used to analyze components and purity of a mixture. • Thin layer chromatography (TLC) is also used to monitor the progress of a reaction.
  • 4. Chromatography What do we need to perform a chromatographic separation? • Adsorbent: Silica gel (silicon dioxide), also called “stationary phase”. • Eluent: solvent used to move your compound trough the silica gel, also called the mobile phase. • Your compound mixture to be separated. • Patience and chemical intuition.
  • 5. Chromatography • More polar molecules “stick” to the adsorbent longer. • Less polar molecule separate more easily from the adsorbent. • When this happens, separation occurs. SiOH SiOH SiOH SiOH SiOH SiOH SiOH SiOH SiOH SiOH SiOH SiOH Stationary phase To be separated O Eluent (mobile phase)
  • 6. Chromatography • More polar solvent move the molecules more efficiently • Less polar move the molecules less efficiently • Separation occurs Most polar Least polar Alkanes Toluene Diethyl ether Chloroform Acetone Ethyl acetate Ethanol Methanol (CH3OH)
  • 7. Column Chromatography Load the silica gel plus eluent into the column…this is called “column packing”
  • 8. Column Chromatography Using a Pasteur pipette, load your compound that was dissolved in a minimum of solvent onto the silica. Your test solution will then add the eluent. Do not let your column run dry!!
  • 9. Thin Layer Chromatography Typical TLC chamber We will use beaker with watch glass or aluminum foil
  • 10. Thin Layer Chromatography Spotting TLC plate •Use different capillary for each solution. • make solution of approx. 1-2 mg of sample in 1 ml of solvent. • Spot 2-3 times •Try to make small spots
  • 11. Insert filter paper to saturate atmosphere with solvent Keep the lid on!! Thin Layer Chromatography preparation of chamber
  • 12. Mark a line about 1 cm from the bottom with pencil It is important to use pencil
  • 13. Place TLC plate in chamber Let things develop! Don’t let the solvent front run off The top of the plate!!
  • 14. Pull it out and mark the solvent front before it evaporates Mark spots with pencil!
  • 15. Good, bad and ugly • First TLC shows ”overloading" due to too much sample. • Second shows good separation. • Third shows almost not enough compound, but OK