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CHROMATOGRAPHY.

BIOCHEMISTRY

mprasadnaidu
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CHROMATOGRAPHY.

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  1. THIN LAYER CHROMATOGRAPHY (TLC) AND COLUMN CHROMATOGRAPHY M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

  2. Overview for today’s experiment • 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. • Patienceand chemical intuition.

  5. SiOH SiOH SiOH SiOH SiOH SiOH O SiOH SiOH SiOH SiOH SiOH SiOH Chromatography • More polar molecules “stick” to the adsorbent longer. • Less polar molecule separate more easily from the • adsorbent. • When this happens, separation occurs. Eluent (mobile phase) Stationary phase To be separated

  6. Chromatography • More polar solvent move the molecules more efficiently • Less polar move the molecules less efficiently • Separation occurs Least polar Alkanes Toluene Diethyl ether Chloroform Acetone Ethyl acetate Ethanol Methanol (CH3OH) Most polar

  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 ChromatographyTypical TLC chamber We will use beaker with watch glass or aluminum foil

  10. Thin Layer ChromatographySpotting 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. Thin Layer Chromatographypreparation of chamber Insert filter paper to saturate atmosphere with solvent Keep the lid on!!

  12. Mark a line about 1 cm from the bottom with pencil It is important to use pencil

  13. Let things develop! Place TLC plate in chamber 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

  16. What to do today? • Using diethyl ether, you will extract a mixture of three compounds from paprika ( Capsanthin, Capsorbin and b-carotene). • The three compounds have different polarities, thus can be separated using chromatography. • You will spot the mixture on a TLC plate, develop using 15% Et2O and 85% heptane. • Calculate the Rf for each spot. Record data in your notebook with the color of the spots. • Using the procedure in your handout you will perform a column chromatography, this time you will increase the polarity of the solvent (eluent) gradually. Asses with TLC

  17. Thank you

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