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Chromatography

Chromatography. TLC. Thin layer chromatography Stationary phase Mobile phase. Separation AND Characterization. Chromatography Basics. Based on different affinities for stationary and mobile phases Silica gel: polar, water-covered surface Compound(s)

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Chromatography

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  1. Chromatography

  2. TLC • Thin layer chromatography • Stationary phase • Mobile phase

  3. Separation AND Characterization

  4. Chromatography Basics • Based on different affinities for stationary and mobile phases • Silica gel: polar, water-covered surface • Compound(s) • Polar: _______ affinity for plate, travels _______ • Nonpolar: _______ affinity for plate, travels ______ • Developing solvent • Polar: higher affinity for plate, travels slower, displaces compound more (compound travels __________) • Nonpolar: lower affinity for plate, travels faster, displaces compound less (compound travels ________)

  5. Test your Understanding • Which spot represents a more polar compound? • What would happen to each spot if a less polar solvent were used? • Why should you ALWAYS report your developing solvent with any TLC data?

  6. Quantitative Characterization • Retention factor • Distance traveled/ solvent front distance • Unitless • For silica gel TLC, based on polarity of the compound(s) Must report solvent!

  7. Solvent effect on Rf • Polar solvents outcompete compounds, drive them up plate

  8. Choosing a Developing Solvent • Adjust solvent to give Rf values around 0.4 • Common mixtures • Ether/Hex • EtOAc/Hex • CH2Cl2/methanol • Determined experimentally

  9. Visualization • Most compounds are invisible on TLC • UV lamp • Stains • Iodine chamber

  10. Application of TLC • Purity • Identity • Reaction Progress Column 1 is your target compound; column 2 is an expected impurity. What can you determine about your reaction (column 3)? What can we determine about the identity of the unknown?

  11. Common Problems • Overspotting • Underspotting • Wrong solvent

  12. Column Chromatography • Similar to TLC in separation • Preparative process • 0.1g to 5 g scale • Purify small quantities of liquids/solids (contrast recrystallization)

  13. Mobile phase: eluent similar to TLC • Stationary phase: silica gel similar to TLC • Column is upside down from TLC, so a larger Rf for a compound means it comes out ____

  14. Practical Considerations • Preparing the column • Loading the sample • Choosing the solvent • Separation capacity

  15. Practical Considerations • Preparing the column • Loading the sample • Choosing the solvent • Separation capacity

  16. Practical Considerations • Preparing the column • Loading the sample • Choosing the solvent • Separation capacity Most common: Hexane/ethyl acetate CH2Cl2/methanol

  17. Practical Considerations • Preparing the column • Loading the sample • Choosing the solvent • Separation capacity • Effect of diameter • Effect of length • Effect of Silica gel grade

  18. Flash Chromatography • Faster separation • Tighter separations

  19. HPLC

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