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Chromatography

Chromatography. The use of chromatography in the organic lab Christian Tortzen. Agenda. Introduction Very little theory Stationary phase Mobile phase Analytical methods Preparative methods Gravitational/Flash chromatography Dry Column Vacuum Chromatography. Theory.

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Chromatography

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  1. Chromatography The use of chromatography in the organic lab Christian Tortzen

  2. Agenda Introduction Very little theory Stationary phase Mobile phase Analytical methods Preparative methods Gravitational/Flash chromatography Dry Column Vacuum Chromatography

  3. Theory Distribution coefficient Theoretical plates HETP Band broadening

  4. Theory

  5. The stationary phase Silica Aluminiumxoid Basic Acidic Activity E vs T Reversed-phase silica Cellulose, florisil, BioBeads, Sephadex

  6. The stationary phase Silica Particle size 63-200 μm - Gravitation/MPLC 40-63 μm - Flash/MPLC 15-40 μm - DCVC 5-15 μm – TLC/HPLC

  7. The mobile phase Not as restricted as the stationary phase Solvent parameters Polarity Selectivity Solvent groups Binary eluent mixtures

  8. The mobile phase

  9. Analytical methods Automated techniques Analytical HPLC LC-MS LC-NMR TLC

  10. TLC Aluminiumoxide - F254 Reversed phase silica – No indicator! Silica - F254 2D-TLC TLC-MS TLC-NMR

  11. TLC Development / visualisation UV 2,4-DNPH Ninhydrin CAN Many more!

  12. Preparative methods Preparative TLC Easy transfer of results from analytical TLC Easy visualisation No need for further TLC-analysis Small amounts per plate

  13. Column Chromatography HPLC Small particle size / High Pressure Very expensive columns Typically reverse phase Elaborate setup, DAD, fraction collector Great resolution Typically several runs needed for prep. scale Pre-columns and extensive wash needed

  14. Column Chromatography Gravitational/classic column chromatography Slurry packed with a solvent reservoir Typically done with 40-63μm or 63-200μm particle size Good resolution possible on “known columns” Time consuming Often done “blindly” Can be done faster with pressure Huge amounts of solvent needed Eluent system and column length often guestimated

  15. Column Chromatography Flash Column Chromatography Very narrow definition 45-63μm particle size Defined flow rate: 5 cm / min Eluent mixture determined by TLC – Rf = 0.35 Crude method - resolution only trivial ΔRf ≥ 0.15 Optimum packed length = 18 cm 1L solvent per gram loaded compound needed Amounts above 10 g are non trivial

  16. Column Chromatography Dry Column Vacuum Chromatography

  17. Column Chromatography Dry Column Vacuum Chromatography Equipment Silica – 15-40μm Solvents Length of packed column Loading amounts - 500 mg/cm² - π*r² Fraction Volume – Follows the column Packing

  18. Column Chromatography Dry Column Vacuum Chromatography Scalability Economy Time saved in post column analysis and evaporation Separations impossible using Flash routinely achievable Shaping of gradient makes separation below TLC resolution possible

  19. Column Chromatography Dry Column Vacuum Chromatography Sample loading Gradient elution Choice of eluent system and gradient Example 25% EtOAc/Heptane give Rf = 0.3 with large ΔRf 5 Steps until this polarity give a 5% gradient (25/5) Compound elutes around fraction 5-6 Limitations

  20. Other types of chromatography Size exclusion chromatography Chromatotron 3D chromatotron Counter current Single run dry column Quartz columns

  21. Modified silica Reverse phase Aromatics Doping Affinity Chromatography Chiral chromatography Self prepared modified silica

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