240 likes | 491 Views
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.
E N D
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 Distribution coefficient Theoretical plates HETP Band broadening
The stationary phase Silica Aluminiumxoid Basic Acidic Activity E vs T Reversed-phase silica Cellulose, florisil, BioBeads, Sephadex
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
The mobile phase Not as restricted as the stationary phase Solvent parameters Polarity Selectivity Solvent groups Binary eluent mixtures
Analytical methods Automated techniques Analytical HPLC LC-MS LC-NMR TLC
TLC Aluminiumoxide - F254 Reversed phase silica – No indicator! Silica - F254 2D-TLC TLC-MS TLC-NMR
TLC Development / visualisation UV 2,4-DNPH Ninhydrin CAN Many more!
Preparative methods Preparative TLC Easy transfer of results from analytical TLC Easy visualisation No need for further TLC-analysis Small amounts per plate
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
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
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
Column Chromatography Dry Column Vacuum Chromatography
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
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
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
Other types of chromatography Size exclusion chromatography Chromatotron 3D chromatotron Counter current Single run dry column Quartz columns
Modified silica Reverse phase Aromatics Doping Affinity Chromatography Chiral chromatography Self prepared modified silica