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

Theory

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

The mobile phase

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 40-63 μm 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 45-63 μm 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

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