1!Affinity Chromatography!Chiral Chromatography, separation of enantiomers (R, S, mirror images, not superimposable)!2!Normal phase!HILIC = mobile phase forms a water-rich layer on the surface of the polar stationary phase (liquid-liquid extraction system). Hydrogen bonding, weak electrostatic interactions.!Reverse phase (3/4 are this style)!Stationary phase: hydrocarbon (C8 or C18 chain)!Mobile phase: water, methanol, acetonitrile, THF!Match polarity of analyte to that of the column!Hydrocarbon<ether<ester<ketones<aldehydes<amides<amines<alcohols!3!Avoid pH < 2.5 and above 7.5!Bonded and cross-linked stationary phases (Prevent bleeding)!4!Match polarity of stationary phase (not identical though!)!5!Longer chains are more retentive!Longer chains make it possible to analyze larger samples!6!Eluent strength: absorption energy of the solvent per unit area of solvent. Alumina!P’ ( Polarity index) by Snyder is based on solubility in three solvents (dioxane, low dipole proton acceptor) (nitromethne a high dipole proton acceptor), and ethyl alcohol (a high dipole proton donor)!Mixed solvents (P’) eq. 28-2 !Two unit change in P’ results in 10-fold change in k.!Eq 28-4!Practice Example 28-1!7!8!1. Water is used as adjusted k to same value!2. The vertices are the solvent + water!For normal phase (ethyl ether, methylene chloride, and chloroform) solvent strenght is done with hexane.!9!10!Pulse flow!Small internal volume 35-400 µl!High output pressure: 10,000 psi!Gradient elution!Large volume!Constant flow rates independent of colum back pressure and solvent
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