1. Created with MindGenius Business 2005® Ion Exchange Chromatography Ion Exchange Chromatography Principles Small K means low affinity – so low RT K is often quoted vs. a common reference ion (e.g. H+) K affected by: Charge of ion:  charge gives  K Size of hydrated ion Uses displacement of an ion from the stationary phase by a solute ion K is dependent on relative affinity of the analyte for the surface over the solvent ions

2. Created with MindGenius Business 2005® Cation Exchange Chromatography Cation Exchange Chromatography Use organic acid functional groups as ionic species “Strong acid” type uses sulphonic acid (RSO 3 H) (more common) “Weak acid” type use carboxylic acids (RCOOH) xRSO 3 H + M x+ (RSO 3 - ) x M x+ + xH + Stationary Mobile Stationary Mobile Ion Series For a strong acid column, (e.g. RSO 3 H) size of K : Ag + > Cs + > Rb + > K + > NH 4 + > Na + > H + > Li + and Ba 2+ > Pb 2+ > Sr 2+ > Ca 2+ > Ni 2+ > Cd 2+ > Cu 2+ > Co2+ > Zn 2+ > Mg 2+

3. Created with MindGenius Business 2005® Anion Exchange Chromatography Anion Exchange Chromatography Use amine groups as ionic species “Strong base” type uses quaternary amines (e.g. RN(CH 3 ) 3 +OH - ) “Weak base” type uses secondary or tertiary amines xRN(CH 3 ) 3 +OH - + A x- (RN(CH 3 ) 3 ) x +A x- + xOH - Stationary Mobile Stationary Mobile Ion Series For a strong base column, affinity series for K is usually : SO 4 2- > Cr 2 O 4 2- > I - > NO 3 - > Br - > Cl - > HCO 2 - > CH 3 CO 2 - > OH - > F -

4. Created with MindGenius Business 2005® Ion Exchange Chromatography (optimisation) Ion Exchange Chromatography (optimisation) Stationary phase: “Strong” or “weak”, different pKa’s and pKb’s of ionic stationary phase groups give different strengths Mobile phase pH: can affect both a) the proportion of protonated stationary phase sites, b) the proportion of ionised analyte, but b) is more influential so  in mobile phase pH gives:  in retention time for cation exchange  in retention time for anion exchange Ionic Strength: (concentration of the mobile phase), mostly increases selectivity between ions of different charge Buffer salt: i.e. the relative position of the competing counter ion in the series for K Organic modifier: resin supports have a non-ionic component so reverse- phase interactions can occur. Affected by adding an organic solvent Temperature: affects buffer solubility, viscosity and mass transfer kinetics