Differences in retention behavior between small and large molecules in ion-exchange chromatography and reversed-phase...

The retention (k') for various biomolecules was studied as a function of the mobile phase composition in reversed-phase chromatography (RPC) and ion-exchange chromatography (IEC). The "elution window" (EW) for a molecule is defined as the mobile phase composition in which 1 less than k' less than 10. The following relations were verified: (i) EW(RPC) less than EW(IEC), and (ii) EW(large Mr) less than EW(small Mr). The results support the theory that in both RPC and IEC larger molecules interact with the stationary phase by multiple-site binding. The results are used to explain why, in both RPC and IEC, larger molecules are best separated on short columns using gradient elution while smaller molecules often require longer columns and isocratic conditions.