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Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

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Contributions to reversed-phase column selectivity. II. Cation exchange.

D H Marchand¹, P W Carr, D V McCalley

¹University of Wisconsin - River Falls, River Falls, WI, USA.

Journal of Chromatography. A

|August 30, 2011

Summary

Cation exchange significantly impacts solute retention in reversed-phase chromatography (RPC) on alkylsilica columns. This study reveals cation exchange occurs even at low pH, contrary to previous assumptions, influencing the separation of ionized bases.

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Area of Science:

Analytical Chemistry
Chromatography

Background:

Reversed-phase chromatography (RPC) is a key separation technique.
Type-B alkylsilica columns are widely used in RPC.
Understanding solute retention mechanisms is crucial for method development.

Purpose of the Study:

To investigate the contribution of cation exchange to solute retention on type-B alkylsilica columns.
To evaluate the influence of mobile phase conditions and column properties on cation exchange.
To refine the hydrophobic-subtraction (H-S) model for RPC selectivity.

Main Methods:

Examination of solute retention (κ') for 87 solutes based on molecular structure.
Analysis of column cation-exchange capacity (C) for 167 type-B alkylsilica columns.
Correlation of column properties (ligand length, concentration, pore diameter, end-capping) with cation exchange capacity.
Investigation of mobile phase variables: pH, buffer concentration, and buffer cation type.

Main Results:

Cation exchange is a significant factor in the retention of ionized bases during RPC.
The extent of cation exchange varies with column acidity, mobile phase pH, buffer concentration, and buffer cation.
Evidence of cation exchange and silanol ionization was observed at pH as low as 3 for most columns.
Solute retention properties (κ') and column properties (C) were quantified and related to molecular structure and column characteristics, respectively.

Conclusions:

Cation exchange plays a more substantial role in RPC retention than previously assumed, particularly for ionized bases.
The hydrophobic-subtraction (H-S) model can incorporate cation exchange effects through solute (κ') and column (C) properties.
These findings provide a more detailed understanding of cationic solute retention in RPC under various separation conditions, challenging the notion that cation exchange is negligible at pH < 7.