Role of the Third Mobile Phase Solvent in Bonded Phase Chromatography

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Abstract
The role of the third mobile phase solvent in bonded phase chromatography is investigated on several different C18 bonded phase columns. Subtle changes in selectivity are demonstrated by the addition of a third solvent to the mobile phase. The changes in selectivity can be varied dynamically by changing the percentage composition of three components in the mobile phase. For example, the use of a third component allows use of the lipophilic side chains of peptide diastereoisomers to enhance separation. In addition, the utilization of a third component allows the selective adjustment of the properties of the stationary phase so as to obtain identical selectivity for column packing materials from different sources. The observed selectivity adjustments lead to further insight in terms of the properties of the diastereoisomers as well as the active role played by the stationary phase in the separation mechanism.

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