Band-spacing in reversed-phase high-performance liquid chromatography as a function of solvent strength: A simple and fast alternative to solvent optimization for method development

Numerous reports have described the use of solvent optimization for isocratic reversed-phase high-performance liquid chromatography method development. Solvent optimization involves the use of different solvents (usually methanol, acetonitrile and tetrahydrofuran) to control band-spacing for maximum resolution of the sample. Here, we examine an alternative approach, based on variation of the concentration of organic solvent in the mobile phase (solvent strength). This procedure is less powerful than classical solvent optimization, but it nevertheless possesses a significant ability to effect changes in band-spacing. It is also much more easily carried out. Many samples do not require solvent optimization, and in these cases, a change in solvent strength may be the more practical approach. The retention data required for solvent-strength optimization are most conveniently collected by using two initial gradient runs. The application of gradient retention data for developing a final isocratic separation is facilitated by the use of commercial software. The advantages and limitations of gradient-retention data for this purpose are examined.