| Liquid chromatography is a popular, analytical technique that is utilized in countless industries. With problems and samples becoming more and more complex, it is important to maximize the separation. In traditional cation-exchange chromatography, charged analytes are separated based on electrostatic interactions between the analytes and the stationary phase. But ion-exchange chromatography is thought to have reached its limit with practicality. One area where this improvement may be beneficial to improve the separation is in Multi-dimensional Protein Identification Technique (Mud-PIT), which is a two-dimensional separation that uses cation-exchange chromatography in the first dimension, to separate peptides based on their charge, and then analyzes them by reversed-phase chromatography in the second dimension. If the cation-exchange separation can be improved, the analysis as a whole will be improved.;Studies have shown that, besides the electrostatic interactions, there are hydrophobic interactions that also affect retention. The electrostatic interactions are the dominating force in overall retention, whereas the hydrophobic interactions are only a minor component of the overall retention. By lowering the dielectric constant of the mobile phase through the addition of an organic modifier, the goal is to reduce the strength of the electrostatic interactions, thereby increasing the relative strength of the hydrophobic interactions. The work presented here will focus on increasing the role hydrophobic interactions play in retention through the addition of acetonitrile to the mobile phase. Most of the previous research concerning the use of organic modifiers in the mobile phase for ion-exchange chromatography has been cursory, and a much deeper understanding of the fundamentals is needed.;In the first section, all work was performed on a commercially available alkylsilica column. The analysis shows that, as the percentage of organic solvent is increased in the mobile phase to 40%, there are decreases in the electrostatic interactions between the analyte and the stationary phase by greater than 50%. Also, the relative contributions of ion-exchange are decreased by as much as 60%, while the relative reversed-phase contributions are increased, with the increased organic solvent in the mobile phase. After some intial concerns, there does not seem to be an issue with the desorbing agent, a tetrabutylammonium cation, binding hydrophobically to the alkylsilica stationary phase.;In the second section, all work was performed on a commercially available polystyrene-divinylbenzene column. The initial results suggested much stronger retention than ion-exchange theory allows. When looking for the additional interactions, it became obvious that pi-pi interactions are present and will affect retention. But after measuring the hydrophobicity of the phase, it became clear that the tetrabutylammonium cations are hydrophobically binding to the polymeric stationary phase, instead of exchanging with the analytes at the fixed anionic group on the stationary phase and changing the overall charge of the stationary phase. |
