Amino acid and peptide partitioning thermodynamics by reversed-phase liquid chromatography

This study shows that Reversed-Phase Liquid Chromatography (RPLC) using stationary phases of high bonded chain density may be a suitable system to model protein stabilities. This is done by measuring capacity factors over a wide temperature range and several different mobile phase compositions with a series of indoles and a series of pentapeptides. These measurements are made on a series of stationary phases two of which are above the critical bonding density and two of which are below the critical bonding density.; Knowing this information, van't Hoff plots are constructed to determine the free energy (DeltaG), entropy (DeltaS) and enthalpy (DeltaH) for the partitioning process of biological solutes between the mobile phase and the stationary phase in the chromatographic system. While variations in free energies with changes in bonding density are minimal, significant variations in entropies and enthalpies reveal a change in the dominant driving force in the retention process. One of the key points in identifying RPLC as a better model for biological partitioning is an understanding of the thermodynamics of partitioning. Bulk phase measurements such as octanol/water partitioning experiments are the standard for measuring biological partitioning, but this method is generally an enthalpy driven process. Since it is thought that biological partitioning is entropy driven, the most suitable thermodynamic model would be one that is also entropy driven.; The indole experiments represent a starting point for making comparisons with data from Jacobs and White [58]. In short, these experiments confirmed the ability to control solute partitioning thermodynamics by changing the packing density of the stationary phase.; In regards to the pentapeptide studies, van't Hoff plots from stationary phases above the critical bonding density using estimated values for the capacity factor in 100% water, k'w, were nonlinear and showed enthalpy as the driving force of retention. This effect was independent of both solute and stationary phase. While the initial van't Hoff plots were nonlinear, through analysis of DeltaH vs. T plots, a set of mobile phase conditions was found that generates linear van't Hoff plots. Even though these conditions varied from solute to solute and from stationary phase to stationary phase all were in the range of 26.2% to 32.2% organic modifier. From analysis of the linear van't Hoff plots, entropy was identified as the dominant driving force of partitioning on stationary phases above the critical bonding density. This suggests that reproducing biological conditions has more do with the representing of the total system correctly as opposed to a set of specific conditions. In other words the microenvironment of the chromatographic system is important.; In addition, investigation into amino acid hydrophobicity scales has been done. (Abstract shortened by UMI.)...