Complex Protein Retention Shifts with a Pressure Increase: An Indication of a Standard Partial Molar Volume Increase during Adsorption?

Studies of protein adsorption on reversed-phase and ion exchange stationary phases demonstrated an increase in retention with increasing pressure, which is interpreted as a standard partial molar volume decrease during the transition of the protein from a mobile to a stationary phase. Investigation of the pressure effect on the retention of lysozyme and IgG on a cation exchange column surprisingly revealed a negative retention trend with the increase of pressure. Further investigation of this phenomenon was performed with β-lactoglobulin, which enabled adsorption to be studied on both cation and anion exchange columns using the same mobile phase with a pH of 5.

Simultaneous separation of insulin and six therapeutic analogues on a mixed mode column: HPLC-UV method development and application.

Human insulin and six most used therapeutic analogues are very similar in terms of retention on a reversed-phase column. Thus, the LC methods prescribed in the European Pharmacopoeia monographs for insulin and insulin analogues include many similar separation methods, which tend to be time consuming when separating individual products of insulins or are inadequate when handling a mixture. In this study, we present a simple, robust, versatile and accessible HPLC-UV separation method for identification and quantification of human insulin and its analogues in one run.

Effect of Pressure Increase on Macromolecules' Adsorption in Ion Exchange Chromatography.

In this study a new method for evaluating the pressure effect on separations of oligonucleotides and proteins on an anion exchange column was developed. The pressure rise of up to 500 bar was attained by coupling restriction capillaries to the column outlet to minimize differences in pressure over the column. Using pH transient measurements it was demonstrated that no shift in ion exchange equilibria occurs due to a pressure increase.

Effect of pressure on the retention of macromolecules in ion exchange chromatography.

Shorter analysis times and greater resolving power are contributing factors for transfer of separation methods from an HPLC to a UHPLC system when performing analysis in biopharmaceutical or clinical research. The effect of pressure on separations in reversed phase chromatography is well described, however such investigations on ion exchange columns were previously not conducted. In this study we describe the effect of pressure on retention properties of proteins, oligonucleotides and plasmid DNA in ion exchange chromatography.