Ultra-high pressure chromatography provides faster separation and lower solvent consumption compared to conventional HPLC systems while improving column efficiency. This can be achieved by reducing the particle size of the column packing and by increasing the linear velocity of the mobile phase. [1] The operation of UHPLC at very high pressures can cause several new difficulties that were relatively insignificant for HPLC systems.[2] These are friction heating of the column and the formation of temperature gradients, the effects on the mobile and stationary phases and the shift of the thermodynamic equilibrium of adsorption and desorption. Among the different chromatographic techniques, ion exchange is one of the most widely used forms of column chromatography. It is used in various research, analyzes and in protein purification. It is based on the interaction of charged molecules in the mobile phase with oppositely charged groups on the stationary phase. Charged functional groups are provided by a variety of amino acids in proteins. [3] It is known that the distribution of surface charge is responsible for the binding of proteins in the column and not the total net charge. In this study, we investigated the effect of pressure on the protein beta-lactoglobulin using ultrahigh pressure ion exchange chromatography. Due to its large dipole moment at wide isoelectric point, we were able to observe the adsorption process on the cation and anion exchange column under the same conditions. A negative retention trend was observed with increasing pressure on the anion exchange column at pH of isoelectric point of protein (5,2), while on the cation this trend was positive. When the pH value dropped below the isoelectric point, the same negative trend also appeared on the cation exchange column, which became more pronounced with increasing protein charge. The same results were obtained on the anion exchange column, where the difference in retention time became larger with increasing charge. We could assume that a higher charge of the protein increases the repulsion between the adsorbed protein molecules, leading to less dense packing on the surface of the column and consequently a higher standard partial molar volume. The presented results indicate the complexity of the interaction of ion exchange and the transition from conventional HPLC systems to the UHPLC system, which have a very large impact on the separation, so the presented mechanism should be studied in more detail.
|
