Effect of Pressure Increase on Macromolecules’ Adsorption in Ion Exchange Chromatography

Abstract

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. Results from isocratic and gradient separations of oligonucleotides (model compounds) were evaluated by stoichiometric displacement and linear gradient elution model, respectively. Both elution modes demonstrated that for smaller oligonucleotides the number of binding sites remained unchanged with pressure rise while an increase for large oligonucleotides was observed, indicating their alignment over the stationary phase. From the obtained model parameters and their pressure dependencies, a thermodynamic description was made and compared between the elution modes. A complementary pattern of a linear increase of partial molar volume change with a pressure rise was established. Furthermore, estimation of the pressure effect was performed for bovine serum albumin and thyroglobulin that required gradient separations. Again, a raise in binding site number was found with pressure increase. The partial molar volume changes of BSA and Tg at the maximal investigated pressure and minimal salt concentration were −31.6 and −34.4 cm3/mol, respectively, indicating a higher rigidity of Tg. The proposed approach provides an insight into the molecule deformation over a surface at high pressures under nondenaturing conditions. The information enables a more comprehensive UHPLC method development.

Adsorption Equilibrium and Thermodynamics of Tea Theasinensins on HP20—A High-Efficiency Macroporous Adsorption Resin

“…The Langmuir adsorption isotherm equation was an ideal model of the surface adsorption for column chromatography packing with HP20 resin. However, in the actual separation and purification process, only a narrow range of adsorption could be recognized on a uniform surface, which indicated that only a narrow range of this equation was applicable [ 26 , 27 ]. Most of it was not applicable.…”

“…The adsorption and separation function of column chromatography mainly relied on intermolecular force, hydrogen bond, coordination bond, hydrophobic interaction, ionic bond, and other interactions to achieve its adsorption and separation function. Kristl et al tested the range of adsorption heat caused by different types of forces [ 27 ], and found that the adsorption heat caused by van der Waals force was 4–10 KJ/mol, that caused by water-dispersing bond force was about 5 KJ/mol, that caused by hydrogen bond force was 2–40 KJ/mol, that caused by coordination bond exchange was greater than 40 KJ/mol, and that caused by chemical bond force was greater than 60 KJ/mol. The adsorption thermodynamic parameters of the HP20 resin for theasinensin A, theasinensin B, and theasinensin C were 10–40 KJ/mol, which belong to hydrogen bond force adsorption.…”

“…Macroporous adsorption resins are an organic polymer adsorbent developed in the 1960s that have been widely used in environmental protection, food, medicine, and other fields. A multicomponent Langmuir isotherm model can be used to describe the adsorptions, and parameters were regressed with high accuracy [ 27 , 28 ]. The adsorptive interaction on resin correlated well with the experimentally measured adsorption affinity and enthalpy [ 29 , 30 ].…”

Pressure-Enhanced Liquid Chromatography, a Proof of Concept: Tuning Selectivity with Pressure Changes and Gradients

“…Conformational changes upon adsorption were reported in ion exchange (IEX) and in hydrophobic interaction chromatography (HIC) as well. 20 − 22…”

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

“… 21 An increase in retention of biopolymers and macromolecules with pressure was also demonstrated on an anion exchange column where up to an 80% increase in retention time and a 40% increase in resolution were observed. To understand these effects on RP or ion exchange (IEX) columns, the change in retention with pressure was described by equations based on basic thermodynamic principles 11 , 22 − 24 where Δ G θ , Δ E θ , Δ V θ , and Δ S θ are changes in the standard Gibbs free energy, internal energy, entropy, and volume of the system at a given temperature ( T ) and pressure ( p ). R is the gas constant, and K is the equilibrium constant.…”