Retention and diffusion characteristics of oligonucleotides in a solid phase with polymer grafted anion-exchanger.

In the chromatographic separation process of oligonucleotides (ONs), mechanistic understanding of their binding and diffusion processes is of significant importance to determine operating conditions in a fast and robust way. In this work, we determined the number of binding sites and the diffusivities of ONs in a polymer grafted anion exchange chromatography through linear gradient experiments (LGE) being carried out at selected four to five gradient slopes. Synthetic poly (T)s with length ranging from 3 to 90-mer were employed as a model of an antisense oligonucleotide with typical lengths of 10 - 30 bases. Comparison of the retention was also conducted between the grafted anion exchanger with a conventional ligand and an anion monolith disk. For the ONs up to 50 bases, the number of binding sites determined can be correlated with the length of ONs, and the grafted resin showed a better diffusion and narrower peak width compared to the nongrafted one. The retention behavior became similar for porous media when the longer ONs (> 50mer) were applied. The results obtained suggest that antisense ONs can be separated with grafted ligands without sacrificing mass transfer properties.

Microcalorimetric Analysis of the Adsorption of Lysozyme and Cytochrome c onto Cation-Exchange Chromatography Resins: Influence of Temperature on Retention.

We studied the adsorption mechanism of two basic proteins, equine cytochrome c (Cyt) and chicken egg-white lysozyme (Lys), adsorbing onto negatively charged chromatography surfaces. In liquid chromatography, the retention volume of Lys was larger than that of Cyt on negatively charged ion-exchange resins. When the temperature increased, the retention volume of Cyt increased, whereas that of Lys clearly decreased. Both Lys and Cyt share similar physical characteristics, so the opposite behavior with increasing temperatures was surprising, indicating a more complex mechanism of adsorption may be involved. We analyzed the adsorption of these proteins by using isothermal titration calorimetry (ITC). The change in adsorption enthalpy determined by ITC allowed the understanding of the reason for and underlying driving forces of protein adsorption that resulted in this opposite behavior. Large exothermic enthalpies of adsorption were observed for Lys (-43.95 kJ/mol), and Lys adsorption was found to be enthalpically driven. On the other hand, endothermic enthalpies were dominant for Cyt adsorption (32.41 kJ/mol), which was entropically driven. These results indicate that dehydration and release of counterions play a more important role in Cyt adsorption and ionic interaction and hydrogen bridges are more significant in Lys adsorption. Understanding of the adsorption mechanism of proteins onto chromatography resins is essential for modeling and developing new, efficient chromatographic processes.

Thermodynamic analysis of polyphenols retention in polymer resin chromatography by van't Hoff plot and isothermal titration calorimetry.

Temperature effect on the adsorption enthalpy of polyphenols was analyzed with van't Hoff plots using the distribution coefficient, K, determined with isocratic and gradient elution chromatography. Catechin and epigallocatechin gallate (EGCG) were used as model polyphenols. The stationary phase was polystyrene-divinylbenzene (PS-DVB) resin particles and the mobile phase was an ethanol-water mixture. The values of adsorption enthalpy determined by chromatography and isothermal titration calorimetry were obtained in the temperature range of 283 and 318 K. The results obtained by van't Hoff plots were consistent with the ones obtained with the isothermal titration calorimetry (ITC). The interaction between PS-DVB particles and the polyphenols was found to be exothermic with negative values of enthalpy, -30.1 and -37.7 kJ/mol for catechin and EGCG, respectively.