High-Performance Membrane Chromatography of Small Molecules

Abstract

High-performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic chromatography of plasmid DNA conformations. However, no study about the separation of small molecules has been performed until now. In this work, we investigated the possibility of gradient and isocratic HPMC of small molecules with Convective Interaction Media disks of different chemistries and tried to explain the mechanism that enables their separation. We demonstrated that it is possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed-phase mode. It was shown that similar peak resolution can be provided in both gradient and isocratic modes.

An Introduction to Monolithic Disks as Stationary Phases for High Performance Biochromatography

“…The single-step desorption separation mechanism, which is currently assumed to be the basis of protein HPMDC is thus less likely for small molecules, which do not differ to such a great degree in adsorption energy. In spite of this, mixtures of small molecules have recently been shown to separate well on monolithic disks, both in the isocratic and the gradient elution mode [21,43] (Figure 6). An explanation for this hitherto unsuspected feature of HPMDC will be attempted below.…”

“…In recent publications [21,43] it was shown that the isocratic separation of oligonucleotides benefits from an increase in the separation layer, but that such separations are nevertheless still possible on extremely thin monoliths (0.75 and 0.3 mm respectively in comparison with the commercially available 3 mm CIM disks of the same material), see Figure 7. Furthermore, since the binding characteristics among small molecules are more homogeneous than in the case of proteins, shallow gradients might be applied to improve the separation.…”

“…(0.75 and 0.3 mm) monoliths [21,43]. Four oligonucleotides with chain lengths of 8, 10, 12, and 14 bases were used as standard mixture.…”

Affinity membrane chromatography for the analysis and purification of proteins

“…The pore-size distribution and the porosity of the rod can be easily controlled by selecting the proper porogen and varying the volume ratio of porogen to monomer. Linear decreases in efficiency due to intraparticle w x diffusion at increasing flow-rates are not observed 22,43,[130][131][132][133][134][135][136][137][138] . A number of affinity-membrane cartridges are commercially available.…”

Short columns with molecularly imprinted monolithic stationary phases for rapid separation of diastereomers and enantiomers

“…Thus, the separation of small molecules is usually carried out on a relatively long column. However, Podgornik and co-workers [28,29] recently reported the separation of small molecules such as organic acids and hydroxybenzoates on a thin monolithic column (3 mm thickness) due to the absence of pore diffusion in the monolithic column. Based on their results, we investigated the effect of column length on separation of enantiomers and diastereomers on the self-made molecularly imprinted monolithic columns with different lengths but almost identical column volume.…”