Synthetic polycation: polynucleotide interactions determined using liquid chromatography with short monolithic columns.

LC on short monolithic columns (Convective Interaction Medium Disks) was applied to investigate several specially synthesized water soluble polycations of different charge type (primary, tertiary, quaternary amine), as well as a copolymer of neutral saccharide and cationic monomers, regarding their ability to form reversible complexes with DNA. For this purpose, two separation modes were used, namely, pseudo-affinity and cation-exchange chromatography. Synthetic polynucleotides, namely, polyriboadenylic acid (poly(rA)) and polyribocytidylic acid (poly(rC)), were used as approximate structural analogues of DNA. In first case, the hypothetical specific binding between dissolved polymers and polynucleotide (poly(rA) or poly(rC)), covalently attached to epoxy-bearing monolithic sorbent, has been studied and compared to the results obtained using cation exchange chromatography. Quantitative parameters of interactions between macromolecules were established using frontal elution method.

Affinity processes realized on high-flow-through methacrylate-based macroporous monoliths.

The technology for preparation of rigid macroporous polymers suggested in the late 1980s has become a powerful instrument for the development of a novel scientific and practical field. At present, monolithic stationary phases are widely used in the processes of bioseparation (chromatography), bioconversion (enzyme reactors) as well as in other processes based on interphase mass distribution (for example, solid phase peptide and oligonucleotide synthesis). Bioaffinity modes of suggested dynamic methods are very promising for their use in different analytical processes (immunological, ecological, medical and other types of analytical monitoring), preparative isolation of blood proteins such as myoglobin, hemoglobin, immunoglobulins, etc. and also recombinant products directly from cell supernatants or lysates. For the first time, it has been shown that bioaffinity pairing with participation of immobilized on carefully designed rigid supports is very fast and the whole process of affinity separation can be realized within second time scale. The principle of bioaffinity recognition is generaly at the construction of biological reactors (for example, enzyme reactors). Improved kinetics of biocatalized reactions is explained by a minimal influence on the surface of the used sorbent. Very perspective field is the use of discussed monoliths for solid phase chemical synthesis of fragments of biological macromolecules (peptides and oligonucleotides). Several examples of these applications will be presented and discussed.

Chromatographic investigation of macromolecular affinity interactions.

High-performance monolithic disk affinity chromatography was applied to the investigation of formation of complexes between (1) complementary polyriboadenylic and polyribouridylic acids, e.g. poly(A) and poly(U), respectively, (2) poly(A) and synthetic polycation poly(allylamine), pAA. Polyriboadenylic acid and poly(allylamine) were immobilized on macroporous disks (CIM disks). Quantitative parameters of affinity interactions between macromolecules were established using frontal analysis at different flow rates.