High-performance liquid chromatography of amino acids, peptides and proteins .149. Synthesis of silica-based heparin-affinity adsorbents

In these investigations, methods for the synthesis of rigid heparin-affinity adsorbents were developed. An optimal binding-site accessibility of the ligand was achieved by end-point attachment of the heparin through reductive amination onto amino-derivatised silica-based support materials. The rigid LiChroprep Si60 and Fractosil 1000 supports were derivatised by treatment with gamma-aminopropyltriethoxysilane, and a range of different heparin contents were immobilised onto these materials through controlled coupling conditions. Thus, heparin-LiChroprep Si60 adsorbents and heparin-Fractosil 1000 adsorbents with heparin contents ranging from 0.6 to 26 mg and 1 to 14 mg heparin/g adsorbent, respectively, were obtained. Due to their radically different pore size and specific surface areas, the heparin-LiChroprep Si60 (pore size 60 Angstrom, 500 m(2)/g) and heparin-Fractosil 1000 (pore Size 1000 Angstrom, 20 m(2)/g) adsorbents possessed markedly different adsorption properties. The small pore size of LiChroprep Si60 largely restricts the immobilisation of heparin to the external surface regions of these silica particles, with interaction with proteins in chromatographic applications mimicking non-porous affinity adsorbents. With the heparin-Fractosil 1000 adsorbents, the larger pore size (1000 Angstrom) enabled both the immobilisation of heparin and the solute interaction to take place within the pores to a much greater extent. Although high ligand densities per unit-accessible-area of the heparin-LiChroprep Si60 adsorbents could be achieved, decreased yield for the amino-group modification due to steric effects was observed. With the heparin-Fractosil 1000 adsorbents, lower ligand densities per unit area were obtained, but the binding-site accessibilities were considerably higher. These differing ligand densities were a function of differences in coupling yields (around 10 to 20% on LiChroprep-NH2, but only 1 to 3% on NH2-Fractosil 1000), and the different (accessible) surface areas. The hydrolytic stability of the aminopropyl-modified supports and heparin adsorbents was also investigated. A new quantitative method, based on absorbance measurements in 96-well microtitre plates, to determine the extent of the aminopropylsilyl group and ligand leakage is described. Although some instability of the heparin adsorbents was observed at pH values below pH 8.0, the ligand loss rate was much lower than the silane leakage on corresponding aminopropylated support materials lacking the heparin coverage. Thus, the heparin-LiChroprep Si60 and heparin-Fractosil 1000 adsorbents typically retained greater than 95% of their initial heparin content when stored in 0.2 M sodium acetate, pH 5.0 at 4 degrees C for eight weeks.