High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent

Abstract

High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed ; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however.Hydroxyapatite (HA) chromatography, ideally used for separation of proteins, nucleic acids and viruses [l-31, is unique in that the geometrical arrangement of the adsorbing sites on the packed particles (i.e. the HA particles) is highly regular in any loci in the column [4, 51. Therefore, the adsorption configuration of a macromolecule on the HA surface can be deduced to be the same in any locus in the column; the elution position of the molecule is determined by how the geometrical arrangement of adsorption groups (i. e. carboxyl, phosphate, &-amino and guanidinyl groups) on a local molecular surface (that can keep in contact with the crystal surface) fits that of adsorbing crystal sites [4, 51.HPLC columns packed with plate-like HA micro-crystals (with thicknesses of about 1-3 pm and diameters of several times 10 pm) were recently developed in our laboratory (KB columns; Japan Biomedical Material Research Center, Koken Co. Ltd, Tokyo, Japan). A high level of chromatographic separation was obtained by selecting from very extensive ranges the optimum overall column length and the optimal slope of the molarity gradient for each particular sample based on chromatographic theory; except in some cases, the chromatographic resolution increases when the slope of the molarity gradient decreases and when the column length increases at the same time [4,5].The purpose of the present paper is to make another independent approach to high chromatographic resolutions by improving the shape of the packed particles in the column; this procedure would considerably reduce the flow heterogeneity occurring within the column. Thus, HPLC columns packed with spherical aggregates (with diameters of 1-10 pm) of extremely small HA micro-crystals (with diameters of the order of 0.1 pm) were prepared and preliminary performance tests were carried out by using several types of protein. Considerably high chromatographic resolutions were obtained even by using very compact columns with lengths of 0.5-3 cm. MATERIALS AND METHODSHA micro-crystals with diameters of the order of 0.1 pm as starting material were precipitated by mixing an aqueous Ca(OH)2 suspension and an aqueous NH4 a H2P04 solution. The aqueous HA suspension obtained above was sprayed; before being captured, the sprayed drips were dried into spherical crystal aggregates with diameters of 1 -10 pm (Fig. 1). These spherical aggregates, which will later be shown to be composed of stoichiometric HA crystals (see Discussion), were packed in SUS316 st...

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

“…Although they gave interesting results [12,13], these materials were clearly not ideal chromatographic supports. New production procedures developed in the 1980s resulted in HPLC compatible HA beads [14][15][16]. A major breakthrough in this context was the development of a sintering process for the production of ceramic HA variants [15].…”

“…In the last case, the SVSV fragment was mainly responsible for the interaction, whereas the MKPSP fragment apparently provided a conformational-dependent component that enhanced the peptide's binding, but by itself did not show any specific affinity. In 2009 our group presented a study of HA-interacting peptides at the 5 th Conference on Hydroxyapatite and Related Products: CHT for Bioprocessing (Oct. [11][12][13][14]2009, Rottach-Egern, Germany). The HA interaction (binding/elution) of several amino acids (glycine, serine, phenylalanine, lysine, aspartic acid), two proteins (BSA (IEP 4.7) and lysozyme (IEP 10.7)), was reported together with that of several peptides.…”

Kinetic study on the protein decomposition by photocatalytic Ti(IV)-doped calcium hydroxyapatite particles

“…5),13) These particles were prepared by the coprecipitation method as described briefly below. 7) Ca(NO 3 ) 2 and Ti(SO 4 ) 2 were dissolved in 1 dm 3 pure water free from CO 2 at an atomic ratio of Ti/(Ca+Ti) (abbreviated as X Ti ) was 0.2. The total amount of Ca and Ti in the solutions was hold at 0.1 mol; 0.060 mol of H 3 PO 4 was added to the solution and the solution pH was adjusted to 9.0 by adding a 15 M NH 4 OH solution.…”

“…It is well known that a synthetic calcium hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 , designated as CaHap, attracts attention as bioceramics, acid-base catalysis in various reactions and adsorbents for separation of biomaterials. 1) 6) One of the authors (M. Wakamura) has developed that the Ti(IV)-doped CaHap (expressed as TiHap), with Ti/(Ca+Ti) atomic ratio (abbreviated as X Ti ) of 0.2 and calcined at 650°C for 1 h, possesses a high photocatalytic activity for decomposition of acetaldehyde and albumin under ultra violet (UV) irradiation.…”

Synthesis and Structural Characterization of Silica-Hybridized Hydroxyapatite with Gas Adsorption Capability

“…A suspension of Ca OH 2 in 100 ml of H 2 O was prepared beforehand, while a Hydroxyapatite dilute phosphoric acid solution in 100 ml of isopropyl alcohol whose tetra-n-butyl orthosilicate =CH 3 CH 2 3 O? 4 Si, Kanto Chemical, Japan was dissolved in a phosphoric acid solution was also prepared so that Si IV could be incorporated into the HAp lattice structure in the range from 0 to 50 mass! against the mass of HAp, when Si was assumed to be present in the formula SiO 2 .…”