THE MINI-COLUMN OF LANTHANUM TREATED HYDROXYAPATITE CERAMIC BEADS FOR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF PROTEIN

Lanthanum treated hydroxyapatite (La-HAP) beads was prepared and applied for the high performance liquid chromatography (HPLC). X-ray analysis of lanthanum on La-HAP was performed. Appearance of La-HAP beads was also observed by a scanning electron microscope. After the treatment of the HAP beads with the lanthanum chloride solution, the small fluffy structure on the surface of beads and then some inside fiber-like structure were observed. The elution behavior on La-HAP mini-column (0.76 I. D. ×0.4cm) was compared with those of HAP mini-column (0.76 I. D. ×0.4cm). Proteins were loaded on a column and eluted by liner gradient system. The relationship between capacity factor (k\u27) of various proteins on La-HAP column and k\u27 on HAP was estimated through the correlation coefficient as 0.93. The elution pattern of proteins on La-HAP column was approximately similar with that on HAP, but the adsorption of proteins on La-HAP was a little stronger than on HAP. After chromatography under acidic conditions, La-HAP beads were observed by electron microscope again. Fiber-like structure remained even after fifty cycles of chromatography. The height equivalent to a theoretical plate number (HETP) was estimated with tryptophan (0.25μg/5μl) in isocratic elution system (pH4.0). The change of HETP by prolonged use under acidic conditions was determined. Half height width of tryptophan peak on La-HAP (approximately 0.5mm) was persistent for 120min and after 120min gradually increased. While under the same conditions, HETP on HAP was remained only for about 40min. The durability of La-HAP seemed to be as three times as larger than that of HAP. It should be studied further that the results described above simply imply whether one-third of surface area of HAP is covered by lanthanum, or the durability of this material under acidic conditions is improved actually. According to the results of chromatography, the treatment with lanthanum chloride solution may not be homogeneous, and the improvement of processing of lanthanum coating of HAP is also remained to be studied. And then, so far studied, lanthanum-hydroxyapatite would be useful for the packing material for HPLC for separation of proteins and other biomacromolecules under fairly acidic conditions even

CHROMATOGRAPHY OF PROTEIN ON HYDROXYAPATITE IMPREGNATED PAPER

Ceramic hydroxyapatite beads for high performance liquid chromatography (HPLC) was already reported to be a cation exchanger in type and proteins were adsorped and eluted with changing the concentration of sodium phosphate buffer solution. Hydroxyapatite crystalline impregnated paper (HAP paper) which was made of micro crystalline and paper fiber, but without sintering process was used for filtration or separation of biological macromolecule materials. A circular sheet of HAP paper was cut into a suitable size and (0.76mm I. D.) and placed in a column for HPLC of protein. Chromatography was performed across the HAP paper sheet. One sheet of HAP paper could adsorbed about 12μg cytochrome c. A pile of these sheets could also be used for the chromatography of protein as well as hydroxyapatite beads column. HAP paper was useful not only for the separation of protein but also for the preliminary sample clean up for the HPLC, electrophoresis or various immunochemical treatment