Separation of double-strand DNA fragments by high-performance liquid chromatography using a ceramic hydroxyapatite column

The liquid chromatographic behavior of double-strand DNA fragments was inve stigated using a column packed with a newly developed ceramic hydroxyapatit e sintered at 950 degrees C (pore size: 1500 Angstrom) or 1050 degrees C (a lmost non-porous). Fragments of pBR322DNA digested by Hae III, pUC19DNA dig ested by Hpa II, pBR322DNA digested by Alu I and phi X174DNA digested by Hi nf I were separated with a linear gradient of 10-400 or 600 mM phosphate bu ffer. These DNA fragments were adsorbed more strongly with hydroxyapatite s intered at 950 degrees C than with that sintered at 1050 degrees C. In comp arison with commercially available Type I and Type II columns, the best sep aration of DNA fragments was achieved with the newly developed ceramic hydr oxyapatite beads. The retention time of each fragment was remarkably increa sed with a slight increase in pH in the range 7.0-7.5. Based on the results of poryacrylamide gel electrophoresis, each of these fragments was eluted from the hydroxyapatite column in the order of fragment length. Furthermore , a good linear correlation between the capacity factor (k') and the logari thm of the number of base pairs was obtained up to 500 bp for each of the D NA fragments tested. With a pH gradient, the retention time of each DNA fra gment decreased in comparison to that with a concentration gradient, while the same separation of DNA fragments was obtained. (C) 1999 Elsevier Scienc e B.V. All rights reserved.