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

Abstract

The liquid chromatographic behavior of double-strand DNA fragments was investigated using a column packed with a newly developed ceramic hydroxyapatite sintered at 950°C (pore size: 1500 Å) or 1050°C (almost non-porous). Fragments of pBR322DNA digested by Hae III, pUC19DNA digested by Hpa II, pBR322DNA digested by Alu I and φX174DNA digested by Hinf I were separated with a linear gradient of 10–400 or 600 mM phosphate buffer. These DNA fragments were adsorbed more strongly with hydroxyapatite sintered at 950°C than with that sintered at 1050°C. In comparison with commercially available Type I and Type II columns, the best separation of DNA fragments was achieved with the newly developed ceramic hydroxyapatite beads. The retention time of each fragment was remarkably increased with a slight increase in pH in the range 7.0–7.5. Based on the results of polyacrylamide 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 logarithm of the number of base pairs was obtained up to 500 bp for each of the DNA fragments tested. With a pH gradient, the retention time of each DNA fragment decreased in comparison to that with a concentration gradient, while the same separation of DNA fragments was obtained.