LIQUID CHROMATOGRAPHIC ASSAY FOR THE SEPARATION OF SINGLE- AND DOUBLE-STRANDED DNA BY USING UV AND UV DIODE-ARRAY DETECTORS AND HYDROXYLAPATITE COLUMN

摘要

A high-performance liquid chromatographic (HPLC) method, using UV and UV diode-array (DA) detection, is reported for the separation of single-stranded (s.s.) and double-stranded (d.s.) DNA molecules. Commercially available calf thymus DNA was used as the standard, to develop and optimize necessary analytical procedures and chromatographic parameters. Bio-Gel(R) hydroxylapatite was used as the column packing and the sorbed polynucleotides on the column matrix were separated by using an ionic strength gradient system consisting of phosphate buffer at pH 6.8. The stationary phase was stable and proved sufficiently reliable in the separation and resolution of s.s, and d.s. DNA molecules in the standard. Pointedly, the DA detector was more sensitive to the analytes than the UV detector. The response of both detectors was higher for the s.s. DNA compared to the d.s. DNA. Minimum quantification limits (MQL) for the s.s. DNA molecules by the DA and UV detectors were, respectively, 0.10 and 0.50 mu g in 10 mu L injections. The corresponding value for the d.s. DNA, using both detectors, was 1.0 mu g. The plot log (mu g of DNA) vs absorbance (mAU) was linear for the d.s. DNA. The MQL, using both detectors, was 0.10 mu g in 10 mu L injection volume. Extension of the method to separate the viral DNA molecules showed some promise. However, problems associated with sample purity and homogeneity, peak characterization, quantification of the analytes etc. were encountered and these drawbacks are discussed.