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

A high-performance liquid chromatographic (HPLC) method, using UV and UV diode-array (DA) detection, is reported for the separation of singl e-stranded (s.s.) and double-stranded (d.s.) DNA molecules. Commercial ly available calf thymus DNA was used as the standard, to develop and optimize necessary analytical procedures and chromatographic parameter s. Bio-Gel(R) hydroxylapatite was used as the column packing and the s orbed 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 s tandard. Pointedly, the DA detector was more sensitive to the analytes than the UV detector. The response of both detectors was higher for t he s.s. DNA compared to the d.s. DNA. Minimum quantification limits (M QL) for the s.s. DNA molecules by the DA and UV detectors were, respec tively, 0.10 and 0.50 mu g in 10 mu L injections. The corresponding va lue 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 MQ L, using both detectors, was 0.10 mu g in 10 mu L injection volume. Ex tension of the method to separate the viral DNA molecules showed some promise. However, problems associated with sample purity and homogenei ty, peak characterization, quantification of the analytes etc. were en countered and these drawbacks are discussed.