Studies on polynucleotides. 146. High-pressure liquid chromatography in polynucleotide synthesis

Abstract

Reverse phase high-pressure liquid chromatography (HPLC) using columns containing microparticulate materials with bonded octadecyl groups was developed as a rapid and efficient method for the separation of nucleosides, nucleotides, and, in particular, of protected oligonucleotides which are standard intermediates in the stepwise synthesis of deoxyribopolynucleotides. Extensive studies of the influence of the different purine and pyrimidine bases, protecting groups, the phosphate groups and of the chain lengths of oligonucleotides on their retention on such columns are reported. The application of HPLC in the stepwise synthesis of an oligonucleotide, d(G-G-A-A-G-C-T-T-A-A-C), was described. The methods, which are herein described, lend themselves to separations on a preparative scale and effect a marked reduction (up to 50%) in the time required for the synthesis of oligonucleotides.