HPLC of nucleotides. II. General methods and their development for analysis and preparative separation. An approach to selectivity control

In order to develop efficient separation methods for nucleotides according to their size and heterocyclic composition, the application of ion‐exchange, reverse‐phase, and normal‐phase adsorption HPLC has been studied. The comparative investigation of retention power and selectivity of various packings (non‐polar bonded‐phase and amino silicas) in relation to nucleotide length and composition yields data which enable suitable packings to be selected and a method of preparing the new packing for a particular separation problem to be formulated. Thus a new anion exchanger with high selectivity and dynamic mass transfer has been prepared for fractionation of large oligonucleotides. The effect of the eluent pH and composition (organic modifier, salt) on retention, selectivity, and resolution in ion‐exchange and reverse‐phase HPLC has been studied. The optimum separation conditions comprise elution with oppositely directed gradients of the salt and the modifier, use of a precolumn packing that provides the best protection for the main column without loss of its efficiency, and the optimum gradient program for the desired retention of the component of interest. The relation between loading and sample concentration has been studied and the system for gradient elution improved. Our work shows that two‐dimensional separation is the most reliable and informative method for preparation of homogeneous oligonucleotides. The hydrophobic‐pair ion‐exchange mechanism is proposed for ion‐pair chromatography. Protected and partially deblocked oligonucleotides, chemically synthesized for genetic engineering studies, have been separated with high selectivity by adsorption (normal‐phase) HPLC which is efficient for gradient elution with isohydric eluents. The analysis of a monomeric composition of nano‐(pico‐) molar amounts of oligonucleotides has been developed; the procedure involves microcolumn digestion of the oligonucleotides with immobilized enzymes followed by microcolumn separation of the nucleoside‐mononucleotide mixture. Also, a new slurry method for packing stable HPLC columns with a tightly consolidated, nonshrinkable bed of particles has been developed.