HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATION OF OLIGONUCLEOTIDES USING DEOXYADENOSINE IMMOBILIZED SILICA-GELS FOR THE STATIONARY-PHASE - EFFECT OF THE DEOXYADENOSINE LIGAND CONCENTRATION

Deoxyadenosine immobilized silica gels with different ligand concentrations were prepared as novel HPLC packing materials for the separation of oligonucleotides. Oligothymidylic acids, which are complementary to the immobilized ligand, were clearly separated from the other oligonucleotides by the formation of specific hydrogen bonding with immobilized deoxyadenosine. In the case of separation with deoxyadenosine immobilized silica gels containing hydrophilic spacer groups, the capacity factor (k') of complementary d(T)(3) or d(T)(4) seemed to be almost constant over a wide range of ligand concentrations from ca. 0.010 similar to 0.10 mmol/g. This result strongly suggests that the interaction between the immobilized ligand and the solute is non-cooperative. On the other hand? an increase in the ligand concentration caused a stronger interaction with complementary solutes, thus retarding the retention time when the separation is performed with deoxyadenosine immobilized silica gel having no hydrophilic spacer group.