Zone formation in ion-pair reversed-phase liquid chromatography. III. Step-gradient elution of...

Step-gradient elution by decrease of the counter-ion concentration or increase of the co-ion concentration was studied in an ion-pair reversed-phase system. As analytes, polyvalently negatively charged oligodeoxyribonucleotides were used. In the co-ion step-gradient technique, the retention volumes can be predicted by use of a simple equation. This technique gives a drastic decrease in peak width, which makes it possible to decrease the separation time by using an optimized composition of the two eluents used. Furthermore, the detectability of organic ions is considerably improved. The time for the introduction of the second eluent, containing the co-ion, into the column after the sample injection is critical.