Reverse-phase chromatography:

Another version of reverse-phase chromatography is ion-pair chromatography which deals with separation of ionized or ionizable species on a reverse phase column. The method can handle samples that are very polar, multiply ionized and/or strongly basic. In ordinary reverse phase HPLC, organic ions show poor peak shapes and inadequate retention. Ion-suppression method is limited to the pH range 2.0 to 7.5 by the instability of stationary bonded phases outside this pH range. In case of ion-pair chromatography, an ion pair reagent, a large organic counter-ion which is ionized, is added at low concentration to the mobile phase. One ion of the reagent is retained and separate organic solute ions of opposite charge by forming a reversible ion-pair complex with the ionized sample as represented by the equilibrium

RCOO^- + R₄N⁺      ↔            [R₄N⁺, ^- OOCR]^o  ion-pair

Thus, electrically neutral compounds are partitioned between the mobile and non-polar stationary phases. Unlike conventional ion-exchange, ion-pair partition can separate non-ionic and ionic compounds in the same sample. First of all separation of the nonionic solutes is optimized and then counterion is added to the mobile phase whereby ionic solutes are retained.