Before introducing reversed-phase ion pair reagents, let us recall ion pair chromatography, which can be viewed as a modified form of reversed-phase chromatography for the purpose of separating ion samples. The only condition that IPC differs from RPC is the addition of ion-pair reagents to the mobile phase, which can interact with the A- of the acidic compound or BH+ of the basic compound during the equilibrium process.
The ion pair reagent
Ion pair reagents are neutral ion pairs formed by strong hydrophilic ions that react on the sample molecule. Therefore, it can be used to separate charged and uncharged molecules at the same time. Reverse phase ion pair chromatography is to add the ion pair reagent to the aqueous mobile phase, the analyzed component ions in the mobile phase and the ion pair of reagents to generate neutral ions without charge, so as to increase the solute and non-polar stationary phase, increase the distribution coefficient, improve the separation effect.
In general, when establishing HPLC separation methods, we recommend starting with RPC, followed by the addition of ion-pair reagents (only when necessary). For example, when we know that a peak corresponds to an acidic, alkaline, or neutral substance, we can accurately predict the effect of the added IPC reagent on solute retention. Therefore, when other conditions of RPC can not achieve proper separation degree, we can improve the separation effect of acidic solute and alkaline solute by using IPC reagents to continuously change their retention behavior.
So, when or when does IPC apply to the separation of what substances would be a suitable separation method? Ion pair reagents can be considered when the sample has the following characteristics:
(1) No or weak retention on the RGC column
(2) The compound has strong ionic functional groups, such as carboxyl group, ammonium group, amino group, etc
(3) The compound has sufficient solubility in the mobile phase of the reversed phase system
Using ion chromatography can result in a change in the retention behavior of the sample similar to changing the pH of the mobile phase, but ion-pair chromatography can better control the retention behavior of the acidic or alkaline solute without the need for extreme mobile phase pH (e.g., pH < 2.5 or pH > 8.0).
Common ion pair reagents
Common ion pair reagents mainly include the following types:
Anion pair reagent: tetrabutylammonium hydroxide, tetrabutylammonium bromide and other basic reagents, suitable for structural formula containing sulfonic acid group, carboxyl and other polar compounds.
Cationic reagent: sodium methanesulfate, sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecyl sulfonate, sodium dodecyl sulfate, suitable for structural formula containing ammonium, amino and other polar compounds.
Other ion pair reagents: sodium perchlorate, trifluoroacetic acid, heptafluorobutyric acid, etc.