Before introducing the reversed-phase ion pair reagent, let’s recall ion pair chromatography (IPC). Ion pair chromatography can be regarded as an improved form of reversed-phase chromatography (RPC) for the purpose of separating ion samples. The only difference between IPC and RPC is that IPC adds ion pair reagents in the mobile phase, which can interact with a – of acidic compounds or BH + of basic compounds in the equilibrium process.

Ion pair reagents

Ion pair reagents are neutral ion pairs formed by strong hydrophilic ions that react on sample molecules. Therefore, it can be used to separate charged and uncharged molecules at the same time. Reversed phase ion pair chromatography is to add the ion pair reagent to the mobile phase containing water flow, and the analyzed component ions and the counter ions of the ion pair reagent form neutral ions without charge in the mobile phase, so as to increase the effect of solute and non-polar stationary phase, increase the partition coefficient and improve the separation effect.

In general, when establishing an HPLC separation method, we recommend starting with RPC and then adding ion pair reagents (only when needed). For example, when we know that a certain peak corresponds to an acidic substance, alkaline substance or neutral substance, we can accurately predict the influence of the added IPC reagent on solute retention. Therefore, when the proper resolution cannot be achieved by changing other conditions of RPC, we can continuously change the retention behavior of acidic and alkaline solutes by using IPC reagents to improve their separation effect.

So, when or what substance is IPC used for separation? We can consider using ion pair reagents when the following characteristics of the sample appear:

(1) No or weak retention on the reversed-phase chromatographic column;

(2) The compound has strong ionic functional groups, such as carboxyl, ammonium, amino, etc.;

(3) The compound has sufficient solubility in the mobile phase of the reversed-phase system.

Using ion chromatography can make the retention behavior of the sample change similar to changing the pH of the mobile phase, but ion pair chromatography can better control the retention behavior of acidic or alkaline solutes, and there is no need to use extreme mobile phase pH ( For example, pH<2.5 or pH>8.0).

Common ion pair reagents

Common ion pair reagents mainly include the following categories:

Anion pair reagents: Alkaline reagents such as tetrabutylammonium hydroxide and tetrabutylammonium bromide, suitable for polar compounds containing sulfonic acid groups and carboxyl groups in the structural formula.

Cation pair reagent: Sodium methane sulfonate, sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecyl sulfonate, dodecane Sodium sulfate, etc., suitable for polar compounds containing ammonium groups and amino groups in the structural formula.

Other ion pair reagents: Sodium perchlorate, trifluoroacetic acid, heptafluorobutyric acid, etc.

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