Introduction to Ion Pair Chromatography

Ion pair chromatography (IPC) is an effective rever-sed-phase liquid chromatographic (RPLC) technique for separation of organic ions and partly ionized organic analytes. The technique utilizes the same types of stationary phases and mobile phases as RPLC; the main characteristic for IPC is that an ion pair reagent is added to the mobile phase. The ion pair reagent is usually an alkylsulfonate, an alkylsulfate or an alkylammonium salt. The high efficiency of RPC columns compared with columns used in ion exchange or ion chromatography also makes IPC a valuable alternative to these techniques.

The purpose of adding an ion pair reagent to the mobile phase is usually to change the retention time of ionic analytes. By varying the mobile phase concentration of the ion pair reagent, the retention factor for an oppositely mono-charged analyte can be continuously increased by a factor of 10-20 compared to the value with no added ion pair reagent. Correspondingly, it is possible to continuously reduce the retention factor for a similarly mono-charged analyte by a factor of 10-20. The retention factor for non-charged analytes is usually more or less unaffected by the presence of the ion pair reagent.

Ion pair extraction, i.e. extraction of ionized solutes into organic phases by adding an oppositely charged ion to the system, has been used for many decades. In this technique, the distribution of an ion into the organic phase is enhanced by the formation of an ion pair between the two oppositely charged ions. The pioneering work in IPC by Schill and co-workers in Uppsala was performed in the liquid-liquid partition mode. The extraction of an ion pair to the organic phase was considered to be the cause of retention, and the name ion pair chromatography originates from this early application. When covalently bonded non-polar stationary phases were introduced, important contributions to the further development of IPC were, among others, made by the research groups of Horvath, Knox, Schill and Haney.

IPC has been applied in almost all areas of analytical chemistry where chromatography is used. Since many drugs are basic or acidic, the driving force for the development of IPC came from the pharmaceutical industry where today it is used on a routine basis.

Of particular current interest is chiral separations of pharmaceutical compounds by using a chiral ion pair reagent. In IPC, water-rich mobile phases can be employed with a variety of buffers and ionic and non-ionic additives and the technique is therefore suitable for separation of important classes of bio-molecules and specifically amino acids, peptides, proteins and nucleic acids. In the food industry, IPC is used for the analysis of water-soluble vitamins, caffeine, theobromine, amines, etc.

For the separation of inorganic cations and anions IPC is often used as an alternative to ion (exchange) chromatography and in these applications it is usually referred to as ion interaction chromatography. In ion interaction chromatography, the ion pair reagent is usually called the ion interaction reagent. The technique has been used, for example, in the area of environmental analysis for the separation and analysis of nitrate and nitrite. Another technique that is closely related to ion pair chromatography is micellar chromatography. Here the concentration of ion pair reagent in the mobile phase is so high that micelles are formed, i.e. the concentration of the ion pair reagent in the mobile phase is above the critical micelle concentration (CMC). Micellar chromatography is treated in a separate article.

When the analyte ion lacks properties that make it easily detectable by commonly used detectors, IPC in the indirect detection mode can be used. The basis of this method is that a constant concentration of a detectable ion pair reagent is added to the mobile phase. In the chromatographic zone, where the non-detectable analyte ion is present, a change from the otherwise constant concentration of the detectable ion pair reagent is induced by the analyte. Depending on the relative properties of the analyte and the ion pair reagent, the concentration of ion pair reagent in the analyte zone may be either higher or lower than in the mobile phase.

Retention and selectivity in reversed-phase IPC are influenced by a large number of experimental parameters. These parameters are given in Table 1 together with a short description of their effect on retention. Briefly, for the separation of a particular set of analyte ions the parameters that are most important are type and concentration of ion pair reagent, type and concentration of organic modifier, and mobile phase pH. By varying these parameters while keeping the others constant within sensible ranges, an acceptable separation is usually obtained. A rational use of IPC is facilitated by a basic knowledge of the impact of the different parameters on retention. A more detailed analysis of the role of the most important parameters is presented below.

Table 1 Effect on retention of increasing the value of different chromatographic variables in reversed-phase ion pair chromatographic systems

Effect on k for oppositely charged solute

Increases the slope of the In kcB vs In cA relation

Slightly decreases the slope of the In kcB vs ln cA relation Increases


Decreases. The slope of the ln kcB vs p relationship becomes steeper compared with the regular RP slope

The slope of the ln kcB vs p relationship decreases Decreases

A parallel shift of the ln kcB vs ln cA relationship occurs for both positively and negatively charged analytes when using different columns


Increasing charge of analyte ion

Increasing concentration of pairing ion (ca)

Increasing hydrophobicity of pairing ion (KA)

Increasing concentration of organic modifier (p)

Increasing polarity of the organic modifier

Increasing ionic strength (I) Type of stationary phase

Eluent pH

Retention of the ionized form increases; it can become even larger than the regular RP retention of the non-ionized form

Effect on k for similarly charged solute Increases the absolute value of the slope of the ln kcB vs ln cA relation

Slightly decreases the absolute value of the slope of the ln kcB vs ln cA relation Decreases


Decreases. The slope of the ln kcB vs p relationship becomes less steep compared with the regular RP slope

The absolute value of the slope of the ln kcB vs ( relationship increases Increases

Retention of the ionized form decreases

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