Ion Pair Chromatography

By: Michael Kiselewsky, Analytix Volume 9 Article 3

Hansjörg Tinner, Michael Kiselewsky

It was the Russian botanist Mikhail Tswett who discovered the principle of chromatography in 1901. More than 100 years later, chromatography is omnipresent in analytical research and far from being out of fashion. Even nowadays, it is part of the latest hi-tech development of instruments with extreme ranges of sensitivity and selectivity. Despite the many modern computerassisted instruments currently available, several traditional variations of chromatography are still widely used. One such chromatographic variation is Ion Pair Chromatography (IPC).

In the past, the approach used to separate charged analytes was ionic suppression. By changing the pH value of the mobile phase, charged analytes become non-ionised. This approach of method development can be time consuming and is better suited for single analytes or simple mixtures where pKA’s of the analytes are close together.

On the other hand, IPC is a more general and applicable approach that allows the separation of complex mixtures of very polar and ionic molecules. The mobile phase is supplemented with an ion-pairing reagent. Ion-pairing reagents consist of large ionic molecules having a charge opposite to the analyte of interest as well as a substantial hydrophobic region that allows interacting with the stationary phase, plus associated counter-ions. In total, IPC results in different retention of analytes, thus facilitating separation. IPC is an established and reliable technique that provides:

  • Reduced separation times
  • Highly reproducible results
  • Sharper peak shapes
  • Simultaneous separation of ionised and non-ionised analytes
  • Wide choice of additives to improve separation


The more sensitive modern instruments become, the easier they will detect any impurity added by auxiliaries in addition to the analytes of interest. Therefore, the purity of any kind of eluent additives will influence performance and accuracy. Only products that have been tested for suitability and that have been carefully analysed for purity will guarantee the quality and performance for your application.

Figure 1 shows an example of polar compounds that have been successfully separated using IPC additives on a Supelco Ascentis® Express C18 column. The analytes are imidazolium and pyridinium derivatives. The column was subsequently rinsed with solvent in order to prevent ion pair reagent agglomeration.

Figure 1.Separation of polar compounds on a Supelco Ascentis® Express C18, 2.7 μm, 7.5 cm x 4.6 mm I.D. (Supelco 53819-U).

Figure 1. Separation of polar compounds on a Supelco Ascentis® Express C18, 2.7 μm, 7.5 cm x 4.6 mm I.D. (Supelco 53819-U). Acetonitrile was used as a gradient with a buffer of 1.1 g Sodium 1-heptanesulphonate monohydrate (51832) and 700 μL Phosphoric acid 85 % in 1 l Water. Sample volume 5 μL, flow rate 1.0 mL/min, T = 25 °C, UV detection = 210 nm, gradient: t = 0 2 % acetonitrile, t = 1 min to 10 min 2 – 20 % and t = 15 min 35 % acetonitrile. Analytes: anions (1), 1-(3-Cyanopropyl)-3-methylimidazolium dicyanamide (2), 1-Methyl-2-vinylpyridinium triflate (3), 1-Butyl-3-methylimidazolium bromide (4), 1-Butyl-2,3-dimethylimidazolium tetrafluoroborate (5), 1-Benzyl-3- methylimidazolium tetrafluoroborate (6), 1-Hexyl-3-methylimidazolium chloride (7)

Sigma-Aldrich® has a long tradition of offering superiorquality analytical reagents. We are proud to offer you a wide range of accurately tested IPC reagent products. For almost 20 years, Sigma-Aldrich has followed a well-established, reliable technique and can provide you with a broad range of products and application notes to help you to resolve your samples. Our reagents are of the highest purity and exhibit minimal extinction in the low UV. They have excellent transparency down to 200 nm, even at high concentrations. In addition, they are tested for the absence of insoluble matter. Non-absorbing impurities like redox-traces, which may interfere with the sample, are also tested. The suitability tests are carefully performed, using a very steep gradient.

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