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Ion Pair Chromatography

By: Hansjörg Tinner, Michael Kiselewsky, Reporter US Vol 28


Hansjörg Tinner, Michael Kiselewsky

Chromatography is omnipresent in analytical research in spite of the development of instruments of extreme ranges of sensitivity and selectivity. Several traditional variations of chromatography are still widely used though many computer assisted advanced analytical instruments are now available. 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-ionized. This approach of method development can be time consuming and is better suited for single analyte or simple mixtures where pKA’s of the analytes are close.

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 interaction with the stationary phase, plus associated counter-ions. In total, IPC results in different retention of analytes to facilitate separation. IPC is an established and reliable technique that provides:

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


Figure 1 presents an example of polar compounds that were successfully separated on a Supelco Ascentis Express C18 column using IPC additives. The analytes are imidazolium and pyridinium derivatives. The column was subsequently rinsed with solvent in order to prevent ion pair reagent agglomeration. A gradient consisting of acetonitrile with a buffer of 1.1 g sodium 1-heptanesulphonate monohydrate (51832) and 700 μL phosphoric acid 85% in 1 L water was used.

Figure 1.Separation of Polar Compounds Using Ion Pair Additives (53819-U)


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 carefully analyzed for purity will guarantee the quality and performance in an application.

Sigma-Aldrich has a long tradition of offering superior quality analytical reagents. We are proud to offer a wide range of accurately tested IPC reagent products. Our reagents are of the highest purity and exhibit minimal extinction in the low UV region. These reagents exhibit 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 that can interfere with the sample are also tested. The suitability tests are carefully performed using a very steep gradient.

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