Technique: ion-exclusion chromatography

Ion exclusion chromatography (IEC or ICE) enables separation of neutral and ionisable compounds depending on their acidity or basicity, their size, and to some extent also their hydrophobicity. Although the ICE technique is rather mature and may not provide the most time-efficient separations, it has found many applications within research & development, and in quality control in sectors such as pharmaceutical, food & beverage, and biotechnology. Polar hydrophilic compounds with high structural diversity, such as organic acids and carbohydrates, are among the most frequently targeted analytes in ICE.

The experienced application team at Diduco can assist with anything from method development to troubleshooting also in this liquid chromatography mode.

Columns and mechanism

The separation process in ICE is governed by neutral and partially ionized molecules having different access to an internal pore space due to Donnan exclusion by charges on the stationary phase being of the same sign as the ionized analytes. Thus, for separation of carboxylic acids that ionize into anions, a cation exchange stationary phase with negatively charged groups is used.

As with all forms of exclusion chromatography, retention in ICE will have an upper and lower limit, ranging from full access to no access into the total pore volume. Neutral molecules with small molecular volume will thus show the highest retention. To provide sufficient stagnant volume for partitioning into, ICE columns typically have larger inner diameter and are longer. On top of this mechanism may secondary interactions with the stationary phase particle surface cause additional retention. Typical examples include molecules with unsaturated functional groups that interact with the organic polymer resins (crosslinked polystyrene) typically used in ICE columns.

Mobile phase conditions

Eluent compositions for ICE of acids and their corresponding anions range from pure water to low concentrations (<10 mM) of strong acids such as sulfuric acid, methane sulfonic acid, hexafluoro butyric acid, but also weaker acids such as carbonic acid and phosphoric acid are used. Admixture of organic solvents at low concentrations is sometimes used in ICE to reduce strong secondary interactions with the stationary phase and reduce retention for analytes suffering from that.


The instrument demands on an ICE separation may be slightly lower than typical HPLC equipment due to that most separations are isocratic, and the eluted peak volumes normally are somewhat larger. Representative detectors in ICE include refractive index (RI), ultra-violet light adsorption (UV), and conductivity (CD), the latter sometimes combined with suppressors to achieve lower backgrounds and higher signals.

Learn more

To learn more, browse the application examples below or contact Diduco to discuss your specific analysis.


Separation of organic acids can be a challenging task due to their hydrophilic nature and structural diversity. One of the more traditional approaches to this analysis is ion exclusion chromatography, which offer complementary selectivity to other separation techniques, especially for the monovalent acids which tend to be more strongly retained in this mode.

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