24
August/September 2011
Evolution of Mixed-Mode Chromatography
Vlad.Orlovsky@sielc.com,
Yury.Zelechonok@sielc.com SIELC Technologies
Mixed-mode chromatography is emerging as a powerful tool in separation of various molecules. Mixed-mode is defined as liquid chromatography in which at least two modes of interactions exist simultaneously, both controllable by mobile phase selection. In the last few years, several companies have introduced new mixed-mode columns with different stationary phases (SIELC, Dionex, Imtakt, Sepax, Agela, etc). Mixed-mode approach emerged due to a need for better retention control for both polar and non-polar molecules. Multiple mechanisms of interactions allow the use of one stationary phase for a much wider range of applications as compared to reversed-phase or ion-exchange approach. Simultaneous analysis of polar and non-polar, ionisable and neutral, and organic and inorganic compounds is possible, adding ability to reduce number of methods for pharmaceutical formulations and other complex mixtures. Modern mixed-mode stationary phases are designed with the purpose of creating controllable multiple interactions, and to provide reproducibility, high efficiency and loadability. Tools are available for efficient method development [1-3]
History of Mixed-Mode Chromatography
Mixed-mode interactions have been known for decades, and for the most part Liquid Chromatography column stationary-phase design has been dominated by the goal of elimination of extra interactions and obtaining a simple and predictable single retention mechanism. Unfortunately, simplification of the retention process limits the range of compounds for simultaneous analysis, and reduces the ability to control elution order of analytes as well as the scope of available applications the system can be used for. As a response to this limitation, hundreds of different reverse-phase columns have been introduced in the past few years, all with slightly different characteristics. While working mainly by hydrophobic mechanism, those columns provide additional weak poorly controllable interactions.
At the same time, there were some limited attempts to commercialise mixed-mode columns. Such columns were introduced by several column manufacturers using different approaches (Figure 1).
In fact, the first generation of mixed-mode columns (Figure 1a) is represented by low- coverage C18 stationary phases. Residual silanol groups provide additional ionic interaction. These groups have acidic properties with pKa of about 5. A problem with this approach is that inclusions, or
Figure 1. Variations of commercially available mixed-mode packing materials.
contaminations, in silica gel can affect acidity of the silanols, and thus cause un-uniformity of ionic sites. Other problems include the consistency of partial coverage, and the narrow range of mobile phase pH to provide the silanol ionisation.
A different approach included physical mixing of different type of silica gels to obtain the “mixed-mode” effect.
Second-generation mixed-mode columns (Figure 1b) combine one of the ion-exchange
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