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Tech Tip — Why do I Lose Retention on my C18 in Highly Aqueous Mobile Phases?

By Rick Lake, Pharmaceutical Marketing Manager

When analyzing polar compounds by conventional reversed phase chromatography on a C18 column, the percent organic (%B) often must be lowered in order to provide adequate retention. This is because low retention (often k'<1) results from polar compounds having a greater affinity for the organic mobile phase than for the hydrophobic C18 stationary phase. However, using a low organic mobile phase is not a universal solution, as it can also cause method problems such as lower efficiency, longer equilibration periods, and decreasing retention over time. More significantly, a conventional C18 stationary phase is prone to de-wetting, which is an expulsion of the highly aqueous mobile phase from the pores of the silica particle [1]. This manifests as a complete loss of retention and typically occurs after de-pressurization of the column.

De-wetting is a function of column pressure, since pressure is required to force the mobile phase into the pores of the particle, as shown in the equation below.

Equation 1:

The pressure required to limit de-wetting is higher for more hydrophobic phases (e.g. pressure requirements are greater for a C30 phase and than for a C1) and for columns constructed using particles with smaller diameter pores. Therefore, with highly hydrophobic phases, like C18s, one of the best ways to create rugged analyses in aqueous mobile phases (less than 5% organic) is to use more polar “aqueous” packings. Aqueous C18 columns are an excellent alternative to conventional C18 columns for the analysis of polar compounds. The Aqueous C18 is a uniquely bonded C18 that resists de-wetting and is completely compatible with 100% aqueous mobile phases. It is more retentive and selective for compounds capable of hydrogen bonding, which ultimately extends the range of compounds that can be analyzed. Beginning method development with a more versatile C18, like the Aqueous C18, can greatly speed up development and lessen method variability.

[1] L.R. Snyder, J.J. Kirkland, J.W. Dolan (Editors), Introduction to Modern Liquid Chromatography. John Wiley & Sons, Hoboken, NJ, 2010, p. 224.

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