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Recent Developments in Type C Stationary Phases: Exploiting the Versatility of Silica Hydride Materials


Joseph J. Pesek*, Department of Chemistry, San Jose State University, San Jose, CA 95192, USA. Email: pesek@sjsu.edu, Tel: 1-408-924-4950, Fax: 1-408-924-4945 Maria Matyska, Microsolv Technology Corporation, One Industrial Way West, Eatontown, NJ 07724, USA Corresponding author*


TYPE C™ silica is a relatively new chromatographic material that has been finding ever-increasing use in the last few years. The properties exhibited by these stationary phases are often significantly different than the ordinary silica used for most commercial products. While all TYPE C phases can be utilised in the reversed-phase, organic normal phase and aqueous normal phase modes, there are some unique capabilities within each retention mode that have resulted in innovative method development strategies with great success. Some of the more challenging separation problems involve polar compounds; two approaches for the analysis of hydrophilic compounds are described in this report.


Introduction


TYPE C silica, based on a surface of Si-H, was introduced many years ago. However, it has only been recently that some of the unique chromatographic features of this material have been discovered and exploited in solving challenging separation problems. This report focuses on the capabilities of these stationary phases for the separation of hydrophilic materials in two modes: aqueous normal phase (ANP) that utilizes high organic content mobile phases and in reversed-phase using high aqueous content mobile phases. For descriptions about the chromatographic properties of TYPE C in the organic normal phase, earlier reports have provided examples of separations utilising this separation mechanism [1,2].


The chromatographic retention and separation of polar compounds continues to be a challenging analytical problem. The versatility and ruggedness of reversed-phase chromatography for separations based on hydrophobic interactions has not been matched by any single method for hydrophilic species. A number of approaches have been developed for polar compound retention but many are limited in their applicability or have other serious drawbacks. For example polar compounds can be derivatised to make them amenable to RP methods, but this is often time- consuming or not very reproducible.


Ion-exchange can be used for some polar compounds, provided they have a permanent charge, but is not applicable to neutral polar compounds like carbohydrates and is also not compatible with mass spectrometry, the most rapidly expanding method of detection. Making polar compounds neutral by the use of extremely high pH mobile phase and more recently, hydrophilic interaction liquid chromatography (HILIC) have been introduced as a means of analysing polar compounds. However, many labs report that HILIC methods have poor reproducibility and systems equilibrate slowly when gradient elution is used.


Also, many of the analytical schemes developed are not compatible with MS detection. Some of the problems reported for HILIC are likely related to the retention mechanism of these materials; generally regarded to be the formation of a water layer near the surface of the stationary phases so that polar molecules partition between it and the more organic-rich surrounding of the mobile phase. TYPE C stationary phases are an entirely different material with a slightly more hydrophobic surface that does not generate a dense water layer at the particle/mobile phase interface. While the mechanism of separation is not yet completely understood, the actual retention and separation capabilities for hydrophilic compounds have been extensively demonstrated [3-7].


The most rapidly growing and the most extensively investigated area of polar compound retention is in the aqueous normal phase (ANP) mode. Under these mobile phase conditions the stationary phases have properties that are similar to the characteristics of HILIC phases (increasing retention with increasing amount of organic component, usually acetonitrile or acetone).


In contrast to many HILIC phases and applications, the TYPE-C silica material is robust (lasts for hundreds of injections), very reproducible from run-to-run (% RSD values generally 0.5% or less) and equilibrates rapidly (five minutes or less) after a gradient method. Applications where the analysis of polar compounds is essential include plant, animal, human and drug metabolomics, clinical analysis, impurity testing, food safety and nutrition, forensics and environmental monitoring.


Figure 1. Analysis of urea in a 100% aqueous mobile phase on a Cogent Bidentate C18 column. Mobile Phase: 100% DI water (isocratic). Column: 4.6 x 150mm. Flow rate: 0.5mL/min. Detection at 210nm. Sample: 1 mg/mL. Injection volume: 10µL.


Reversed Phase


All TYPE C stationary phases display some reversed-phase behaviour. Even the unmodified material can retain nonpolar compounds because the hydride surface is slightly hydrophobic. As the hydrophobicity of the stationary phase is increased by having greater surface coverage of bonded organic moieties, retention of nonpolar compounds increases as with all other reversed-phase materials. Selectivity for common phases such as C18 and C8 is somewhat different because of the underlying hydride surface and the resulting lack of water on this material.


For retention of hydrophilic compounds under RP conditions with TYPE C materials, mobile phases are used that typically contain 90-100% (v/v) water. Stationary phases based on silica hydride are especially suited to these conditions since they do not undergo ‘dewetting’, sometimes referred to as phase collapse or phase fold back. In highly aqueous environments, many hydrophobic bonded phases such as C18 will try to minimise their contact with the polar environment by forming highly associated packets of organic moieties. This process reduces the total hydrophobic surface available for solutes to interact with and thus retention can drop drastically in high aqueous mobile phase environments. A few examples utilising high aqueous content mobile phases will be presented to illustrate this capability.


Figure 1 shows five overlaid chromatograms of a sample containing urea obtained on a C18 TYPE-C stationary phase using a 100% aqueous mobile phase. This highly polar compound is adequately retained under these conditions (k ≈ 1.5) demonstrating that the TYPE-C materials are particularly useful for hydrophilic compounds even in this mode. In addition, there is a remarkable degree of repeatability in these five runs which is another feature of the TYPE C stationary phases. Since this column material has an octadecyl bonded moiety, it can function like other reversed-phase stationary phases and can be used for the analysis of a wide range of hydrophobic compounds as well.


Another example of the retention capability for hydrophilic compounds in the reversed- phase mode is the analysis of guanidine shown in Figure 2. Guanidine is a strong base and is protonated at all pH values below 12. The low molecular weight of guanidine, its positive charge, and lack of a significant chromophore, make the analysis very


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