Stationary silanol activity

Reversed-phase liquid chromatography shape-recognition processes are distinctly limited to describe the enhanced separation of geometric isomers or structurally related compounds that result primarily from the differences between molecular shapes rather than from additional interactions within the stationary-phase and/or silica support. For example, residual silanol activity of the base silica on nonend-capped polymeric Cis phases was found to enhance the separation of the polar carotenoids lutein and zeaxanthin [29]. In contrast, the separations of both the nonpolar carotenoid probes (a- and P-carotene and lycopene) and the SRM 869 column test mixture on endcapped and nonendcapped polymeric Cig phases exhibited no appreciable difference in retention. The nonpolar probes are subject to shape-selective interactions with the alkyl component of the stationary-phase (irrespective of endcapping), whereas the polar carotenoids containing hydroxyl moieties are subject to an additional level of retentive interactions via H-bonding with the surface silanols. Therefore, a direct comparison between the retention behavior of nonpolar and polar carotenoid solutes of similar shape and size that vary by the addition of polar substituents (e.g., dl-trans P-carotene vs. dll-trans P-cryptoxanthin) may not always be appropriate in the context of shape selectivity. 

Temperature, pressure, and density may also influence SFC selectivity in other ways. For example, water solubility in superaitical fluids generally increases with temperature, causing a shift the equihbrium of the number of water-deactivated silanol groups to carbon-dioxide-deactivated groups [1]. Therefore, the solubihty of analytes in the mobile phases inaeases but so does retention for polar analytes due to increased stationary-phase activity. 

In this section, we explain the principles that influence the selectivity of a reversed-phase column. The first parameter is the hydrophobicity of the stationary phase, which can be measured with purely hydrophobic probes. The second value describes the silanol activity, which is of special importance for basic analytes. Naturally, the silanol activity is best measured using a basic compound, using a correction for the hydrophobic contribution of the structure of the analyte to its retention. The third value is the polar selectivity, which measures the formation of hydrogen bridges between analytes and the stationary phase. With these values, one can create selectivity charts that can help in the selection of the best stationary phase for a particular separation problem. These charts help in method development, whether one would like to find a stationary phase that is drastically different or one that is rather similar to one that is available or in use. At the end of the chapter, we briefly touch on the subject of stationary phase reproducibility. 

Fig. 1. Plot of silanol activity versus hydrophobicity of the stationary phase. Designations (1) Nova-Pak CN HP, (2) Waters Spherisorb CN RP, (3) Hypersil CPS CN,...

These comments demonstrate that the classification of the stationary phases is in line with expectation and is consistent with practical experience. The reproducibility of the values surely depends on the phase and the manufacturer. Therefore, one should expect some variation of the values given here. For the retention factor of a hydrophobic probe, one should expect today a relative standard deviation of around 5%. Kele [16] has demonstrated that the relative standard deviation of the batch-to-batch reproducibility achieved with Symmetry Cjg is as low as 1.3%. The silanol activity can have a standard deviation as high as 15% in... 

The fundamental reason for this phenomenon is the fact that the selectivity of a separation arises from a combination of the influence of the stationary phase and the influence of the mobile phase. If the composition of the mobile phase is drastically different from the test conditions, one can expect a different position of the different columns relative to each other. It remains correct that Symmetry Cj8 has a lower silanol activity than Spherisorb ODS-2, but whether a Lima Cjg(2) or a YMC-Pack Pro Cjg has a higher hydrophobicity or a lower silanol activity surely depends on the details of the measurement conditions. 

In general, one can be sure that the hydrophobidty of the stationary phase will play the major role, no matter what the analyte is. If the analyte bears basic functional groups, then the silanol activity will surely affect the retention. However, if the analyte does not contain basic functions, then the silanol activity is not very important see also Chapter 2.1.1. The same is true for the polar selectivity. The similarities and differences of stationary phases depend upon the nature of the analytes. For example, the stationary phases Hypersil Elite Cjg, 89, and Synergi MAX RP, 78, are practically identical with respect to hydrophobicity and polar selectivity, but the silanol activity at neutral pH is much lower for the Hypersil Elite Cjg column. Therefore, we would expect very similar separations on both columns if the silanol activity does not play a significant role, but more different separations if the opposite is true. On the other hand, if one is working on a new separation and would like to efficiently exploit the selectivity differences of different columns in an automated method development scheme, then the selectivity charts presented here will certainly be useful in the selection of columns of different selectivity properties. 

In view of the previous observations (Fig. 4), chromatographic data should be separately treated in each mobile phase to obtain a fine distinction of stationary phases in relation to their silanol activity (pH 7.0) or hydrophobicity (pH 3.0). 

Column evaluations obtained in mobile phases 1 and 2 were very similar at first sight and both seem appropriate for the evaluation of stationary phases in relation to their silanol activity. Results obtained with the two mobile phases indicated that a slightly better batch variability and asymmetry were achieved when using methanol as organic modifier (data not shown). 

The most widely used particulate support is diatomaceous earth, which is composed of the silica skeletons of diatoms. These particles are quite porous, with surface areas of 0.5-7.5 m /g, which provides ample contact between the mobile phase and stationary phase. When hydrolyzed, the surface of a diatomaceous earth contains silanol groups (-SiOH), providing active sites that absorb solute molecules in gas-solid chromatography. 

Silica gel and aluminium oxide layers are highly active stationary phases with large surface areas which can, for example, — on heating — directly dehydrate, degrade and, in the presence of oxygen, oxidize substances in the layer This effect is brought about by acidic silanol groups [93] or is based on the adsorption forces (proton acceptor or donor effects, dipole interactions etc) The traces of iron in the adsorbent can also catalyze some reactions In the case of testosterone and other d -3-ketosteroids stable and quantifiable fluorescent products are formed on layers of basic aluminium oxide [176,195]... 

Retention in RP chromatography is based on the interaction of the hydrophobic part of the analyte with the hydrophobic section of the stationary phase. This interaction can be modulated with the type and the concentration of the organic modiher in the mobile phase. The selectivity is mainly inflnenced by the interaction of the polar fnnctional gronps of the analyte with constituents of the mobile phase (bnffer, salts, etc. in the aqneons part) and with the amonnt and activity of residual surface silanols, which are, of course, also modihed by mobile phase constituents. 

This assay is altogether more difficult since three active ingredients are involved and several excipients interfere in the analysis, including one major excipient (methylparaben), which is not removed in the extraction process. In addition the active ingredients are bases which have a tendency to interact with any uncapped silanol groups in the stationary phase and it is essential to use a column which is deactivated with respect to the analysis of basic compounds. The three active ingredients are all at different concentrations in the formulation so that attention has to be paid to selection of a detection wavelength at which each component can be detected. In this particular assay a DAD would be useful. 

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