Column Selectivity in Reversed-Phase Liquid Chromatography

Column Selectivity in Reversed-Phase Liquid Chromatography 

The selectivity of reversed-phase liquid chromatography (RP-LC) columns is known to vary, even columns with the same ligand (e.g., Cjg). Column selectivity can also vary from batch to batch for columns claimed to be equivalent by the manufacturer. For different reasons, it is sometimes necessary to locate a replacement column for a given assay that will provide the same separation as the previous column. In other cases, as in HPLC method development, a column of very different selectivity may be needed - in order to separate peaks that overlap on the original column. For each of these situations, means for measuring and comparing column selectivity are required. Until recently, no such characterization of column selectivity was able to guarantee that two different columns can provide equivalent separation for any sample or separation conditions. 

The present chapter describes the hydrophobic-subtraction model of RP-LC column selectivity, for characterizing columns in terms of five fundamental column properties (H, S, A, B, C). Because the hydrophobic-subtraction model can predict retention (values of k) within a few percent, coltunns with sufficiently similar values of H, S, etc. should provide equivalent separation for any sample. Similarly, columns with very different values of H, S, etc. should provide quite different separation. Several examples of the use of values of H, S, etc. for the selection of similar columns have been reported, some of which are discussed below. The present chapter also compares the selectivity of different column types (e.g., alkyl-silica columns, phenyl columns, cyano columns, etc.) and summarizes values of H, S, etc. for several commercial Cjg columns. 

Some of the above (and other) contributions to column selectivity (for neutral solute molecules) have been incorporated into the solvation equation model [6] for RP LC retention  

Over the past few years, an alternative method for specifying column selectivity has been proposed [9-19], which will form the basis for the following discussion. 

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Gilroy, J. J., Dolan, J. W, Carr, P. W. and Snyder, L. R., Column Selectivity in Reversed-phase Liquid Chromatography V. Higher Metal Content (type-A) Alkyl-silica Columns,/. Chromatogr. A, 1026 77—89,2004. 

J. J. Gilroy, J. W. Dolan, and L. R. Synder, Column selectivity in reversed-phase liquid chromatography IV. Type-B alkyl-silica columns, /. Chromatogr. A 1000 (2003), 757-778. 

Wilson, N.S., Dolan, J,W, Snyder, L.R., Carr, P.Wand Sander, L.C. (2002) Column selectivity in reversed-phase liquid chromatography. 111. The physicochemical basis of selectivity. J. Chromat., 961, 217-236. 

The maximum retention factor (kQ) is related to the log P value and k and k are the retention factors of the cationic and anionic forms, respectively. The pKa values are known, and the retention factor in a given eluent can therefore be predicted in reversed-phase liquid chromatography using an alkyl-bonded silica gel or polystyrene gel column. The separation conditions can be adjusted according to their logP and pKa values by the selection of a suitable organic modifier concentration and the pH of the eluent.3,4... 

Additionally, the separation of diastereomers by reversed-phase liquid chromatography is preferred over normal-phase liquid chromatography, because the former method does not require the need to switch to chirally selective columns. The diastereomers used in reversed-phase liquid chromatography are relatively large molecules whose optimized energies vary widely. Their molecular shapes can be used to derive the differences in their contact surface areas using a model phase. 

Deyl Z, Macek K, and Janak J (1975) Liquid Column Chromatography. Amsterdam Elsevier Scientific. Heinisch S, Riviere P, and Rocca J-L (1991) Computer routine for the selection and the optimization of multisolvent mobile phase systems in reversed phase liquid chromatography. Chromatographia 32 559-565. 

Figure 13.7 Selectivity effected by employing different step gradients in the coupled-column RPLC analysis of a surface water containing 0.40 p-g 1 bentazone, by using direct sample injection (2.00 ml). Clean-up volumes, (a), (c) and (d) 4.65 ml of M-1, and (b) 3.75 ml of M-1 transfer volumes, (a), (c) and (d), 0.50 ml of M-1, and (b), 0.40 ml of M-1. The displayed cliromatograms start after clean-up on the first column. Reprinted from Journal of Chromatography, A 644, E. A. Hogendoom et al, Coupled-column reversed-phase liquid chromatography-UV analyser for the determination of polar pesticides in water , pp. 307-314, copyright 1993, with permission from Elsevier Science.

Normal-phase liquid chromatography is thus a steric-selective separation method. The molecular properties of steric isomers are not easily obtained and the molecular properties of optical isomers estimated by computational chemical calculation are the same. Therefore, the development of prediction methods for retention times in normal-phase liquid chromatography is difficult compared with reversed-phase liquid chromatography, where the hydrophobicity of the molecule is the predominant determinant of retention differences. When the molecular structure is known, the separation conditions in normal-phase LC can be estimated from Table 1.1, and from the solvent selectivity. A small-scale thin-layer liquid chromatographic separation is often a good tool to find a suitable eluent. When a silica gel column is used, the formation of a monolayer of water on the surface of the silica gel is an important technique. A water-saturated very non-polar solvent should be used as the base solvent, such as water-saturated w-hexane or isooctane. 

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. 

Macek et al. [120] developed a method to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The method is based on the protein precipitation with acetonitrile and a reversed-phase liquid chromatography performed on an octadecylsilica column (55 x 2 mm, 3 /im). The mobile phase consisted of methanol-10 mM ammonium acetate (60 40). Omeprazole and the internal standard, flunitra-zepam, elute at 0.80 0.1 min with a total rim time 1.35 min. Quantification was through positive-ion made and selected reaction monitoring mode at m/z 346.1 —> 197.9 for omeprazole and m/z 314 —> 268 for flunitrazepam, respectively. The lower limit of quantification was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. The method was applied to the analysis of samples from a pharmacokinetic study. 

In normal-phase liquid chromatography on silica and alumina the hydrogen bonding of fullerenes with surface hydroxyl groups of adsorbent is the most important. On separation of Ceo and C70 on a column packed by alumina Alusorb N 200 from n hexane the selectivity is 1.80 (Fig. 2 a.). The reversed-phase adsorbents such as silica with bonded diphenylsilyl... 

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