Reverse phase liquid chromatography mixtures

Glajch, J. L., Kirkland, J. J., Squire, K. M., and Minor, J. M., Optimization of solvent strength and selectivity for reversed-phase liquid chromatography using an interactive mixture-design statistical technique, /. Chromatogr., 199, 57, 1980. 

Gray, M.J., Dennis, G.R., Slonecker, P.J., Shalhker, R.A. (2004). Comprehensive two-dimensional separations of complex mixtures using reversed-phase liquid chromatography. J. Chromatogr. A 1041, 101. 

A comprehensive 2D HPLC can be carried out with two very similar columns in reversed-phase liquid chromatography (Ikegami et al., 2005). A mixture of water and tetrahydrofuran was used as a mobile phase in the lst-D separation, and a mixture of water and methanol (CH3OH) in the 2nd-D separation with a common Ci8 stationary phase. 

From the theoretical viewpoint, acetonitrile is the most suitable solvent to study the correlation of retention times and log P values of analytes, since the dipole moment (2.44) is nearly equal to that of water (2.55) (Figure 4.4). The electron donor effect can therefore be eliminated, and the elution order is not changed on modification of the acetonitrile-water mixture ratio. The first choice of an eluent should therefore be an acetonitrile-water mixture for non-ionic compounds in reversed-phase liquid chromatography. Methanol, acetone, THF, or DMF can then be added to improve the resolution. 

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. 

The retention time of an organic compound in reversed-phase liquid chromatography is heavily influenced by the activity coefficient of the compound in the mobile phase, which commonly consists of a CMOS/water mixture. 

The use of reversed-phase liquid chromatography is growing in applications for separating mixtures of peptides. In this type of chromatography, the stationary phase is nonpolar, whereas the mobile phase is polar. The stationary phase is normally porous silica with bonded n-alkyl chains, mainly octadecyl but also octyl, hexyl, butyl, and propyl chains. 

More satisfactory separations were obtained when reverse-phase liquid chromatography was used. The separation of the standard dimeric mixture was carried out on an LC-18 column with refractometry as the mode of detection and acetonitrile/acetone (1 1) as the mobile phase (system II). Separation proceeded according to the polarity of the various dimers, and complete separation of all dimers, except those of the thermal dimer of methyl linoleate and the dehydrodimer of methyl oleate, were obtained at a flow rate of 0.5 ml/min. The resolution of the two unresolved peaks would be increased by using another LC-18 column in series, but a sacrifice in the analysis time would have to be made. 

Reverse-phase liquid chromatography is now virtually the only method used in the analysis of the TG mixtures. The first paper on TG-HPLC analysis was published in 1975 by Pei et al. (81). Triglycerides were separated on a VYDAC reverse-phase (35 - 44 /xm) column and eluted with methanol-water (9 1). Since Pei et al. first applied RP-HPLC to the separation of triacyl-glycerols, a number of reverse-phase systems have been developed as rapid and efficient resolution of complex triacylglycerol mixtures can be achieved. 

Brinkman et al. [35,36] used a silica gel column which elutes the higher chlorinated PCBs in the normal phase. This system produced a reasonable separation of the lower chlorinated PCBs present predominantly in the commercial mixture Arochlor 1221 but was less efficient in separating the more highly-chlorinated PCBs present in Arochlors 1254 and 2160). Kaminsky and Fasco [37] investigated the potential reversed phase liquid chromatography to the analysis of PCB mixture in environmental samples. They used mixtures of water and acetonitrile as the mobile phase to achieve analysis of 49 different PCBs and of samples of Arochlor 1221, 1016, 1254 and 1260. 

Competitive ionization may be avoided by varying the pH conditions or the matrix, through chemical derivatization of the peptides contained in this mixture, or through the partial fractionation of the mixture through reversed-phase liquid chromatography so that each fraction contains peptides of a similar hydrophobicity. 

Reverse phase liquid chromatography has typically been used for the separation of PFCs, employing either Cg or Cig columns [96], although the use of perfluoroalkyl columns has also been reported [115]. Mobile phases are typically mixtures of methanol-water or acetonitrile-water and are often modified with ammonium acetate to improve chromatographic separation and MS sensitivity. Both isocratic and gradient elution methodologies have been employed [96]. LC-MS/MS methods [116, 117] have also been developed for the separation of PFSA and PFCA isomers and generally employ linear perfluorooctyl stationary phases and acidified mobile phases. 

Figure 4 Depiction of shot-gun proteomics using multidimensional protein identification technology a complex mixture of peptide fragments in the digest are resolved by a combination of ion-exchange and reversed-phase liquid chromatography.

Figure 4.6 illustrates the use of the IAS model to account for the competitive isotherm data of a ternary mixture of benzyl alcohol (BA), 2-phenylethanol (PE) and 2-methyl benzyl alcohol (MBA) in reversed phase liquid chromatography. The RAS model accounts for the nonideal behaviors in the mobile and the stationary phases through the variation of the activity coefficients with the concentrations. Figures 4.6d and 4.6e illustrate the variations of the activity coefficients in the stationary and the mobile phases, respectively. The solutes exhibit positive deviations from ideal behavior in the adsorbed phase and negative deviations from ideal behavior in the mobile phase. 

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