Reversed-phase chromatography effects

These sorbents may be used either for selective fixation of biological molecules, which must be isolated and purified, or for selective retention of contaminants. Selective fixation of biopolymers may be easily attained by regulation of eluent polarity on the basis of reversed-phase chromatography methods. Effective isolation of different nucleic acids (RNA, DNA-plasmid) was carried out [115, 116]. Adsorption of nucleosides, nucleotides, tRN A and DNA was investigated. It was shown that nucleosides and nucleotides were reversibly adsorbed on... 

To assess the trapping of biological nucleophiles, the pyrido[l,2-a]indole cyclopropyl quinone methide was generated in the presence of 5 -dGMP. The reaction afforded a mixture of phosphate adducts that could not be separated by reverse-phase chromatography (Fig. 7.16). The 13C-NMR spectrum of the purified mixture shown in Fig. 7.16 reveals that the pyrido [1,2-a] indole was the major product with trace amounts of azepino[l,2-a] indole present. Since the stereoelec-tronic effect favors either product, steric effects must dictate nucleophilic attack at the least hindered cyclopropane carbon to afford the pyrido[l,2-a]indole product. Both adducts were stable with elimination and aromatization not observed. In fact, the pyrido [1,2-a] indole precursor (structure shown in Scheme 7.14) to the pyrido [l,2-a]indole cyclopropyl quinone methide possesses cytotoxic and cytostatic properties not observed with the pyrrolo [1,2-a] indole precursor.47... 

Zhou, N. E., Mant, C. T., and Hodges, R. S., Effect of preferred binding domains on peptide retention in reversed-phase chromatography amphipathic... 

Reverse-phase chromatography is used mainly for the separation of nonionic substances because ionic, and hence strongly polar, compounds show very little affinity for the non-polar stationary phase. However, ionization of weak acids (or weak bases) may be suppressed in solvents with low (or high) pH values. The effect of such a reduction in the ionization is to make the compound more soluble in the non-polar stationary phase but the pH of the solvent must not exceed the permitted range for bonded phases, i.e. pH 2-8. 

While the technique of ionic suppression (or ionization control) is only effective with weakly ionic species, ion-pair chromatography has been developed for strongly ionic species and again utilizes reverse-phase chromatography. If the pH of the solvent is such that the solute molecules are in the ionized state and if an ion (the counter-ion) with an opposite charge to the test ion is incorporated in the solvent, the two ions will associate on the basis of their opposite charges. If the counter-ion has a non-polar chain or tail, the ion-pair so produced will show significant affinity for the non-polar stationary phase. 

Wise, S.A. and May, W.E., Effect of C18 surface coverage on selectivity in reversed phase chromatography of polycyclic aromatic hydrocarbons. Anal. Chem., 55, 1479, 1983. 

Fnhanced-fluidity liquid reversed-phase chromatography has numerous applications including the separation of nonpolar and polar compounds. For example, EFLC and nonaqueous reversed-phase HPLC are the common means of achieving effective separations of high molecular weight homologous compounds. 

Eluents used in reversed-phase chromatography with bonded nonpolar stationary phases are genei ly polar solvents or mixtures) of polar solvents, such as acetonitrile, with water. The properties of numerous neat solvents of interest, their sources, and their virtues in teversed-phase chromatography have been reviewed (128). Properties of pure solvents which may be of value as eluents are summiuized in Table. VII. The most significant properties are surface tension, dielectric constant, viscosity, and eluotropic value. Horvath e/ al. 107) adapted a theory of solvent effects to consider the role of the mobile phase in determinmg the absolute retention and the selectivity found in reversed-phase chromatography. 

In the preceding sections we have considered the role of the solvent in determining the solubility of a molecule taken from the gas phase. However, we are interested in a quantitative evaluation of the solvent effect on phenomena which involve more than one molecule, such as the equilibrium distribution process in reversed-phase chromatography using hydro-carbonaceous bonded phases. 

Pic. 53. Schematic illustration of the effect obtained when a neat aqueous eluent is used instead of a hydrooiganic mixture in iompair reversed-phase chromatography. Reprinted with permission from Horvfith el al. ( 4), Anal. Chem. Copyright 1977 by the American Chemical Society.

FIGURE 5.4 Effect of the gradient dwell volume, V7>. the elution volume, Vj, in reversed-phase chromatography. Solute neburon, retention equation (Equation 5.7) with parameters a=A, m = 4. Linear gradients 2.125% methanol/min (a) from 57.5% to 100% methanol in water in 20min ( i = 50) (b) from 75% to 100% methanol in water in 11.75 min (k = 10). Vg uncorrected calculated from Equation 5.8, Vg + Vg, Vg, added to Vg uncorrected, Vg corrected calculated from Equation 5.21. (A) A conventional analytical C18 column, hold-up volume y ,= ImL flowrate l.OmL/min. (B) A microbore analytical C18 column, hold-up volume y = 0.1mL flow rate 0.1 mL/min. 

Hydrophobic Effects and Solvophobic Considerations for the Isolation of Peptides by Reversed-Phase Chromatography Methods... 

Influence of van der Waals Effects and Other Weak Forces on Peptide Interactions in Reversed-Phase Chromatography... 

Particle Size, Pore Diameter, and Ligand Density Effects in the Reversed-Phase Chromatography Separation of Peptides... 

There remains little more for the operator to decide. Sometimes, alternative but similar solvent mixtures that have a lower viscosity or higher solute diffusivity could be selected. For example, a n-hexane/methanol mixture might be chosen as an alternative to the more viscous n-heptane/isopropyl alcohol mixture as it has similar elution properties. However, it will be shown later, that if a fully optimized column is employed the viscosity of the mobile phase does not seem to effect the column performance as it is taken into account in the optimization procedure. The operator would, under some circumstances, be free to choose less toxic or less costly solvents for example, in reverse phase chromatography the operator could select methanol/ water solvent mixtures as opposed to acetonitrile/water mixture on the basis of lower cost or less... 

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