Polarity, in reversed-phase chromatography

Elution in reversed-phase chromatography is often carried out using a gradient, produced from water and some water-miscible organic solvent. The solute components are thus distributed between the stationary and mobile phases mainly on the basis of their polarities. In reversed-phase chromatography hydrophilic compounds elute before hydrophobic ones. 

Adsorption chromatography The process can be considered as a competition between the solute and solvent molecules for adsorption sites on the solid surface of adsorbent to effect separation. In normal phase or liquid-solid chromatography, relatively nonpolar organic eluents are used with the polar adsorbent to separate solutes in order of increasing polarity. In reverse-phase chromatography, solute retention is mainly due to hydrophobic interactions between the solutes and the hydrophobic surface of adsorbent. Polar mobile phase is used to elute solutes in order of decreasing polarity. 

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an -octyl (Cg) or -octyldecyl (Cig) hydrocarbon chain. Most reverse-phase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5. 

To retain solutes selectively by dispersive interactions, the stationary phase must contain no polar or ionic substances, but only hydrocarbon-type materials such as the reverse-bonded phases, now so popular in LC. Reiterating the previous argument, to ensure that dispersive selectivity dominates in the stationary phase, and dispersive interactions in the mobile phase are minimized, the mobile phase must now be strongly polar. Hence the use of methanol-water and acetonitrile-water mixtures as mobile phases in reverse-phase chromatography systems. An example of the separation of some antimicrobial agents on Partisil ODS 3, particle diameter 5p is shown in figure 5. 

In reverse phase chromatography, the polar mixture components would elute first since they would be attracted by the polar mobile phase and repelled by the nonpolar stationary phase. In normal phase chromatography, nonpolar mixture components would elute first since they would be attracted by the nonpolar mobile phase and repelled by the polar stationary phase. 

In recent years, also the number of articles concerning HILIC stationary phases has enormously increased, especially as regards the hydrophilic interactions that resolve some important problems separation and resolution of less retained compound in reversed phase chromatography. With this novel stationary phase, where the silica surface is covered with cross-linked diol groups to increase polar selectivity in hydrophilic conditions, is possible obviate to the use of normal phase with high water content. This allows facilitating the interfacing with sensible and selective detection instruments, such as mass spectrometer with ESI source. The HILIC stationary phase was often chosen to interface the mass spectrometry detector, because it would be... 

In reverse-phase chromatography, the stationary phase is nonpolar (often a hydrocarbon) and the mobile phase is relatively polar (e.g., water, methanol, and acetonitrile). The most polar components elute first, and increasing the mobile phase polarity (i.e., decreasing the organic solvent concentration) increases elution time. 

The liquid liquid partition chromatography (LLPQ method involves a stationary liquid phase that is more or less immobilized on a solid support, and a mobile liquid phase. The analyte is therefore distributed between the two liquid phases. In conventional LLPC systems, the stationary liquid phase is usually a polar solvent and the mobile liquid phase is an essentially water-immiscible organic solvent. On the other hand, in reversed-phase chromatography (RPQ, the stationary liquid is usually a hydrophobic... 

Adsorption chromatography on polar stationary phases is especially suited for the separation of nonpolar to medium polar substances that have some solubility in solvents immiscible with water. More polar substances, which are soluble in pojar solvents such as alcohols or water, of course, can be, and have been, separated on polar stationary phases, too. However, as nonpolar stationary phases are now easily available and widely used in reversed phase chromatography, such substances are preferentially separated on such phases because of better reproducibility and the convenience offered by this technique. (/)... 

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 reversed-phase chromatography more polar compounds migrate faster through the system than do nonpolar materials, which experience hydrophobic interaction with the solid matrix. 

Mobile phases used in reversed-phase chromatography are frequently composed of mixtures of methanol and water or acetonitrile and water. Increasing the proportion of water causes an increased retention of the more hydrophobic solutes relative to the more polar solutes. The surface tension of the mobile phase plays a major role in governing solute retention, so an increase in temperature, by reducing viscosity, increases column efficiency and shortens retention times. 

The mobile phases used in reversed-phase chromatography are based on a polar solvent, typically water, to which a less polar solvent such as acetonitrile or methanol is added. Solvent selectivity is controlled by the nature of the added solvent in the same way as was described for normal-phase chromatography solvents with large dipole moments, such as methylene chloride and 1,2-dichloroethane, interact preferentially with solutes that have large dipole moments, such as nitro- compounds, nitriles, amines, and sulfoxides. Solvents that are good proton donors, such as chloroform, m-cresol, and water, interact preferentially with basic solutes such as amines... 

C X chains). Consequently, the mobile phases need to be more polar (e.g., CH3CN—H20, MeOH—H20) than the coated surface. During elution, separation of constituents of an applied sample mainly relies upon the differences in distribution coefficients between the mobile phase and the hydrocarbon coatings of the stationary phase. For all these reasons, the more polar constituents of a mixture in reversed phase chromatography are eluted before nonpolar components. Note also that although water is more polar than acetonitrile or methanol, it is a poorer eluent in reversed phase chromatography because of the alkyl surface character of the stationary phase. 

In a series of experiments designed to explore further the role of polarity in affecting retention time in reversed-phase chromatography, we developed chemical procedures for the condensation of molecules of known polarity,... 

P. Zhuang, R. Thompson, and T. O Brien, A retention model for polar selectivity in reversed phase chromatography as a function of mobile phase organic modifier type, /. Liq. Chrom. Rel. Technol. 28 (2005), 1345-1356. 

Kennedy 1990). This form of chromatography uses hydrophilic gel-based material as a matrix, which has been partially substituted on the surface with non-polar alkyl (e.g., methyl or octyl) or aryl (e.g., phenyl) groups (Table 4-3). Similar materials are used in reverse-phase chromatography (RPC), but the degree of substitution used for HIC chromatography (10-50 pmol ml-1 gel) is much lower than that used in RPC (100-500 pmol ml-1 gel). The solvent used for elution in HIC is low ionic strength buffer and not organic solvents which are characteristic of RPC. 

In normal chromatography the mobile phase is usually less polar than the solutes being eluted as they need to be retained on the column by polar forces to achieve the separation. Thus, the presence of a solute in the mobile will, in general, increase the dielectric constant of the mobile phase. In reversed phase chromatography, however, the solute is usually less polar than the solute and, consequently, the dielectric constant of the mobile phase is reducedhy the presence of a solute. It follows that if a device is situated at the end of the column, which responds to changes in dielectric constant, such a device would act as a chromatography detector. In practice the sensor often takes the form... 

---