Reversed-phase chromatography stationary phases

Sentell, K.B. and Dorsey, J.G, Retention mechanisms in reversed-phase chromatography. Stationary-phase bonding density and solute selectivity, J. Chromatogr., 461, 193, 1989. 

Figure 4.10. Plot of the retention factor as a function of the volume fraction (% v/v) of organic solvent in reversed-phase chromatography. Stationary phase is an octadecylsiloxane-bonded silica sorbent with methanol-water as the mobile phase. Solute identification 1 = naphthalene 2 = bromobenzene 3 = acetophenone 4 = 2-phenylethanol and 5 = benzamide.

Capillary gas chromatographic methods are described for the analysis of chemically bonded reversed-phase HPLC stationary phase materials. Procedures for acid digestion of the packing material producing stable and volatile derivatives easily analyzed by gas chromatography are compared for precision and accuracy. The methods were used to analyze a variety of commercial phases, possessing widespread monofunctional silane chemistries and bonding consistencies. 

In contrast to reversed-phase, the stationary phase in normal-phase chromatography is polar, usually silica or alumina, and uses nonpolar solvents, e.g., hexane and ethylacetate, that are not compatible with the API processes nsed in LC-MS. In normal-phase chromatography compounds elute progressively from the least to the most polar. The technique is not applicable to the highly polar compounds encountered... 

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. 

Table 8.1 Typical stationary and mobile phases for normal and reverse phase chromatography ...

HPLC requires a mobile phase in which the analytes are soluble. The majority of HPLC separations which are carried out utilize reversed-phase chromatography, i.e. the mobile phase is more polar then the stationary phase. In these systems, the more polar analytes elute more rapidly than the less polar ones. 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Compared with liquid column chromatography, in PLC there is a certain limitation with respect to the composition of the mobile phase in the case of reversed-phase chromatography. In planar chromatography the flow of the mobile phase is normally induced by capillary forces. A prerequisite for this mechanism is that the surface of the stationary phase be wetted by the mobile phase. This, however, results in a Umitation in the maximum possible amount of water applicable in the mobile phase, is dependent on the hydrophobic character of the stationary RP phase. To... 

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

In reversed-phase chromatography (RPC), the mobile phase modulator is typically a water-miscible organic solvent, and the stationary phase is a hydrophobic adsorbent. In this case, the logarithm of solute retention factor is commonly found to be linearly related to the volume fraction of the organic solvent. 

The term chemistry in interphases was first introduced in the field of reverse-phase chromatography [41], In 1995 Lindner et al. transferred the concept to the area of transition metal catalysis [42] and in a recent review the concept is explained in detail [43], The interphase is defined as a region within a system in which the stationary and a mobile component penetrate on a molecular level without the formation of a homogeneous mixture. In these regions the reactive centre on the stationary phase... 

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. 

Reverse phase chromatography describes a system with a non-polar stationary phase and a polar mobile phase. 

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. 

The influence of various structural and physicochemical parameters of the stationary and mobile phases on the tailing of a cationic dye in reversed-phase chromatography has been studied in detail. Measurements were performed in a C8 reversed-phase column (80 X 4.6 mm). The isocratic mobile phase was ACN-0.01 M aqueous HC1 (90 10, v/v). Analyses were carried out at 20°C and the flow rate was 1-5 ml/min. The concentration of the cationic dye, l,l -didodecyl-3,3,3, 3 -tetramethylindocarbocyanine perchlorate (Dil) in the model solutions varied between 0.9-309 pM. The dependence of the chromatographic profile of the dye on the injected concentration is illustrated in Fig. 3.112. Calculations and mathematical modelling indicated that the peak tailing of the dye can be... 

Reversed-phase chromatography employs a nonpolar stationary phase and a polar aqueous-organic mobile phase. The stationary phase may be a nonpolar ligand, such as an alkyl hydrocarbon, bonded to a support matrix such as microparticulate silica, or it may be a microparticulate polymeric resin such as cross-linked polystyrene-divinylbenzene. The mobile phase is typically a binary mixture of a weak solvent, such as water or an aqueous buffer, and a strong solvent such as acetonitrile or a short-chain alcohol. Retention is modulated by changing the relative proportion of the weak and strong solvents. Additives may be incorporated into the mobile phase to modulate chromatographic selectivity, to suppress undesirable interactions of the analyte with the matrix, or to promote analyte solubility or stability. 

The mechanism of reversed-phase chromatography arises from the tendency of water molecules in the aqueous-organic mobile phase to self-associate by hydrogen bonding. This ordering is perturbed by the presence of nonpolar solute molecules. As a result, solute molecules tend to be excluded from the mobile phase and are bound by the hydrophobic stationary phase. This solvophobic... 

Figure 4.2 Protein transformations in reversed-phase chromatography for a two-state model. The native folded state can exist in either the mobile phase (Fm) or the stationary phase (Fs), as can the unfolded state (Um, Us). The equilibrium constants (k) for interconversions of the four species are indicated. (Reproduced from X.M. Lu, K. Benedek, and B.L. Karger, J. Chromatogr., 359 19 [1986]. With permission from Elsevier Science.)...

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