Stationary phases in reversed- phase chromatography

Hanson, M., Unger, K.K., Schomburg, G. Non-poros polybutadiene-coated silicas as stationary phases in reversed-phase chromatography, J. Chromatogr., 1990, 517, 269. 

In normal-phase chromatography, polar compounds are separated on a polar stationary phase. In reversed-phase chromatography, nonpolar compounds are separated on a nonpolar stationary phase. The latter is more common ... 

The stationary phase in reversed-phase chromatography is poorly defined with a fluid structure, composition and volume that depends on the equilibrium mobile phase composition, the identity and bonding density of surface-restrained ligands, the number and type of accessible silanol groups, and temperature [108,109,240,243-... 

The most commonly used stationary phase in reversed phase chromatography consists of octadecylsilane groups (ODS) covalently bonded to silica however, a number of different types of ODS packings of varying chromatographic character are utilised and these... 

Fig. 2.4. Surface groups used for stationary phases in reversed phase chromatography range from ethyl silane (C2) to n-octadecyl silane (Cig).

As opposed to the usual polar stationary phase and a less polar or non-polar mobile phase, the stationary phase in reverse phase chromatography is hydrophobic (hydrophobic bonded phase usually possessing Cjg or Cg functional groups) and the mobile phase is polar (fully or partially aqueous). In this case, polar substances will interact more with the polar mobile phase and elute first. As the non-polarity of the solute components increases, their retention times will also increase since they will interact more with the non-polar stationary phase.The reverse phase system is therefore very useful for separation of non-polar solutes. Water, an extremely polar solvent becomes the weakest eluent here. Methemol and acetonitrile are stronger eluents than water. Solvents of intermediate eluting strength cem be obtained by mixing one of these solvents with water. 

Michael Hanson, Klaus K. Unger, Renaud Denoyel, and Jean Rouquerol Interactions of lysozyme with hydrophilic and hydrophobic polymethacrylate stationary phases in reversed phase chromatography (RPC), J. Biochem. Biophys. Methods, 29(3-4) (1994) 283-294... 

In reversed-pViase 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. 

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. 

In classical column chromatography the usual system consisted of a polar adsorbent, or stationary phase, and a nonpolar solvent, mobile phase, such as a hydrocarbon. In practice, the situation is often reversed, in which case the technique is known as reversed-phase Ic. 

H. hth, R. Tocklu, K. Welten, G. J. de Jong, U. A. Th Brinkman and R. W. Frei (1989), Trace-level determination of 3 -azido-3 -deoxythymidine in human plasma by precon-centi ation on a silver (I)-tliiol stationary phase with on-line reversed-phase liigh-perfor-mance liquid chromatography , J. Chromatogr. 491 321-330 (1989). 

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. 

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. 

The stationary phase matrices used in classic column chromatography are spongy materials whose compress-ibihty hmits flow of the mobile phase. High-pressure liquid chromatography (HPLC) employs incompressible silica or alumina microbeads as the stationary phase and pressures of up to a few thousand psi. Incompressible matrices permit both high flow rates and enhanced resolution. HPLC can resolve complex mixtures of Upids or peptides whose properties differ only slightly. Reversed-phase HPLC exploits a hydrophobic stationary phase of... 

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. 

Emenhiser C., Sander L.C., and Schwartz, S.J., Capability of a polymeric C30 stationary phase to resolve cis-trans carotenoid isomers in reversed-phase liquid chromatography, J. Chromatogr. A, 101, 105, 1995. 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

Not only in HPLC, but also in modem thin-layer chromatography, the application of reversed-phase stationary phases becomes increasingly important. The advantage of the hydrophobic layers in comparison with the polar, surface-active stationary phases is the additional selectivity and a reduced hkehhood of decomposition of sensitive substances. 

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 most common technique used for agrochemicals is reversed-phase SPE. Here, the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon (e.g. C4-C18) or styrene-divinylbenzene copolymer. This technique operates in the reverse of normal-phase chromatography since the mobile phase is polar in nature (e.g., water or aqueous buffers serve as one of the solvents), while the stationary phase has nonpolar properties. 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

---