Reversed-phase Materials

Carbon has a strong affinity to high molecular mass substances and has traditionally been used for purification/decoloration in synthetic organic chemistry. The porous graphitic carbon (PGC), which is used for chromatography, behaves as a hydrophobic adsorbent, stronger than reversed-phase materials, but with strong dipolar and electron lone pair interactions for additional retention of polar analytes. 

Porous graphitic carbon particles are used with aqueous mobile phases, but are probably more valuable for solid-phase extraction in small columns than in ordinary HPLC columns. 

Typical mobile phases with PGC are mixtures of alcohols and aqueous buffers, as 

---

Alhedai et al also examined the exclusion properties of a reversed phase material The stationary phase chosen was a Cg hydrocarbon bonded to the silica, and the mobile phase chosen was 2-octane. As the solutes, solvent and stationary phase were all dispersive (hydrophobic in character) and both the stationary phase and the mobile phase contained Cg interacting moieties, the solute would experience the same interactions in both phases. Thus, any differential retention would be solely due to exclusion and not due to molecular interactions. This could be confirmed by carrying out the experiments at two different temperatures. If any interactive mechanism was present that caused retention, then different retention volumes would be obtained for the same solute at different temperatures. Solutes ranging from n-hexane to n hexatriacontane were chromatographed at 30°C and 50°C respectively. The results obtained are shown in Figure 8. 

The ionic or polar substances can be seperated without any reaction on specially treated chromatographic columns and detected refractometrically. This is necessary because alkyl sulfosuccinates show only small absorption in the UV-visible region no sensitive photometric detection can be obtained. Separation problems can arise when common steel columns filled with reverse phase material (or sometimes silica gel) are used. This problem can be solved by adding a suitable counterion (e.g., tetrabutylammonium) to the mobile phase ( ion pair chromatography ). This way it is possible to get good separation performance. For an explanation of separation mechanism see Ref. 65-67. A broad review of the whole method and its possibilities in use is given in an excellent monograph [68]. 

Purification of double-stranded DNA on micropellicular anion exchange and reversed-phase materials has been reviewed.43 Micropellicular phases adsorb only at the surface and have no internal pores. For this reason, the surface area and hence the capacity of micropellicular phases tends to be low. Using small particles (1-3 p in diameter) increases the surface area but may be impractical for preparative work above the mg scale. 

Li, J., Hu, Y. and Carr, P.W., Fast separations at elevated temperatures on polybutadiene-coated zirconia reversed-phase material, Anal. Chem., 69(19), 3884, 1997. 

In this design, on-column sample enrichment is incorporated into the sheathless interface (Janini et al., 2003). A miniaturized solid-phase extraction (mSPE) cartridge, made of reversed-phase material, was attached to the CE capillary near the injection end as shown in Fig. 16.1. 

The polarity values of binary acetonitrile/water and methanol/water mobile phases used in RPLC were measured and compared with methylene selectivity (acH2) for both traditional siliceous bonded phases and for a polystyrene-divinylbenzene resin reversed-phase material [82], The variation in methylene selectivity for both was found to correlate best with percent organic solvent in methanol/water mixtures, whereas the polarity value provided the best correlation in acetonitrile/water mixtures. The polymeric resin column was found to provide higher methylene selectivity than the siliceous-bonded phase at all concentrations of organic solvent. 

Olsson, M., Sander, L.C., and Wise, S.A., Comparison of the liquid chromatographic behaviour of selected steroid isomers using different reversed phase materials and mobile phase compositions, 7. Chromatogr.,531,13, 1991. 

Horak, J., Maier, N.M., and Lindner, W., Investigations on the chromatographic behavior of hybrid reversed-phase materials containing electron donor-acceptor systems ii. Contribution of pi-pi aromatic interactions, J. Chromatogr. A, 1045, 43, 2004. 

The former polymer system represents a reversed-phase material, providing C4-alkylchains, which has most frequently been employed for protein separation [53], whereas the latter carries reactive moieties that can easily be converted in order to yield the desired surface functionalities. 

Polyst3rrene (Figure 4), which is a pol)rmeric support is also imsuitable in its original form for affinity separations due to the highly hydrophobic character. Native polyst3rrene, which is often used as a reversed-phase material, must be first rendered hydrophilic by one of various surface-coating techniques before used in other chromatographic methods [8]. 

Comparable to the other protein-coated supports are the Ultrabiosep and the BioTrap phases. The former are composed of C4, Cg, or Cis reversed-phase silica supports covered with a biological polymer which is not described in the literature (135). The latter are commercially available as Bio Trap Acid or Biotrap Amine precolumns (136). They are Cis-modified silica supports covered with -1-acid glycoprotein as a biocompatible layer. Due to the immobilized protein, this type of reversed-phase material also possesses weak ion-exchange properties. 

In liquid chromatography, reversed-phase materials such as Cig and Cg are the most commonly used sorbents (429, 430, 434, 438, 446, 447, 453, 454). Examples of baseline separations with reversed-phase columns of several groups of anabolics including stilbenes, resorcyclic acid lactones, and other, frequently used anabolics have been reported (463-466). In addition to reversed-phase separations normal-phase separations of anabolics using either Hypersil (467) and Brownlee (456) silica or diol-modified silica have been reported. Although not all analytes were completely separated, the latter column could be efficiently used to differentiate between estrogenic and androgenic compounds within a mixture of 15 anabolics and their metabolites (468). 

Liquid chromatographic separation of sedatives and -blockers is usually performed using reversed-phase columns. The preferred type of reversed-phase material is Cig-bonded silica (Table 29.16), but phenyl-bonded silica has also been employed for separation of xylazine and its major metabolite (525). Ion-pair liquid chromatography has also been suggested for separation of carazolol and xylazine residues, by addition to the mobile phase of dodecyl sulfate (522) or heptanesulfonate (520) pairing ions, respectively. 

The separation and purification of ethyl esters of EPA, DHA, and the heretofore-minor unreported polyunsaturate octadecatetraenoic acid (C18 4 3, OTA) on a preparative scale by modification of an analytical RP-HPLC procedure has been described by Beebe et al. (48). They used a liquid chromatograph equipped with a differential refractometer as detector operated at room temperature and an ST Macrobore column (350 X 4.6-mm ID) of C18 reverse-phase material, 25-yum particle size. 

The column packing consists of Vydac reversed-phase material (chemically bonded ODS). The fluorescamine derivatives are separated by gradient elution with 10% methanol in buffer (pH 8.0), followed by a linear increase in proportion to 40% methanol in buffer (pH 8.0). The separation of several diamine derivatives with this system is shown in Fig.4.51. The limits of detection are ca. 2S-S0 pmoles of amine using a fluorimeter with a microflow cell. Both amino groups of the diamines react with fluorescamine so that a minimum of a 2 1 molar ratio of fluorescamine to diamine is required for the best results, a ratio of 3 1 or 4 1 is preferred. 

In the last entry in Table III more than 50 samples were extracted in an experiment surveying the performance of laboratory robotic equipment. These included 2.0 gram samples of soils extracted with pure C02,2.5 gram samples of a reverse phase material extracted with C02 and mixtures of C02 with various modifiers, 2.0 gram samples of ground coffee plus aliquots of modifiers dispersed on the samples within the sample thimble extracted with pure CO and 20 microliter aliquots of performance evaluation standard (PES, octadecane in isooctane) on simple matrices extracted with pure C02. The variety of analytes and complex matrices along with the number of different runs clearly show that the instrumentation is reliable. 

Soils/reverse phase material/ ground coffee/simple standards >50... 

The HPLC was carried out at room temperature on a 0.8 i.d. x 25 cm stainless steel column, laboratory-packed with Spheri— sorb 0DS (Phase Separations Ltd, Clwyd, U.K.), a reverse-phase material. 

The following table provides a summary of the general characteristics of the most popular stationary phases used in modem high-performance liquid chromatography.1 7 The most commonly used phases are the bonded reverse phase materials, in which separation control is a function of the mobile (liquid) phase. The selection of a particular phase and solvent system is an empirical procedure involving survey analyses. The references provided below will assist the reader in this procedure. 

Solid-phase extraction (SPE) using reversed-phase materials... 

For reverse phase HPLC, removal of volatile organic compounds from water can be especially troublesome. The problem may be diminished by filtering the water through activated charcoal. Pumping the aqueous mobile phase through a scrubber column (packed with reverse phase material) located between the pump and injector also has been useful. 

The buffer system is a combination of buffer for electrophoresis and eluent for the particular chromatographic mode being employed. Figure 5.14 shows the separation of a group of neutral molecules using a capillary packed with a reversed-phase material.38 The buffer was a mixture of 4 mM sodium tetraborate (pH 9.1) and acetonitrile (20 80, v/v). The separation was compared with a micro-HPLC separation in which the same capillary was used but the eluent was pressure driven. As can be seen in Figure 5.14, sharper peaks were obtained with the EOF-driven system. 

---