Chromatography with Chemically Bonded Phases

Section 7.5 described the chemical derivatization of silica. The stationary phases obtained may have any polarity. Because of their importance the nonpolar products have already been described in Chapter 10, so this chapter is devoted to some high and medium polarity phases, the discussion being limited 

The separations obtained with these stationary phases are often similar to those on silica, although the selectivity may be different (depending on type), a fact the analyst should take advantage of. These phases have the following advantage over silica  

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Martire, D.E. and Boehm, R.E., Unified theory of retention and selectivity in liquid chromatography. 2. Reversed phase liquid chromatography with chemically bonded phases, J. Phys. Chem., 87, 1045, 1983. 

J. J. Kirkland, High speed liquid-paitition chromatography with chemically bonded organic stationaiy phases, 7. Chromatogr. Sci. 9 206 (1971). 

If retardation is caused by adsorption on granular solids or other fixed surfaces, the technique is called adsorption chromatography, as noted above. If the solid surfaces merely act as a scaffold to hold an absorbing liquid (which may be of a chosen polarity) in place—perhaps within the pores of solid particles—we have partition chromatography. Many cases lie between these extremes this occurs whenever the solid acts as a support for liquid but retains some adsorptive activity [7]. A special case exists with chemically-bonded phases (CBPs), which usually consist of a one-molecule thick layer of hydrocarbon (often C18) chemically bonded to the solid surface. (These nonpolar phases are frequently used for nonpolar solutes in RPLC.) Partitioning into such a thin layer is affected by the nearby surface, especially since the configuration and motion of each attached molecule is restricted by its fixed anchor to the surface [8]. 

The utility of ion suppression techniques in the analysis of ionisable molecules by reverse phase chromatography is limited to samples of weakly basic or acidic compounds. The analysis of stronger acids (pATj < 3) or stronger bases (pA j, > 8) would require eluant of pH, <2 or >8, respectively. Reverse phase chromatography with chemically bonded stationary phases is, however, restricted to eluant pH >2 and <8 for reasons previously discussed. 

Crude chloroform-methanol-water (30 60 8, v/v) extracts of immunostainedTLC bands were analyzed without further purification by nanoelectrospray low-energy mass spectrometry. The authors showed that this effective PLC/MS-joined procedure offers a wide range of applications for any carbohydrate-binding agents such as bacterial toxins, plant lectins, and others. Phenyl-boronic acid (PBA) immobilized on stationary support phases can be put to similar applications. This technology, named boronate affinity chromatography (BAC), consists of a chemical reaction of 1,2- and 1,3-diols with the bonded-phase PBA to form a stable... 

The influence of the bonded organic moiety on solute retention has not yet been elucidated and only a very small number of papers discuss the properties and use of such phases so far. The numerous advantages of chemically bonded phases make the application of polar chemically bonded phases with nonpolar eluents quite attractive even if the standardization of these phases may pose problems 106) similar to those encountered in the standardization of aidsorbents as well as of polymeric liquid phases in gas chromatography. A detailed discussion of the properties and chromatographic use of bonded stationary phases is given by Melander and Horvath (this volume). 

Some problems associated with conventional LLC (e.g., the loss of the liquid stationary phase through dissolution in the mobile phase) may be obviated by chemically bonding the liquid stationary phase to the support medium. This type of liquid-liquid chromatography is designated bonded phase chromatography (BPC)(11). Since the properties of bonded phases may differ substantially from those of coated phases, BPC separation characteristics may differ from those of conventional LLC. Many phases have exhibited increased efficiency when bonded to the support medium. Most current reverse phase HPLC work involves the use of stationary phases bonded to microparticles. 

In reversed phase liquid chromatography (RPLC) silylated silicas are preferred. The surface of these silicas is covered with chemically bonded non-polar groups such as alkyl chains or polymeric layers (Chapter 3.2.3). Silica modified with medium polar groups such as cyano, diol or amino might be used in NP as well as RP mode. Alternatively, cross-linked polymers such as hydrophobic styrene divinyl benzene-copolymers can be used (Chapter 3.2.4). Polymer packings show stability in a pH range 2-14 while silica based packings show limited stability for pH > 7. 

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