Bonded phase materials

Several other methods have been published using RP-HPLC for the determination amphetamines and related derivatives. Studies have shown the determination of amphetamine and related derivatives in plasma, urine, and hair by RP-HPLC with precolumn derivatization and either UV/VlS or fluorescence detection. Various methods are employed by SPE technologies using Cl8 cartridges for sample cleanup prior to derivatization. The derivatized compounds were separated on analytical columns of various Cl 8 bonded phase materials. The methods generally used water/acetonitrile mobile phases operated in gradient mode. All studies reported extraction recoveries of 85-102% for all the analytes, with LLOQs ranging from 5 to 60 ng/ml (Tedeschi et al., 1993 Ealco et al., 1996 Hernandez et al., 1997 Al-Dirbashi et al., 1997 Al-Dirbashi et al., 2000 Soares et al., 2001). 

Occasionally, silica gel and bonded-phase material may be leached from the column and be introduced into the purified sample. This may be removed by centrifugation, filtration, or extraction of the sample with nonpolar solvents, such as hexane. 

Porous silica is most widely used as adsorbent, but bonded phase materials with polar groups or crosslinked acrylonitrile39> have also been tested. Silica requires painstaking control of activity. In the separation of poly(styrene-co-methyl methacrylate) samples with dichloroethane—chloroform mixtures, clearer results were obtained with a silica column previously rinsed with methanol40. Continuously decreasing activity of silica columns was observed in the elution of poly(styrene-co-methyl acrylate) with CCU-methyl acetate mixtures38). 

ODS—Octadecylsilyl bonded phase material or column in which the material bound to silica is an 18-carbon saturated hydrocarbon chain. See Ci8.)... 

Good, T.J., Applications of bonded-phase materials, American Laboratory, July 1981, p. 36. 

Monofunctional reagents such as alkyldimethylmonochlorosilanes yield monomeric packings (first reaction in Fig. 1.8A). Such bonded-phase materials are well defined as one silanol group reacts with one silane molecule and exhibit high efficiency because of low mass-transfer resistance due to fast diffusion of molecules into the flexible fur - or brush -like structure of the alkyl chains on the silica surface. 

A common method to prepare bonded-phase materials is to use silica gel as the solid support and to react functionalized organosilanes onto the surface hydroxyl groups of the silica gel. Of course, other solid support materials can be used provided that they present surface functional groups (e.g., OH, COOH, and NH2) that can participate in modification reactions. The flexibility offered by this approach leads to stationary phase materials with myriad functionalities. Fig. 3 illustrates commonly used organosilane reactants and two example reaction paths that lead to bonded-phase silica. For more discussion on bonded-phase materials, see Ref.. ... 

Fig. 4.4.9. Separation of aromatic solutes on two different bonded phase materials. Chemically bonded ODS silica and perfluorodecylsilyl silica respectively. Reprinted from Ref. 13 with permission.

As mentioned previously residual silanol groups present on the surface of bonded phase materials affect the separation and the peak shape, in particular when using... 

The considerations on the choice of eluents for straight phase separations on polar bonded phase materials do not differ much from those used in adsorption chromatography and in conventional liquid-liquid chromatography. Non-polar solvents with the addition of polar modifiers are used, and eluotropic series as in Table 4.4.1 are useful in the adjustment of eluting strength. 

The synthetic methods used for the preparation of bonded phase materials are illustrated in Figure 6.35. One of the first reported bonded phases, the alkoxy silanes (1) also referred to as silicate esters, was prepared by the direct esterification of silanol groups with alcohols. The major disadvantage of this packing material was its hmited hydrolytic stability, as it is readily hydrolysed by aqueous alcohol eluants. 

Of the other stationary phases, the amino phases have been used extensively to separate sugars and peptides the nitrile phase has found application in the separation of porphyrins. An important consideration in the use of polar bonded-phase materials is an awareness of the reactivity of the terminal functional group, for example, aminoalkyl bonded phase should not be used for the chromatography of carbonyl compounds due to possible condensation reactions and formation of Schiff s bases. 

A major advantage of underivatized silica for LC-MS(/MS) is the absence of ligands that may detach from the surface and show up as spurious peaks in the mass spectra. Recently introduced bonded phase materials seem to have addressed this issue, so that functionalized silica gels are as common today as bare silica in HILIC-MS applications. 

Column packings for normal phase columns are mainly porous silica (spherical or irregular) particles of 5-10 micron average diameter and of tight particle size distribution. For reverse phase, bonded phase materials... 

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