Silica-bonded phases, isolation

It is notable that small peptides were retained on a cross-linked PEI column [37] but polypeptides over 20 residues were weakly retained or not at all. Possibly, small peptides were able to penetrate into the depth of the bonded phase while larger peptides could not. These packings were more useful for the separation of oligonucleotides, i.e. more acidic compounds. Lawson et al. [39] have shown the applicability of PEI-silicas for assessing the purity of precursor blocks, monitoring the chemical synthesis and isolating reaction products after synthesis. 

SFC has played an important role in the extraction and isolation of fatty acids [355,356]. Underivatised fatty acids and methyl esters of fatty acids are surprisingly easy to elute using a bonded phase or a silica based packed column and pure C02, probably due to the long hydrocarbon tails on the molecules [357]. On the other hand, most aromatic and polysubstituted acids will not elute. Triglycerides with saturated fatty acids can be analysed faster with pSCF-ELSD than with GC-FID and do not require sample preparation [358]. Using... 

Extraction discs (0.5 mm thick, 25 to 90 mm diameter) constitute a variation of column-based SPE. These discs allow rapid extraction of large volumes of sample, which is not possible using a small column. The discs are made of bonded-phase silica particles, a few micrometres in diameter, trapped in a porous Teflon or glass fibre matrix. The discs are operated in a similar way to a paper filter on a vacuum flask. After extraction, the analyte is recovered by percolating a solvent through the filter. The major application of this technique is the isolation of trace amounts of compound dispersed in an aqueous medium. 

Selectivity, the ability to isolate a particular analyte or separate a number of components within a mixture, has improved dramatically through the development of bonded-phase column packing materials for reversed-phase, ion-exchange and ion-pair chromatography. More recently, forensic laboratories have been introducing polymeric packing materials because of their selectivity and other physical and chemical properties which are more desirable than those of silica-based materials. 

The rapid development of hydrocarbonaceous bonded phases (nonpolar stationary phases with hydrocarbon chains covalently attached to the silica support) in the 1970s and 1980s for the popular re-versed-phase chromatographic (RPC) technique has, however, eclipsed this technique somewhat, lately. Nevertheless, LLPC has become one of the most powerful separation techniques for the isolation of natural products and biopolymers. 

Apparently, trifunctional reagents will react with adjacent silanol groups and derivatize them, which in turn creates a more hydrophobic surface on the silica gel. The greater stability of the sorbent at low pH is an important consideration for the isolation of acidic compounds. For example, organic acids require that the pH be lowered to two pH units below the for adequate sorption thus, the pH must be lowered to -2.0 for most carboxylic acids with a pAT of about 4. In this case, the trifunctional-derivatized silicas are recommended because trifunctional SPE bonded phases will be more stable at acid pH. 

The types of nonbonded phases used for normal-phase SPE are silica, alumina, and magnesium silicate (Florisil). The most popular phase is silica. Several bonded phases may also be used for normal-phase SPE, including aminopropyl, cyanopropyl, and propyldiol (Table 1.1, Fig. 2.7). Water is not used in the mobile phase in normal-phase SPE because it will sorb to the active sites of the sorbent and reduce the interaction between analyte and sorbent. Typically, normal-phase SPE is used as a clean-up procedure for organic extracts of water, soil, food, or other materials. Normal-phase SPE is also used for the isolation of analytes from organic liquids, such as oils. 

While there are other solvents that may also be used effectively with reversed-phase sorbents, these solvents are capable of dissolving the range of compounds that are generally isolated by SPE. If more hydrophobic solvents are used in bonded-phase SPE, then the sorbent must be carefully dried by vacuum to remove all traces of water in the silica matrix in order that the eluting solvent can interact with all areas of the sorbent and not be stopped by residual water trapped in the pores. If this is not done, then the hydrophobic solvent (let us say, methylene chloride) will not effectively wet the surfaces of the C-18 bonded phase and poor recoveries will result. If methanol, acetonitrile, or ethyl acetate is used, then complete drying of the sorbent is not required because the solvent will either be miscible with water (methanol and acetonitrile) or displace the water from the silica (ethyl acetate). 

Silica, and to a lesser extent alumina, are the most common stationary phases used for the separation of low molecular mass organic compounds. Chemically bonded silica sorbents are used for the separation of polar organic compounds in the normal-phase and reversed-phase modes. Wide-pore, chemically bonded sorbents, are used for the separation of biopolymers [18,22]. Some separations require specially prepared stationary phases, such as silica gel impregnated with silver nitrate for the isolation of unsaturated compounds capable of forming charge transfer complexes with silver [23] (section 10.6.1), or silica and chemically bonded phases coated with cellulose tris(3,5-... 

Solid-phase extraction (SPE) using small, disposable cartridges, columns, or disks is employed for isolation and cleanup of pesticides from water and other samples prior to TLC analysis, especially using reversed-phase (RP) octa-decyl (C-18) bonded silica gel phases. Microwave-assisted extraction (MAE) is a time- and solvent-saving method for removing residues from samples such as soils. Supercritical fluid extraction (SEE) has been used for sample preparation in the screening of pesticide-contaminated soil by conventional TLC and automated multiple development (AMD). Ultrasonic solvent extraction (USE) and videodensitometry have been combined for quantification of pesticides in sod. Matrix solid-phase dispersion (MSPD) with TLC and GC has been used to determine diazinon and ethion in nuts. 

Another approach in taxoid preparative separation included solid-phase extraction (alumina, silica, or RP-8 cartridges) followed by preparative TLC on silica gel plates with quaternary mobile phase consisting of dichloromethane-dioxane-acetone-methanol (83 5 10 2, v/v). In this way, 10-DAB III, paclitaxel, and cephalomannine as well as two further taxoids could be easily isolated with relatively high efficiencies from yew materials (Fig. 2). Multiple development technique or fiuther separation of the isolated taxoid fractions (especially less polar ones) on RP-2 silica bond stationary phase with methanol-water mixtures as mobile phases was applied for purification of the compounds isolated. 10-DAB III isolated in this way was relatively pure, as was shown in reversed-phase (RP)-HPLC analysis (Fig. 3). 

Lambert and DeLeenheer (1992) contributed a review on the TLC analysis of K vitamins. They noted that although HPLC is usually the method of choice for analysis, there is some interest in HPTLC-densitometric analysis of the K vitamins. Their review covers isolation, extraction, and cleanup methods uses of polar inorganic solvents uses of modified silica (i.e., nonpolar bonded phases such as C2, Cg, C18, and phenyl) uses of mobile phases and methods of detection and quantification of the K vitamins. Madden and Stahr (1993) assayed vitamin K in bovine liver by reversed-phase TLC with dichloromethane-methanol (7 3)... 

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