Silica-octadecyl chains

R stands for the ligand after which the stationary phase is named. A common example is the octadecyl chain, usually abbreviated as Cjg . Due to steric hindrance, not all silanols on a silica surface can react. With modern, high-quality stationary phases, the surface coverage with a Cjg ligand is usually between 3 and 3.5 J,mol/m on a silica surface. 

Several different analytical and ultra-micropreparative CEC approaches have been described for such peptide separations. For example, open tubular (OT-CEC) methods have been used 290-294 with etched fused silicas to increase the surface area with diols or octadecyl chains then bonded to the surface.1 With such OT-CEC systems, the peptide-ligand interactions of, for example, angiotensin I-III increased with increasing hydrophobicity of the bonded phase on the capillary wall. Porous layer open tubular (PLOT) capillaries coated with anionic polymers 295 or poly(aspartic acid) 296 have also been employed 297 to separate basic peptides on the inner wall of fused silica capillaries of 20 pm i.d. When the same eluent conditions were employed, superior performance was observed for these PLOT capillaries compared to the corresponding capillary zone electrophoresis (HP-CZE) separation. Peptide mixtures can be analyzed 298-300 with OT-CEC systems based on octyl-bonded fused silica capillaries that have been coated with (3-aminopropyl)trimethoxysilane (APS), as well as with pressurized CEC (pCEC) packed with particles of similar surface chemistry, to decrease the electrostatic interactions between the solute and the surface, coupled to a mass spectrometer (MS). In the pressurized flow version of electrochromatography, a pLC pump is also employed (Figure 26) to facilitate liquid flow, reduce bubble formation, and to fine-tune the selectivity of the separation of the peptide mixture. 

The great majority of CEC—MS applications is run in the reversed-phase mode using alkyl-bonded silica stationary phases [12,14,24,26,38,45,81,82,98-104], The dual functionality concept is represented in these stationary phases by the alkyl chains, most frequently octadecyl chains, that constitute the top retentive layer, and residual silanol groups on the surface, that dissociate at pH values higher than 3-4 and... 

One system with these desirable features is a suspension of small a 50 nm) silica particles onto which octadecyl chains have been densely grafted (Stober et al. 1968 van Helden et al. 1981 Woutersen and de Kruif 1991). The octadecyl chains have a theta point near room temperature in various solvents, including benzene, dodecane, and hexadecane. 

Figure 7-6 shows the viscosity of thermoreversible dispersions (discussed in Section 7.2.4) of a 50-nm silica particles onto which octadecyl chains have been densely grafted in benzene at particle volume fractions 4> — 0.088-0.133, as a function of temperature T (Woutersen and de Kruif 1991). For T > 9 — 316 K, the viscosity relative to that of the solvent, rir = r /f]s, is independent of temperature, and its dependence on volume fraction 0 is exactly as expected for hard spherical particles without attractive interactions. As T is lowered below 9, however, the viscosity rises rapidly, because of the onset of attractive interactions. 

Reversed-phase PLC precoated plates are based on silica gel matrices with chemical modifications in such a manner that the accessible polar, hydrophilic silanol groups at the silica gel surface are replaced by nonpolar, hydrophobic alkyl chains via silicon-carbon bonds. For preparative purposes, up to now only PLC precoated RP plates with C-18 modification are available. This abbreviation is often also designated as RP-18, meaning that an octadecyl alkyl chain is chemically bonded to the silica gel surface. 

Although open tubular columns have been used for RPLC,42 most applications use packed columns. The most widely used column packings are formed by chemically bonding butyl (C4), octyl (C8), or octadecyl (C18) chains to a silica surface. Phenyl (Ph), cyano (CN), and amino (NH2) functionalities are also used. Polymeric packings which are finding broad acceptance include alkyl-grafted poly(methylmethacrylate) and alkyl-grafted or unmodified... 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Reverse phase HPLC describes methods that utilize a polar mobile phase in combination with a nonpolar stationary phase. As stated above, the nonpolar stationary phase structure is a bonded phase—a structure that is chemically bonded to the silica particles. Here, typical column names often have the carbon number designation indicating the length of a carbon chain to which the nonpolar nature is attributed. Typical designations are C8, C18 (or ODS, meaning octadecyl silane), etc. Common mobile phase liquids are water, methanol, acetonitrile (CH3CN), and acetic acid buffered solutions. 

The use of reversed-phase liquid chromatography is growing in applications for separating mixtures of peptides. In this type of chromatography, the stationary phase is nonpolar, whereas the mobile phase is polar. The stationary phase is normally porous silica with bonded n-alkyl chains, mainly octadecyl but also octyl, hexyl, butyl, and propyl chains. 

Silica can be drastically altered by reaction with organochlorosilanes or organoalkoxysilanes giving Si—O—Si—R linkages with the surface. The attachment of hydrocarbon chains to silica produces a non-polar surface suitable for reversed-phase chromatography where mixtures of water and organic solvents are used as eluents. The most popular material is octadecyl-silica (ODS-silica) which contains C g chains, but materials with Cj, C, Cg, and C22 chains are also available. 

The recent popularity of HPLC as an analytical technique in biochemical and biomedical research can be attributed to the development and introduction of microparticulate reversed-phase packings in which the hydrocarbon chain (octadecyl-, ocyl-, or di-) moieties are chemically bonded to a silica base (K5). At the present time, it is estimated that approximately 80% of all HPLC separations are performed in the reversed-phase mode. Reversed-phase high-performance liquid chromatography (RPLC) has many advantages over other modes of HPLC ... 

Oligomers of glycine show little appreciable retention on octyl silica with 20 mM phosphate buffers over the pH range 2.10-7.83 (46), or on octadecyl silica with 100 mM phosphate buffer, pH 2.1 (33), or 5 mM phosphate buffers, pH 2.1, containing hydrophobic alkyl sulfonates (30). It is thus likely that the peptide chain proper makes only a very small contribution to the retention of peptides under these conditions. Based on partition coefficient considerations, oligomers of alanine, and the other nonpolar amino acids, should show a linear dependence of log A on the number of residues. This, in fact, has been observed. For example, the plot of log A versus the number of alanine residues shows (33) a linear dependence with a uniform log A increment due to the methyl group of the aliphatic side chain (Fig. 2), i.e., the effect is additive (45a, 46a). 

The most common R group is the octadecyl group (C-18), which leaves the silica particles coated with hydrocarbon chains. The silica particles are very small (—40 p dia. = 4 x 10 cm dia.) and very uniform in size. With very small, uniform particles, a molecule in the solvent can rapidly diffuse to the surface of the packing and undergo partitioning between the station-... 

K. Nagy, A. Jakab, J. Fekete, K. Vekey, An LC-MS approach for analysis of very long chain fatty acids and other apolar compounds on octadecyl-silica phase using partly miscible solvents. Anal. Chem., 76 (2004) 1935. 

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