C8 bonded phase

Phases with phenyl groups are less retentive than C8 bonded phases. They show a pronounced selectivity and retention toward solutes with aromatic ring systems, attributed to n-n interactions. These n-n interactions can be improved when nitro substituted phenyl groups are bonded to silica. The strongest interactions have been reported with tetra nitro or tetra chloro phthalimide substituents. 

Other workers have successfully applied these principles to the optimization of their separations, and their papers attest to the value of this method Antle67 has separated steroids by normal phase on an amine bonded phase, and Lehrer68 has separated phenols and cresols by reverse phase on a C8 bonded phase. Both provide interesting chromatograms. 

For RPLC, the three solvents chosen were methanol from group 2, acetonitrile (ACN) from group 6, and tetrahydrofuran (THF) from group 3. These three solvents are not as widely separated from each other in the diagram as those for normal phase are, but they are sufficiently different to produce good separations. The (polar) carrier solvent used was water, and the stationary phase was a C8 bonded phase. The procedure... 

Ambelline Detection Amaryllidaceae C8 bonded phase 150x5 MeOH-H,0(3 2) containing a trace of NH.0H ... 

A) plasma is applied to a Bond-Elut C8 SPE cartridges, washed with 2 ml 0.15 mM phosphate buffer of pH 3.5, dried under vacuum and eluted with 0.4 ml mobile phase (B) plasma applied to the SPE cartridges packed with 40 p,m C8-bonded phase, washed with 1 ml 75 mM phosphate buffer of pH 3.5 and eluted with mobile phase ... 

Silica gel-based materials for RP chromatography with non-polar (most often C8 or C18 alkyls) or moderately polar stationary phases covalently bonded via Si-O-Si-C bonds are prepared by chemical modification of the silanol (Si-OH) groups on the silica gel surface by chloro-silane or alkoxy-silane reagents, and are relatively stable to hydrolysis. The retention in RP increases with increasing surface coverage and length of the bonded alkyl chains, so that C18 phases show greater retention than C8-bonded phases. 

As with the amino-bonded phase, the organosilane reagent used for bonding the CN phase has a trimethylene (-(CH2)3-) between the silicon atom (with its three leaving groups for bonding) and a cyano group. This thin layer can be used in the RP mode with polar mobile phases or in the NP mode with nonpolar mobile phases. As mentioned, it has less activity compared to a silica gel layer and also a less nonpolar character than C18- or C8-bonded phases. 

The separation was performed using a packing material with a nonpolar C8 bonded phases. The column temperature was set above ambient to ensure reproducible conditions. Analytical HPLC injection volumes are typically an order of magnitude smaller than GPC injection... 

HPLC has also used to determine taxol and related compounds in biological fluids [13]. After evaluating C8-, cyanopropyl-, and phenyl-bonded phases, Riley and his co-workers selected a C8-bonded phase column with a mobile phase of MeOH NaOAc buffer (0.02 M, pH 4.5, 35 65 v/v) as their system for analysis of taxol in human plasma and urine. 

Tetracycline antibiotics have been determined in bovine liver, kidney, and muscle, and in milk by solid-phase extraction followed by TLC7MS with FAB mass spectrometry (45,46). A reverse-phase C8 bonded phase silica TLC plate was used. Adjacent lanes of standards provided Rf values for the compounds of interest. This area of the chromatogram was cut into a trapezoidal shape, and additional solvent concentrated the sample in one end of the shape. That portion of the chromatogram was then placed on the FAB probe of a high-performance mass spectrometer. Then, the FAB support matrix (thioglycerol) was added to the plate. A detection limit of 0.1 microgram of sample per spot was reported for most of the tetracycline antibiotics. The trapezoidal slice from the TLC plate used to concentrate the sample for TLC/MS analysis was also used in an application of fast atom bombardment mass spectrometry to identify and quantitate the drug midazolam (a depressant and anaesthetic) in plasma extracts by Okamoto et al. (47). 

The most popular bonded phases are, without doubt, the reverse phases which consist solely of aliphatic hydrocarbon chains bonded to the silica. Reverse phases interact dispersively with solvent and solute molecules and, as a consequence, are employed with very polar solvents or aqueous solvent mixtures such as methanol/water and acetonitrile/water mixtures. The most commonly used reverse phase appears to be the brush type phase with aliphatic chains having four, eight or eighteen carbon atom chains attached. These types of reverse phase have been termed C4, C8 and Cl8 phases respectively. The C8... 

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. 

FIGURE 6.1 Groups present on the surface of a typical endcapped alkyl bonded phase (C8). 

The nomenclature of the RP is not consequent. The RP most often used contains octyl (RP C8) or octadecyl (RP C18) groups. There is no differentiation even when two methyl groups are introduced additionally with the silane (as with monofunctional silanes) or only one (difunctional) or none (trifunctional silane). Some manufacturer use silanes with bulky side groups (e.g., isopropyl groups) to improve the hydrolytic stability of the bonded phases, but here also, only the longest alkyl group is used in nomenclature. RP C8 and RP C18 are the work horses in HPLC. Shorter chains (RP4) are used in protein separations, and special selectivity can be obtained with bonded phenyl, cyano, amino or fluoro groups. 

The physical structure of the stationary phase depends on the compatibility of the solvent with the bonded n-alkyl chains. Compatible nonpolar solvents tend to promote extension of the chains, allowing full penetration by the solvent. Conversely, fairly polar solvents tend to promote collapse of the chains upon each other, allowing negligible solvent penetration. The stationary phase therefore has the ability to adjust itself to maintain a relatively nonpolar character (113). Retention on monomeric bonded phases with octyl (C8) or longer chains are dominated by a partitioning-like mechanism (114). 

Reversed-phase chromatography is the most popular mode of analytical liquid chromatography for phenolic compounds. In most cases, the reported systems for the separation of phenolics and their glycosides in foods are carried out on reversed-phase chromatography on silica-based Cl8 bonded-phase columns. Occasionally, silica columns bonded with C8 were applied in the analysis of phenolic acid standards and coumarins (7), and C6 columns for the analysis of ferulic acid in wheat straw (8). 

OS Si-O-Si-C Octyl, n-C8 Bonded, reverse Octylsilane lower resolution than the octadecyl bonded phase useful when separations involve... 

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