Surface Reverse Phase Supports

In conU ast to the internal surface reversed-phases described above, Kima-ta s internal surface reversed-phases are synthesized without use of enzymes. These Ci, Cg, Cig, and phenylethyl-modified porous silica supports are treated with HCI at 100 C for 5 h to cleave preferentially the ester bonds at the external surface (127). Hydrolysis inside the pores is extfemely slow because of hydrolysis products that are enriched in the pores, low wettability, and the presence of air bubbles. The generated silanol groups are reacted with 3-glycidoxypropyl-trimethoxysilane, which is then hydrolyzed to hydrophilic diol groups. Several variations of Kimata s supports with different hydrophobicity exist, so these phases can be used for analyzing a broad spectrum of analytes. 

Figure 9.36. Representation of internal surface reverse phase (ISRP) type support. Courtesy of Regis Chemical.

Since these ligates are bonded to the surface of the silica and will not wash off, it is no longer necessary to choose a mobile phase with a polarity opposite to that of the bonded phase. That is to say, a reverse phase support like ODS could be used with a relatively nonpolar organic mobile phase, thus establishing a system that does not fit into either category— reverse phase or normal phase. Some workers have given such a system the name nonaqueous reverse phase, or NARP. 

In 1985, Hagestam and Pinkerton94 published a report on a new type of stationary phase they synthesized. They called it an internal surface reverse phase (ISRP) support, and it is also known as a Pinkerton column. The patented idea has been licensed exclusively to Regis Chemical Company. 

The retention of polar compounds (mono-, di-, and tri-substituted benzenes) on PGC, silica-based, and apolar copolymer supports was performed using unbuffered methanol water as eluent. The relationship between log k and the volume fraction of methanol was calculated separately for each solute. It was found that porous graphitic carbon retains polar compounds fairly well under reversed-phase conditions, while the retention factor increased with an increase in the number of polar substituents. In particular, the retention behavior of polar solutes on PGC supports is mainly governed by several polarity parameters (Hammett s constant, proton-donor capacity, and steric effects of substituents) and is quite different from that observed with other reversed-phase supports. Thus it was concluded that charge-induced interactions between the graphite surface as well as steric... 

The number and nature of unreacted surface silanols affects the character of a stationary phase. Initially free, geminol or associated silanols are minimized through a process known as endcapping, which bonds various species to the residual silanols. Hydrophilic endcaps or bulky steric endcaps that separate the hydrocarbon chains and prevent analyte interaction with the silica surface can be used. If residual silanols are left unreacted (and some always are), the analyte will be separated based on a combination of interactions with both the reverse-phase support and the highly polar silanol groups. Increased retention, changes in elution order, and tailing will result for basic compounds. 

Hagestam, I.H. and Pinkerton, T.C. Internal Surface Reversed-Phase Silica Supports for Liquid Chromatography, Anal. Chem. 57, 1757-1763 (1985). 

Haginaka, J. Wakai, J. Yasuda, H. Kimura, Y. Determination of anticonvulsant drugs and methyl xanthine derivatives in serum by liquid chromatography with direct ii jection column-switching method using a new internal-surface reversed-phase silica support as a precolumn. J.Chromatogr., 1990, 529, 455-461... 

Two types of micellar chromatography are possible. For neutral solutes, a normal micelle with nonpolar interior and ionic surface, in an aqueous mobile phase against a reversed-phase solid support is used (10, 130, 402). The solute components, which may not be water soluble and would be adsorbed irreversibly on the reversed-phase support, are transported by partitioning into the nonpolar interior of the micelle. This type of micellar chromatography has been successful in separating pesticides, polynuclear aromatic compounds, and other model solutes (10, 130, 402). 

Gels made in this way have virtually no usable porosity and are called Jordi solid bead packings. They can be used in the production of low surface area reverse phase packings for fast protein analysis and in the manufacture of hydrodynamic volume columns as well as solid supports for solid-phase syntheses reactions. An example of a hydrodynamic volume column separation is shown in Fig. 13.2 and its calibration plot is shown in Fig. 13.3. The major advantage of this type of column is its ability to resolve very high molecular weight polymer samples successfully. 

In contrast, the mono-layer of methanol is built up much more slowly and is not complete until the concentration of methanol in the aqueous mixture is about 35%w/v. The behavior of methanol on the reverse phase is reminiscent of the adsorption of chloroform on the strongly polar silica gel surface. The complementary nature of the silica gel surface and that of the reverse phase is clearly apparent. It is also clear that strongly dispersive solvents might form bi-layers on the reverse phase surface just as polar solutes form bi-layers on the highly polar surface of silica gel. In fact, to date there has been no experimental evidence furnished that would support the formation of bi-layers on the surface of reverse phases, although their formation is likely and such evidence may well be forthcoming in the future. 

The problems of stationary phase erosion can be largely overcome by solvent-generated LLC, where the stationary liquid phase is generated dynamically by the mobile phase, in this approach, one of the phases of an equilibrated liquid-liquid system is applied as a mobile phase to a solid support which is better wetted by the other phase of the liquid-liquid system. The support is usually silica when the stationary phase is aqueous or a polar solvent and a reversed-phase chemically bonded support when the stationary phase is a nonpolar solvent. Under these Conditions a multimolecular layer is formed on the surface of the solid support which has the properties of the liquid phase in... 

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