Reversed-phase chromatography, wetting

Compared with liquid column chromatography, in PLC there is a certain limitation with respect to the composition of the mobile phase in the case of reversed-phase chromatography. In planar chromatography the flow of the mobile phase is normally induced by capillary forces. A prerequisite for this mechanism is that the surface of the stationary phase be wetted by the mobile phase. This, however, results in a Umitation in the maximum possible amount of water applicable in the mobile phase, is dependent on the hydrophobic character of the stationary RP phase. To... 

Reversed-phase chromatography The stationary phase here is nonpolar in most cases it is derivati-zed silica that carries Cig (i.e., C18H37) or Cg (i.e., CgHiy) groups. The mobile phase is polar, in most cases a mixture of water (or buffer solution) with methanol, acetonitrile, or tetrahydrofuran. Such an eluent cannot wet the surface of the stationary phase and the solutes are retained owing to an energy gain... 

A novel development for HPLC is something called bonded reversed-phase columns, where the stationary phase is a nonpolar hydrocarbon, chemically bonded to a solid support. You can use these with aqueous eluents, usually alcohol-water mixtures. So you have a polar eluent and a nonpolar stationary phase, something that does not usually occur for ordinary wet-column chromatography. One advantage is that you don t need to use anhydrous eluents (very small amounts of water can change the character of normal phase columns) with reversed-phase columns. 

A Lapvetelainen, JA Bietz, FR Huebner. Reversed-phase high-performance liquid chromatography of oat proteins application to cultivar comparison and analysis of the effect of wet processing. Cereal Chem 72 259-264, 1995. 

Isolation and purification. Carotenoids were extracted with CHCl /MeOH from the wet cells of aerobic or semi-aerobic ciilture, or from those of aerobic culture inhibited by DPA or nicotine. The polar carotenoid group was purified by silica gel column chromatography, SEP-PAK NH2 and reversed phase TLC. The others were purified with silica gel and DEAE-Sepharose CL-6B column chromatography. The purity was analyzed with the reversed phase HPLC system using water (20-0%, v/v) in methanol as eluent [3>4] ... 

One major drawback to soap chromatography is the slow rate of mass transfer across the micellar interface. Poor wetting of the reversed-phase support by an aqueous mobile phase also slows mass transfer at the mobile phase/sta-tionary phase interface and, hence, reduces column efficiency. It has been recently shown that adding an appropriate organic modifier - 1-propanol, for example - to the mobile aqueous phase in as low a concentration as 3% greatly enhances the mass transfer and increases column efficiency to a level comparable to that attained with adsorption chromatography (86). 

The natural ryanoids are conveniently isolated from ryania by wet chloroform extraction and rotary chromatography on silica gel with chloroform/methanol/ aqueous methylamine followed by reverse phase HPLC with aqueous methanol (2,7,8). Ryanodine 0 and dehydroryanodine 0 are the major ryanoids, each making up 480-700 ppm (w/w) relative to the stem-wood, with seven other ryanoids contributing 10-64 ppm (w/w) each (8). 

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