Normal-phase chromatography column packing

Early PG analysis using HPLC techniques was carried out as adsorption chromatography on normal-phase (NP) columns packed with silica or alumina. The nonpolar mobile phase comprizing of organic solvents (hexane, toluene, ethyl acetate, and HOAc) allows separation of PGs which are unstable in aqueous media (e.g., PGH2 on cyano- or phenyl-bonded phases). Usually, the injection medium must be fairly polar to dissolve the PGs. This is achieved by the addition of... 

Kovat s retention index (p. 575) liquid-solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554)... 

Bonded-phase column packings for use in normal-phase chromatography are available in which the stationary phase is a polar functional group chemically bonded onto the silica surface. One... 

In normal-phase chromatography, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample. Therefore, the stationary phase is usually silica and typical mobile phases for normal phase chromatography are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. In reverse phase the packing is nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as Ci8, Q, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water. In the strictest interpretation, normal and reverse phase are terms which only relate to the polarity of the column and mobile phase with respect to the sample as shown in Table 3-3 and drawn schematically in Figure 3-14. 

Polar bonded phases such as propylamino-, cyano-, or diol packings used in normal-phase chromatography can be tested exactly like silica columns. Amino-bonded phases used for hydrophilic interaction chromatography are best tested with a carbohydrate sample in 70% acetonitrile 30% water. 

The equilibration of the columns with the aqueous mobile phases is rapid in most cases only a few colunm volumes are n ed. This is in stark contrast to normal-phase chromatography, where the equilibration of the column with the ubiquitous water is a steady challenge. For the majority of analytes, good peak shapes and reproducible retention times are obtained without difficulty. For some compounds, there are some difficulties due to secondary interactions, but these secondary effects are well understood and can be dealt with using proven procedures. Furthermore, reversed-phase packings have steadQy improved, and some of these difficulties have been reduced substantially. 

In order for the separation to take place, a more polar solvent (than the original sample matrix) will be used to effect the desorption of the analyte molecules from the packing material. Examples of packing material include silica bonded with cyano, amine, and diol groups, as with the stationary phase of columns in normal phase chromatography (see Chapter 4 for further explanation). 

Normal phase chromatography systems use a polar stationary phase with a nonpolar mobile phase. Generally, the packing materials within the normal phase columns are composed of unmodified silica spherical beads (cyano, amine, or diol packing materials can also be used) with the mobile phase consisting of nonpolar organic solvents such as ethanol, chloroform, propanol, or hexane. Table 4.3 outlines the main differences between typical normal phase packing materials. 

Normal-phase chromatography is usually employed when separating a lipid extract into polar lipid classes. Columns packed with silica, diol-modified silica, polyvinylalcohol (PVA)-silica or other materials have been successful in separating all kinds of polar lipids (Arnoldsson and Kaufman, 1994 ... 

In our laboratory we make our own columns for normal-phase chromatography (diol-modified silica). We have noticed that by packing the columns under a high pressure, that is, 950-1000 bar, instead of at the recommended 500-600 bar, we obtained far better separation of the phospholipid classes. Accordingly, this way of packing columns may be a way to achieve better separation of intact polar lipids, if choice of column packing, solvent mixture gradient and sample clean-up have already been optimized. 

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