Bonded stationary phases methanol-water mixture

Variations in retention and selectivity have been studied in cyano, phenyl, and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline, and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile, or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns were correlated with polar group selectivities of mobile-phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers [84],... 

The use of nonpolar chemically bonded stationary phases with a polar mobile phase is referred to as reverse-phase HPLC. This technique separates sample components according to hydrophobicity. It is widely used for the separation of all types of biomolecules, including peptides, nucleotides, carbohydrates, and derivatives of amino acids. Typical solvent systems are water-methanol, water-acetonitrile, and water-tetrahydrofiiran mixtures. Figure 3.15 shows the results of protein separation on a silica-based reverse-phase column. 

This protocol focuses on the analysis of chlorophyll a and b, and the more nonpolar derivatives, including pheophytins and pyropheophytins. An octadecyl-bonded, reversed-phase stationary phase is used with a methanol/water mixture and ethyl acetate mobile phases in a gradient elution to provide rapid and complete separation of the major chlorophyll derivatives in 25 to 30 min. This is coupled with traditional UV/visible spectrophotometric detection at 654 nm to selectively screen these photosynthetic pigments in food and plant tissues. 

Another approach in taxoid preparative separation included solid-phase extraction (alumina, silica, or RP-8 cartridges) followed by preparative TLC on silica gel plates with quaternary mobile phase consisting of dichloromethane-dioxane-acetone-methanol (83 5 10 2, v/v). In this way, 10-DAB III, paclitaxel, and cephalomannine as well as two further taxoids could be easily isolated with relatively high efficiencies from yew materials (Fig. 2). Multiple development technique or fiuther separation of the isolated taxoid fractions (especially less polar ones) on RP-2 silica bond stationary phase with methanol-water mixtures as mobile phases was applied for purification of the compounds isolated. 10-DAB III isolated in this way was relatively pure, as was shown in reversed-phase (RP)-HPLC analysis (Fig. 3). 

To retain solutes selectively by dispersive interactions, the stationary phase must contain no polar or ionic substances, but only hydrocarbon-type materials such as the reverse-bonded phases, now so popular in LC. Reiterating the previous argument, to ensure that dispersive selectivity dominates in the stationary phase, and dispersive interactions in the mobile phase are minimized, the mobile phase must now be strongly polar. Hence the use of methanol-water and acetonitrile-water mixtures as mobile phases in reverse-phase chromatography systems. An example of the separation of some antimicrobial agents on Partisil ODS 3, particle diameter 5p is shown in figure 5. 

Bonded phases are the most useful types of stationary phase in LC and have a very broad range of application. Of the bonded phases, the reverse phase is by far the most widely used and has been applied successfully to an extensive range of solute types. The reverse phases are commonly used with mobile phases consisting of acetonitrile and water, methanol and water, mixtures of both acetonitrile and methanol and water, and finally under very special circumstances tetrahydrofuran may also be added. Nevertheless, the majority of separations can be accomplished using simple binary mixtures. 

Most stationary phases used in bonded-phase chromatography in its reversed-phase mode are based on octadecylsilane functionality (C18 columns). The mobile phases typically used in this context are water, aqueous buffers of a given pH and ionic strength, and mixtures of water and a miscible organic modifier, such as methanol or acetonitrile. 

A more or less opposite goal was pursued by de Smet et al. (574], who attempted to reduce the number of stationary phases to a single one, by choosing a cyanopropyl bonded phase of intermediate polarity, which can be used in both the normal phase and the reversed phase mode (see figure 3.8). Furthermore, because of a clever choice of modifiers, the total number of solvents required was restricted to six n-hexane, dichloromethane, acetonitrile and THF for NPLC and the latter two plus methanol and water for RPLC. A variety of drug samples could be separated with a selected number of binary and ternary mobile phase mixtures. 

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. 

Fig. 2 Variation of the available capaeity Cj as a function of the solute concentration in the mobile-phase Q (logarithm scales). In the case of reversed-phase chromatography, the stationary phase is n-octyl-bonded silica Lichroprep R.R8 with 11.6% of carbon, the mobile phases are water-methanol mixtures, and the solute is phenol. 

In reversed-phase chromatography (RPC), a relatively nonpolar stationary phase is used, with a polar mobile phase such as methanol, acetonitrile, tetrahydro-furan, water, or usually a mixture of water with one of the organic solvents. The organic solvent is called the modifier, and acetonitrile is the most common one. The water content is varied for adjusting the polarity. Methanol is used for acidic compounds and acetonitrile for basic compounds. Tetrahydrofuran is used for those with large dipoles. These solvents are UV transparent and have low viscosity. The most common bonded phases are n-octyldecyl (Cig) or n-decyl (Cg) chains, or phenyl groups. Polar reversed-phase columns such as polyethylene glycol (PEG)... 

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