Bonded stationary phases acetonitrile-water mixture

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. 

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. 

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. 

Reversed-phase chromatography employs a nonpolar stationary phase and a polar aqueous-organic mobile phase. The stationary phase may be a nonpolar ligand, such as an alkyl hydrocarbon, bonded to a support matrix such as microparticulate silica, or it may be a microparticulate polymeric resin such as cross-linked polystyrene-divinylbenzene. The mobile phase is typically a binary mixture of a weak solvent, such as water or an aqueous buffer, and a strong solvent such as acetonitrile or a short-chain alcohol. Retention is modulated by changing the relative proportion of the weak and strong solvents. Additives may be incorporated into the mobile phase to modulate chromatographic selectivity, to suppress undesirable interactions of the analyte with the matrix, or to promote analyte solubility or stability. 

Eluents used in reversed-phase chromatography with bonded nonpolar stationary phases are genei ly polar solvents or mixtures) of polar solvents, such as acetonitrile, with water. The properties of numerous neat solvents of interest, their sources, and their virtues in teversed-phase chromatography have been reviewed (128). Properties of pure solvents which may be of value as eluents are summiuized in Table. VII. The most significant properties are surface tension, dielectric constant, viscosity, and eluotropic value. Horvath e/ al. 107) adapted a theory of solvent effects to consider the role of the mobile phase in determinmg the absolute retention and the selectivity found in reversed-phase chromatography. 

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. 

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)... 

To study the solvent behavior in the presence of a bonded moiety, it is necessary to use deuterated solvents since deuterium is a quadrupolar nucleus whose relaxation is controlled by the molecular correlation time, a measure of molecular motion [37]. The C-18 bonded phase has been studied in water/methanol and water/acetonitrile mixtures of varying composition [38]. Figure 10 illustrates one part of this study where the change in the deuterium relaxation time in D2O is monitored as a function of % organic modifier in the mobile phase for methanol and acetonitrile using two different bonding density C-18 stationary phases. These results show that little change in the deuterium... 

Reversed-phase chromatography of A/-nitrosamines has been used in connection with UV, fluorimetric, amperometric, and MS detection. In many cases these methods are based on pre- or postcolumn denitrosation, and derivatization of the denitrosation products. Chemically bonded octadecylsilane, Cig, is the most often reported stationary HPLC phase. Isocratic or gradient elution is usually performed with mixtures of water with acetonitrile, methanol, ethanol, or propanol. In general, the mobile phase is acidified by the addition of acetic acid, ammonium acetate, or phosphate buffers. The HPLC conditions used for the separation and detection of A/-nitrosamines are shown in Table 12.3. 

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