Methanol, solvent, RPLC

Tile possibility of using short wavelengths will depend on the nature and the purity of the solvents. From this point of view, acetonitrile may be preferred to methanol for RPLC. However, as discussed above, methanol-water gradients offer the possibility to estimate the isocratic retention behaviour fairly accurately from a single gradient run, because of the validity of eqn.(3.46). In mixtures of THF and water eqn.(3.46) is only approximately observed, whereas it is completely invalid in mixtures of acetonitrile and water (see table 3.1). 

The isocratic reversed phase solvent system consists of water (polarity, p = 10.2), the most polar solvent in RPLC, as a primary solvent to which water-miscible organic solvents such as methanol (p = 5.1), acetonitrile (p = 5.8), or tetrahydrofuran (p = 4.0) are added. In order to optimize the speed of separation for an analyte pair, the proportions of water to nonpolar solvent are chosen such that the capacity factor of the last-eluting analyte of interest has a value of about 2.13... 

The most commonly used solvents for RPLC are methanol, acetonitrile, and tetrahydrofuran, used in binary, ternary, or quaternary combinations with water. The effect of solvent strengths can be seen in... 

The polarity values of binary acetonitrile/water and methanol/water mobile phases used in RPLC were measured and compared with methylene selectivity (acH2) for both traditional siliceous bonded phases and for a polystyrene-divinylbenzene resin reversed-phase material [82], The variation in methylene selectivity for both was found to correlate best with percent organic solvent in methanol/water mixtures, whereas the polarity value provided the best correlation in acetonitrile/water mixtures. The polymeric resin column was found to provide higher methylene selectivity than the siliceous-bonded phase at all concentrations of organic solvent. 

In summary, the use of RPLC is ideal for pharmaceutical analyses because of the broad range of commercially available stationary phases because the most common RPLC mobile phases (buffers with acetonitrile or methanol) have low UV cut-off wavelengths, which facilitate high sensitivity detection for quantitation of low-level impurities and because selectivity can readily be controlled via mobile phase optimization. Additionally, the samples generated for selectivity screening (as detailed above) are typically aqueous based. In subsequent phases of pharmaceutical development, aqueous-based sample solvents are ideal for sample preparation and are, under limited constraints, compatible with MS detection required to identify impurities and degradation products. 

The extent to which retention in RPLC can be made to vary with the composition of the mobile phase is enormous. For almost all solutes retention will be impractically low in some pure organic solvent (methanol, THF) and impractically high in pure water. Hence, to achieve reasonable retention times, a mixture of water and an organic solvent (a so-called modifier) is usually required. 

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. 

For RPLC, the three solvents chosen were methanol from group 2, acetonitrile (ACN) from group 6, and tetrahydrofuran (THF) from group 3. These three solvents are not as widely separated from each other in the diagram as those for normal phase are, but they are sufficiently different to produce good separations. The (polar) carrier solvent used was water, and the stationary phase was a C8 bonded phase. The procedure... 

NPC is ideally suited for the analysis of compounds prone to hydrolysis because it employs nonaqueous solvents for the modulation of retention. An example of the use of NPC in the analysis of a hydrolysable analyte was demonstrated by Chevalier et al. [28] for quality control of the production of benorylate, an ester of aspirin. A major issue in benorylate production is the potential formation of impurities suspected of causing allergic side effects therefore monitoring of this step is critical to quality control. The presence of acetylsalicylic anhydride prohibited the use of RPLC since it can be easily hydrolyzed in the water-containing mobile phase. However, an analytical method based on the use of normal-phase chromatography with alkylnitrile-bonded silica as the stationary phase provided an ideal solution to the analysis. Optimal selectivity was achieved with a ternary solvent system hexane-dichloromethane-methanol, containing 0.2 v/v% of acetic acid to prevent the ionization of acidic function and to deactivate the residual silanols. The method was validated and determined to be reproducible based on precision, selectivity, and repeatability. 

In RPLC-APCl-MS, where the mobile phase consists of a mixture of water and methanol or acetonitrile, and eventually a buffer, the formation of protonated water clusters can be considered as a starting point in a series of even-electron ion-molecule reactions. The protonated water clusters transfer their proton to any species in the gas mixture with a higher proton affinity (Table 6.1). The mass spectrum of acetonitrile (MeCN)-water mixture shows protonated MeCN-water clusters, [(MeCN), (HjO) + H]", with /w-values of 1-3, and -values of 0-1. The addition of aimnonium acetate to MeCN-water results in the observation of mixed solvent clusters, e.g., [(MeCN), + and [(MeCN) , (HjO) +... 

Pure organic mobile phases, e.g., mixtures of hexane and methanol, dioxane, or isopropanol, are readily compatible with LC-APCI-MS. Because some users dislike the use of hexane, ethoxynonafluorobutane has been suggested as an inflammable alternative [91]. Nonaqueous RPLC with a propionitrile-hexane solvent gradient in combination with positive-ion APCI was performed in the LC-MS analysis of triacylglycerols (TAGs) [92] (Ch. 21.3.1). Another example is the LC-MS analysis of polychlorinated -alkanes on bare silica and with chloroform as the mobile phase [93]. Under these conditions, chloride-enhanced APCI can be applied to generate [M+C1] adduct ions, which suppresses the loss of Cl from the polychlorinated -alkanes. 

Nagy et al. [11] reported the use of RPLC-MS on a Cig column using a stepwise gradient of partly miscible solvents, methanol/water and methanol/n-hexane, for the analysis of TAG in blood and/or plant oil. 

The modulator (in NPLC, a strong, polar solvent, or in IXC, a buffer) or the organic modifier (in RPLC, methanol, acetonitrile, or THE) may affect the retention of the components of the sample in different possible ways. In the most classical case, such as in ion-exchange chromatography or in normal phase HPLC, the... 

Improvements in stationary phase design have advanced to a more coherent technology for achieving, at least in practical terms, well defined sorptive effects. Equally important, similar progress has been made toward improved practical understanding of liquid phase compositions needed to achieve chemical selectivity. Preliminary solvent selection has been reduced to the use of solvent triads, one for aqueous and another for non-aqueous systems (14). Thus, aqueous mixtures for reversed phase HPLC, or RPLC, are prepared with methanol, acetonitrile, and/or tetrahydrofuran as... 

Mobile Phase—A solvent that carries the sample through the column. Typical mobile phases in RPLC are mixtures of water with acetonitrile or methanol. 

Figure 10 Plot of the logarithm of the retention factor versus the number of methylene groups in the side chain of n-alkylbenzenes. The data were obtained at three different solvent compositions (0%, 10%, and 20% water in methanol) and at various temperatures around room temperature. The plot shows the linear relationship of the logarithm of the retention factor with the number of methylene groups and the common focal point of the lines frequently found in RPLC. (Data from Rdi 5.)...

The addition of small percentages of 1-propanol to micellar mobile phases was first recommended by Dorsey et al. [13], to enhance the chromatographic efficiency and decrease the asymmetry of chromatographic peaks. Since then, several organic solvents have been studied as modifiers in MLC. Of these, short and medium chain alcohols (z. e., methanol, ethanol, propanol and butanol) have shown to be the most suitable. Less frequent has been the use of pentanol [14, 15], Only a few rqports have appeared on the MLC behavior of solutes in the presence of other organic solvents commonly employed in conventional RPLC, such as acetonitrile [13, 16, 17], and tetrahydrofiiran [18, 19]. Micellar mobile phases allow the use of organic solvents in aqueous solution at molar concentrations well above their normal solubility limit in water alone. For example, the water solubility of pentanol is ca. 0.30 M, whereas in 0.285 M SDS micellar medimn, it increases to ca. 0.94 M [17]. 

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