Acetonitrile, solvent, RPLC

Acetonitrile, solvent, RPLC, 83 Adsorption chromatography, C02-based SFC, 158-63... 

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. 

Eqn.(3.73) suggests that any mixture of two solvents with the same ° value (iso-eluotro-pic solvents) will also have the same eluotropic strength. This would allow the application of a similar strategy for the definition of iso-eluotropic multicomponent mobile phase mixtures as was used for RPLC in section 3.2.2.1. In practice, the situation in LSC has proved to be more complicated, because an effect described as solvent localization limits the validity of eqns.(3.72) and (3.73) if polar components (such as acetonitrile or methyl t-butyl ether) are present in the mobile phase. This makes it difficult to calculate the composition of iso-eluotropic mixtures for LSC with sufficient accuracy for optimization purposes [360-363]. 

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). 

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... 

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) +... 

In RPLC, the influence of pressure on the chromatographic behavior is related to the hydrophobic interactions involved in the retention mechanism and to the change upon adsorption in the numbers of acetonitrile and water molecules in the solvent shells of the protein molecule and of the bonded layer. The importance of the changes in the retention factor and the saturation capacity with a change in the average column pressure will thus depend on the retention mode used and will vary with the hydrophobicity of the molecule [128]. In RPLC, it is larger with polymeric than with monomeric bonded phases [126]. 

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... 

Figure 2.10. Six RPLC chromatograms illustrating the effect of mobile phase solvent strength on solute retention and resolution. LC conditions were column Waters Symmetry C18, 3pm, 75x4.6 mm, 1 mL/min, 40°C, Detection at258nm. Mobile phase is mixture of acetonitrile (ACN) and water. Solutes were nitrobenzene (NB) and propylparaben (PP).

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

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