Analyte retention acetonitrile/water eluent

Overall analyte retention in acetonitrile/water eluent is the superposition of different processes partitioning and adsorption. The volume of acetonitrile adsorbed layer is also dependent on the eluent composition (v/v% acetonitrile). This essentially may provide the explanation for the nonlinear behavior of the logarithm of the retention factors as a function of the eluent composition for acetonitrile as opposed to methanol, which forms only monomolecular layer and analyte retention factors generally show linear logarithmic dependence on the eluent composition (v/v% methanol). 

From the theoretical viewpoint, acetonitrile is the most suitable solvent to study the correlation of retention times and log P values of analytes, since the dipole moment (2.44) is nearly equal to that of water (2.55) (Figure 4.4). The electron donor effect can therefore be eliminated, and the elution order is not changed on modification of the acetonitrile-water mixture ratio. The first choice of an eluent should therefore be an acetonitrile-water mixture for non-ionic compounds in reversed-phase liquid chromatography. Methanol, acetone, THF, or DMF can then be added to improve the resolution. 

In a binary eluent system (acetonitrile-water), an adsorbed organic phase with finite thickness and composition different from the bulk mobile phase is preferentially accumulated near the surface of the bonded phase. The organic layer accumulated near the bonded ligands could behave as a liquid stationary phase in reversed-phase HPLC, and it contributes to the overall analyte retention process. 

Enhanced hydrophobicity of hypercrosslinked polystyrene, combined with additional 7r-interactions with aromatic moieties, explains the unusually strong retention of phenol and its derivatives from aqueous solutions. This makes it possible to pre-concentrate traces of phenols and chloro- and nitrophenols from water samples directly on the top of the analytical HPLC column (with MN-200 disintegrated to 15 im particles) [199] and then analyze the mixture on the same column with an aqueous-acetonitrile RP eluent. This approach increases the sensitivity of the determination because the whole amount of analytes initially present in the sample appears in the column and arrives at the detector, contrary to the situation with the off-line solid phase extraction (SPE) pre-concentration approach. 

Most studies on the direct introduction of water involve the on-line coupling of micro-LC and GC because the LC peak volumes and, consequently, the amount of water-containing eluent that has to be transferred, will then be small. For exam pie, Cortes et al. [7], who used an on-column interface and a nondeactivated retention gap, introduced up to 20 p.1 of acetonitrile water (50 50, v/v) and even pure water without serious distortion of the peaks of components eluting near the fully concurrent solvent evaporation (see Chap. 1) transfer temperature. One application dealt with the determination of the toxic bacteriostat, N-Serve, in corn. In another study, chlorpyrifos was determined in well water after a direct large-volume (20 Ltl) injection into the GC system [8]. However, the risk remains that more polar analytes will be adsorbed on the inner wall of the nondeactivated retention gap, which will result in bad peak shapes. 

In LC-LC and SPE-LC, the presence of water is commonly no problem at all. Actually, the reverse is true because eluents in RPLC are typically water-methanol or water-acetonitrile mixtures, and a high water content is mandatory during trace enrichment in order to ensure strong retention. However, when such an SPE precolumn or analytical column is coupled to a GC system, the introduction of water should be avoided completely or, at best, be permitted under strictly controlled conditions (see above). It will be clear that on-line trace enrichment (and clean-up) by SPE... 

Gradients of aqueous and organic mobile phases are typically used for LC-MS/MS analysis of drug compounds and metabolites. The most common aqueous solvents are water with 0.1 % formic acid or 0.1 % acetic acid (v/v) or volatile buffers like 5 mM ammonium-acetate or ammonium-formate. Often adjusted to a certain pH value with the corresponding acid or base (the pH of the eluents will have to be optimized with respect to the polarity of the analytes, since ionic species will have very low or no retention on the reversed pahse LC-columns). Other volatile buffers can be used as well. Phosphate buffers should be avoided, since they will cause suppression of the ionization and thus lead to very bad analytical performance (Venn 2000). Reagents like triethyl-amine should also be avoided as mobile phase or as part of mobile phases. They induce ion suppression as well. In terms of the organic solvents, methanol and acetonitrile are very widely used and they are very well suitable for LC-MS. Other solvents can be used as well, as long as they are compatible with the materials used in the LC-MS system. 

Figure 4-54. Schematic of the retention mechanism of basic analyte on reversed-phase material in water/acetonitrile eluent in the presence of liophilic ions (PFe ).

In liquid chromatography, the complexity of parameters contributing to retention is high. This is due to numerous different interactions between the stationary phase and the analyte on the one hand and the mobile phase and the analyte on the other. Moreover, the mobile phase interacts with the stationary phase, thus modulating its properties. Within this multifaceted entity, the stationary phase plays the central role. A schematic illustration of a Cjg reversed-phase system in a water/acetonitrile eluent is depicted in Fig. 1. 

Provided that the system (regression) parameters for a chromatographic method are known and the molecular descriptors for a new analyte are available, retention and thus selectivity should be predictable with the help of the LFER approach. Correlations of the calculated and the measured selectivities for nine pairs of solutes under six eluent compositions on a Merck Lichrospher 100 RP-18e column are shown in Fig. 6a for water/acetonitrile eluents and in Fig. 6b for water/ methanol eluents (taken from [14]). This plot, however, does not illustrate a real prediction of solute retention, since the depicted measured retention data served at the same time as a basis for the calculation of the theoretical selectivities. Even under these less critical conditions, relative deviations (expressed as the distances from the best linear fit in the x-coordinate) reach values of up to 20%, due to the significant residues from the multivariate regression (see confidence ranges in Fig. 4). To discuss the impact of these uncertainties on the quality of a possible prediction, a real example is discussed here. Starting from a selectivity of... 

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