Type of Organic Modifier

The principal difference in the behavior of acetonitrile and methanol, the most common eluent modifiers, was recently shown [50] where acetonitrile and THF forms a thick multimolecular adsorbed layer on the surface of reversed-phase adsorbent (C1-C18 and phenyl phases), while methanol is adsorbed only in monomolecular fashion. This brings a principal difference in the analyte retention mechanism in these two hydro-organic systems. Different retention mechanisms and their theoretical description are discussed in the Chapter 2. 

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. 

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). 

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In addition to temperature and flow rate, the retention and selectivity in reversed phase are controlled by (i) the concentration and type of organic modifier and (ii) the type, concentration and pH of the buffer. 

The most common method for varying the chromatographic selectivity for neutral molecules in RPC is to change the type of organic modifier in the mobile phase. In numerous studies using binary mobile phases, equation (4.15) has been shown to describe reasonably well the variation of solute retention with the volume fraction of organic solvent in an aqueous-organic mobile phase... 

Type of organic modifier Retention decreases with increasing solvent strength. 

The adduct formation can be largely controlled and directed into the formation of a single selected species by adequate choice of the ionisation mode (possibly at the expense of sensitivity), the eluent composition (buffer addition, pH adjustment, type of organic modifier) and by optimisation of the ion source parameters influencing the stability of individual (adduct) ions. In contrast to the variations in adduct or cluster formation, which principally can be diagnosed by recording more than one (adduct) ion in SIM mode, the occurrence of ion suppression requires more careful diagnosis. 

The same RP-TLC investigations were carried out by using ethanol, THF and dioxane as organic modifiers. The parameters of regression analyses are compiled in Table 3.6. It was established that the type of organic modifier exerts a negligible effect on the lipophilicity but influences considerably the specific hydrophobic surface area of the dyes. 

Regarding the type of organic modifier, there has not been any particular reason for selecting methanol or acetonitrile in the preparation of the mobile phase. Adjusting the mobile phase at a pH value lower than 3.0, the ionization of the carboxylate moiety is suppressed and increased retention of the analytes can be achieved. Under these conditions, however, problems may appear due to the high sensitivity of some penicillins at these pH values. 

A sensitive and rapid chromatographic procedure using a selective analytical detection method (electrospray ionization-mass spectrometry in SIM mode) in combination with a simple and efficient sample preparation step was presented for the determination of zaleplon in human plasma. The separation of the analyte, IS, and possible endogenous compounds are accomplished on a Phenomenex Lima 5-/rm C8(2) column (250 mm x 4.6 mm i.d.) with methanol-water (75 25, v/v) as the mobile phase. To optimize the mass detection of zaleplon, several parameters such as ionization mode, fragmentor voltage, m/z ratios of ions monitored, type of organic modifier, and eluent additive in the mobile phase are discussed. Each analysis takes less than 6 min. The calibration curve of zaleplon in the range of 0.1-60.0 ng/ml in plasma is linear with a correlation coefficient of >0.9992, and the detection limit (S/N = 3) is 0.1 ng/ml. The within- and between-day variations (RSD) in the zaleplon plasma analysis are less than 2.4% (n = 15) and 4.7% (n = 15), respectively. The application of this method is demonstrated for the analysis of zeleplon plasma samples [14]. 

In principle, the type of organic modifier(s) may be used as a parameter to optimize the selectivity of the RP-IPC system. However, because of the wide choice of other parameters, this possibility has not yet been extensively investigated. 

A similar study was performed on packed columns at HPLC conditions, and it was found that at any composition of the mobile phase, regardless of the type of organic modifier (THF, methanol or acetonitrile), the volume of the bonded layer corresponds to the densest arrangement of bonded ligands [60]. 

Variations in the selectivity are sometimes observed with the change in the type of organic modifier due to the specifics of the analyte-solvent interactions (solvation) and the specific adsorption behavior of the organic modifier. In the following example the effect of type and concentration of methanol and acetonitrile modifiers on the retention of acidic, basic, and neutral analytes is discussed. 

Because different forms of analyte usually show different affinity to the stationary phase, secondary equilibria in HPLC column (ionization, solvation, etc.) can have a significant effect on the analyte retention and the peak symmetry. HPLC is a dynamic process, and the kinetics of the secondary equilibria may have an impact on apparent peak efficiency if its kinetics is comparable with the speed of the chromatographic analyte distribution process (kinetics of primary equilibria). The effect of pH of the mobile phase can drive the analyte equilibrium to either extreme (neutral or ionized) for a specific analyte. Concentration and the type of organic modifier affect the overall mobile phase pH and also influence the ionization constants of all ionogenic species dissolved in the mobile phase. 

Overall, liophilic ions (usually small ions capable for dispersive interactions) provide a useful means for selective alteration of the retention of basic analytes. Influence of these ions on the column properties is fully reversible, and equilibration requires minimal time (usually less than an hour, or about 10 to 20 column volumes). On the other hand, the mechanism of their effect is very complex and is dependent on the type of organic modifier used and on the concentration applied. Theoretical description and mathematical modeling of this process is a subject for further studies. 

The obtained analytical results can be directly transferred to a preparative chromatography column. For the standard method depicted in Figure 3.51, a combination of acetonitrile and methanol is used as the organic modifier. Therefore, some additional experiments, investigating the effect of each type of organic modifier individually, are advisable because, very often, large differences in selectivity can be observed. 

As pointed out before, the polymer may be functionalized with polar groups to enhance compatibility with the modified clay. Bellucci et al. [92] have reported that the formation of polymer-clay nanocomposites depends mostly on the polymer properties, clay characteristics, and type of organic modifier. 

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