Retention in IPC

Figure 3.28 reduces to the simple mechanism of figure 3.27 if both Kx and K Y are very small. If Kx is small (i.e. the solute molecule is mainly in the mobile phase) but K Y is large (the pairing ion is mainly absorbed into the stationary phase), then the mechanism of retention in IPC becomes similar to that of IEC. Typical ion-pairing as well as typical ion-exchange mechanisms may play a role in practical IPC systems. 

Figure 3.31 Example of the effect of the chain length of the pairing ion on retention in IPC. Pairing ions alkyl-benzyl-dimethyl ammonium with different lengths of the alkyl chain. 

As in 1EC, the counterion concentration has a considerable effect on the retention in IPC. In IPC the counterion is charged similar to the solute molecules, but opposite to the pairing ion. For example, for the separation of anionic solutes, the pairing agent may be a sodium sulfonate, in which the sulfonate is the pairing ion and sodium the counterion. The addition of a buffer salt (e.g. sodium phosphate) and a neutral salt (e.g. sodium bromide) may also contribute to the concentration of the counterion. Because of the similar retention mechanism, the counterion concentration has a similar effect on retention and selectivity in RP-IPC as in IEC. 

In Chapter 3 the significant consequences of salting effects on charged and neutral analyte retention in IPC will be treated in detail. 

The temperature dependence of retention in IPC is much more complicated. As explained above, retention results from at least four interdependent equilibria, each characterized by its own and A5°. The retention factor under IPC conditions (see Equation 3.19 and meanings of symbols and abbreviations in Section 3.1.2) is ... 

Table 11.1 summarizes the influences of the most important factors on analyte retention in IPC and condenses the concept expressed in Chapters 3 to 11. 

Several theoretical models, such as the ion-pair model [342,360,361,363,380], the dyneuaic ion-exchange model [342,362,363,375] and the electrostatic model [342,369,381-386] have been proposed to describe retention in reversed-phase IPC. The electrostatic model is the most versatile and enjoys the most support but is mathematically complex euid not very intuitive. The ion-pair model emd dynamic ion-exchange model are easier to manipulate and more instructive but are restricted to a narrow range of experimental conditions for trtilch they might reasonably be applied. The ion-pair model assumes that an ion pair is formed in the mobile phase prior to the sorption of the ion-pair complex into the stationary phase. The solute capacity factor is governed by the equilibrium constants for ion-pair formation in the mobile phase, extraction of the ion-pair complex into the stationary phase, and the dissociation of th p ion-pair complex in the... 

The temperature will affect both retention and efficiency in IPC, but not to the same extent as it does in IEC. IPC is usually a much more efficient technique (in terms of plate counts) than is IEC and therefore ambient temperatures usually yield satisfactory results. 

TABLE VIII. Comparison of the General Effect of Variables on Retention in Reversed-Phase Ion-Pair (RP-IPC) and Micellar Liquid Chromatography (MLC)... 

The retention model by Cecchi and co-workers also quantitatively faced the prediction of the retention behavior of neutral and zwitterionic analytes in IPC. According to the electrostatic models, at odds with clear experimental data [1,50,52,53], the retention of a neutral solute is not dependent on the presence and concentration of a charged IPR in a chromatographic system. Equation 3.23 is very comprehensive if Ze is zero [50], it simplifies since ion-pairing does not occur (C2= C3 = 0). Adsorption competition models the retention patterns of neutral analytes in IPC and the slight retention decreases of neutral analytes with increasing HR concentration may be quantitatively explained [50,53]. 

In Equation 3.30, c, and c c n be obtained, respectively, from Equations 3.26 and 3.29 and C2 is related to the molecular dipole [54], or alternatively to the fractional charge [61] the higher it is, the stronger the retention increase upon HR addition. Equation 3.30 can be valuable in IPC of life science samples that require keeping peptides at their isoelectric points (zwitterions) to avoid denaturation. 

The first effort to use LSERs in IPC relied on a retention equation based on a mixture of stoichiometric and electrostatic models. Several approximations were made [1-3]. First, ion-pairing in the eluent was neglected, but this is at variance with clear qualitative and quantitative experimental results [4-13]. In Chapter 3 (Section 3.1.1), the detrimental consequences of this assumption were clarified and danonstiated that extensive experimental evidence cannot be rationalized if pairing interactions in the eluent are not taken into account. Furthermore, in the modeling of A as a function of the analyte nature, the presence of the IPR in the eluent was assumed not to influence the retention of neutral analytes. This assumption is only occasionally true [14,15] and the extended thermodynamic retention model of IPC suggests the quantitative relationship between neutral analytes retention and IPC concentration in the eluent [16]. 

Two other reasons ILs have not attracted wider interest as putative organic modifier replacers are (1) their poorer UV transparency and (2) higher cost. Conversely, the successful use of these ionic compounds in IPC relies on the multiplicity of roles they can play simultaneously. The use of ILs as silanol suppressors (described in Chapter 5) and as IPRs (discussed in Chapter 7) demonstrates that they are versatile reagents in IPC as confirmed by their ability to control retention of ionized sample without including the organic modifier in the eluent. For this reason, they were proposed as environmentally friendly mobile phases [20],... 

The retention of ionogenic analytes in RP-HPLC is obviously dependent on the eluent pH. The pH value is an important optimization parameter because it controls the extent of ionization of the solute and hence the magnitude of electrostatic interactions. Varying the acidity of the mobile phase can lead to extreme changes in selectivity. Many interdependent parameters can be modulated in IPC and their optimization requires a theory-driven procedure. 

Equation 10.12 is algebraically correspondent [15] to the final relationship that describes analyte retention under IPC conditions (Equation 3.21). It upgrades the parallel stoichiometric equation of the model by Kazakevich and co-workers [16] that is inherently inadequate because it cannot predict the decrease of retention for analytes similarly charged to the chaotropic reagent and the electrostatic tuning of the retention of the unpaired analyte in the presence of the electrified stationary phase. It also upgrades electrostatic models [17,18] that disregard the role played by the ion-pair complex (final term in Equation 10.12). 

Examples of linear van t Hoff relationships can be found in the literature. Almost linear relationships between InA versus 1/T were reported for alkaloids under IPC conditions at a temperature range of 10 to 50°C with a linear decrease of retention with the increase of temperature [10]. A linear van t Hoff plot was also obtained in IPC of impurities in 2,4-disulfonic acid benzaldehyde di-sodium salt [21], even if nonlinear dependencies were also reported [22],... 

An increase in the alkyl chain length of the counter-ion increases retention in reversed-phase IPC by up to 2.5 times per added -CH2- group in the counterion. 

The nonsteric interactions in ipc depend on the chemical structure of the analyte, and also on nature of stationary and mobile phases. In normal- or reversed-phase hplc, neutral solutes are separated on the basis of their polarity. In the former case, polar stationary phases are employed (eg, bare sihca with polar silanol groups) and less polar mobile phases based on nonpolar hydrocarbons are used for elution of the analytes. Solvent selectivity is controlled by adding a small amoimt of a more polar solvent, such as 2-propanol or acetonitrile or other additives with large dipole moments (methylene chloride and 1,2-dichloroethane), proton donors (chloroform, ethyl acetate, and water), or proton acceptors (alcohols, ethers, and amines). Correspondingly, the more polar the solute, the greater is its retention on the column, yet increasing the polarity of the mobile phase results in decreased solute retention. 

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