Ionizable analytes

Reversed-phase IPC has quickly gained widespread acceptance- as a versatile and efficient method for the separation of ionized and easily ionizable analytes [342,355,360-364]. It is complementary to ion-exchange (section 4.5.8) and ion... 

Ionization Analyte Working Ionizing agent General features... 

Conductivity detection, which involves measuring changes in the conductivity of an aqueous solution between two electrodes, is employed in ion chromatography for the detection of ionized analytes. 

APPI was introduced by Robb et al. in 2000 as a complementary technique to ESI and APCI for broadening the range of ionizable analytes by API techniques. This ionization... 

EKC is not restricted to the separation of neutral analytes, as it is widely employed for the simultaneous separations of charged and neutral analytes as well as of ionizable compounds having similar electrophoretic mobility. The separation of ionizable analytes by EKC is governed by differences in the partitioning between the pseudostationary phase and the surrounding electrolyte solution as well as electrophoretic mobility. For these analytes, the retention factor can be described by the following mathematical model ... 

Bombardment Ionization Analytes Sample Ionization Mode Matrix Ref. 

Sorption of the IL cation anion partners also modifies the stationary phase which can introduce an ion-exchange type of retenhon. Further, either of the IL partners in the bulk mobile phase can serve as an ion-pairing agent for ionized analytes [44]. The extent to which any of these roles contribute to overall retention likely depends on the structure of the analytes as well as the lipophilicity of the cahon, charge diffusivity of the anion, and concentration of the IL in the mobile phase. 

Other evidence for the contribution of electrostatic interactions to retention on SOIL phases may be found in a study by Sun and Stalcup [67]. In this work, it was reported that while the LSFER approach successfully accounted for intermolecular interactions responsible for retention of nonpolar solutes, inclusion of ionizable solutes such as pyridine or nitrophenol isomers seriously degraded the correlation between experimental and predicted retention. Successful global application of an LSFER approach for a training set which includes ionizable analytes required incorporation of an additional descriptor to account for the degree of ionization [68] of the analytes as well as to account for the impact of electrostatic interactions. The additional descriptor incorporated the mobile-phase pH as well as the acid dissociation constant of the analyte. 

The main argument for making MIP CEC is to combine the selectivity of the MIPs with the high separation efficiency of CEC. This argument appears to fail, however, if the adsorption isotherm of the MIP is nonlinear, which seems to be the rule. In the case of nonlinear isotherms, the peak shapes depend mainly on the isotherm, particularly so if the separation system is otherwise very efficient (has low theoretical plate height, see Fig. 1). In the case of ionized analytes the situation is more complex. If an ionized analyte is not adsorbed at all on the MIP, then it is separated only due to electrophoresis, and its peak will not be widened due to the nonlinear effect. In this case, however, the MIP is merely behaving like an inert porous material. In intermediate cases an ionized analyte may participate in both separation mechanisms and for this case we do not have exact predictions of the peak shape. 

Fabrizzi L, Lichelli M, Rabaioli G, Taglietti A. The design of luminescent sensors for anions and ionizable analytes. Coord Chem Rev 2000 205 85-108. 

This radical cation may then ionize a solvent molecule by proton transfer, if the proton affinity of the solvent molecule is higher than that of the deprotonated radical cation. It seems that the solvent acts as aggregates, having then a higher proton affinity. These protonated solvent molecules may then ionize analyte molecules by proton transfer if these last have a higher proton affinity than the solvent molecules ... 

While a hydrophobic ion-pair is retained on hydrophobic stationary phases better than an ionized analyte, the retention of the duplex on normal phases is easily predicted to be lower than that of the ionized analyte because polar interactions are reduced. Actually the trend of k versus IPR concentration under normal phase IPC is the opposite of reversed phase IPC [34]. An aminopropyl, a cyanoethyl, and a silica stationary phase were compared for the analysis of alcohol denaturants. The cyanoethyl phase was selected and anionic IPRs were used to reduce retention of cationic analyte, suppressing their interactions with negatively charged silanols... 

The left-hand side of equation (2-33) expresses the presence of the gradient of the analyte in the column cross section. In the case of an ionizable analyte, there are two forms of the analyte present, and using expression (2-73) the left-hand side of equation (2-33) should be written in the form... 

The expression (dxldt) is the linear velocity of the analyte in the column and we get the final equation for the retention volume of ionizable analyte using the same conversions as in equations (2-33)-(2-39). 

Equation (2-79) is the general form describing retention of ionizable analytes. Since it was derived with the assumption that injected analyte does not noticeably disturb the eluent adsorption equilibrium in the column, it is only applicable for very low analyte concentrations. At these low analyte concentrations, the slope of the excess adsorption isotherm is assumed to be constant and we can substitute the derivatives of the excess adsorption functions for both forms of the analyte with corresponding Henry constants (K and bh) ... 

Each constant in the equation above represents single equilibrium process, which is assumed to be independent on other equilibria in the column. Equation (2-93) describes the retention of basic ionizable analytes in reversed-phase chromatographic system with binary eluents and liophilic counteranions added. Similar expression could be derived for the behavior of anionic analytes in the presence of liophilic countercation. 

Chemical Stability. Hydrolytic stability of base material is the most important parameter because most reversed-phase HPLC separations are performed in water/organic eluents with controlled pH. Selection of the mobile phase pH is mainly dictated by the properties of the ionizable analytes to ensure that they are in one predominate ionization state. 

Variation of the mobile-phase pH is one of the most powerful tools in controlling the separation for ionizable analytes. The main drawback of silica-based HPLC packing materials is their narrow applicable pH range. The other limitations are surface activity (or polarity), which for specific applications (such as separation of proteins or biologically active compounds) could play a major role. All these factors are the driving force for the search in alternative base materials for HPLC packings. 

Table 3-4 shows the relative standard deviation in the retention of the same analyte on all columns. For ionized analytes eluted very close to the void volume, the retention difference between all columns is significant, but for analytes retained on the column, the RSD of is much smaller than k and essentially within the experimental error range. 

Figure 4-39. Effect of temperature on ionizable analyte retention at pH 7.8 nsing a ODS3V column in temperature range 30-60°C. (1) Benzylamine 8.96,30°C). (2) BteN (quaternary amine), (3) berberine chloride, (4) qninine (4p.K), 8.3,30°C) (5) protriptyline 10.0, 30°C) (6) nortriptyline Q,pKa 9.1, 30°C). Flow rate ImL/min.

In reversed-phase HPLC with water/organic eluents, ionic interactions always play an important role in regard to analyte retention, solvation, ionic equilibria, and other processes. To some extent, chromatographic effects and practical use of ionic interactions have been discussed in the previous sections of this chapter. In this section the influence of the ionic additives in the mobile phase on the retention of ionic or ionizable analytes will be discussed. 

U. D. Neue, C. H. J. Phoebe, K. Tran, Y. Cheng, and Z. Lu, Dependence of reversed-phase retention of ionizable analytes on pH, concentration of organic solvent and silanol activity, /. Chromatogr. A 925 (2001), 49-67. 

Figure 8-12. Effect of pH and buffer on the peak shapes of ionizable analytes. (Reprinted from reference 14, with permission.)...

In what form will the molecule be analyzed (neutral or ionized) For this particular molecule we want to analyze the molecule in its neutral form. The pKa shift of the ionizable analyte. For this example, since the analyte is basic, the downward pK shift for basic analytes must be accounted for. The working pH should be at least 2 pH units above the basic analyte pKa to be fully neutral. One pH unit could also be used (analyte is approximately 90% in neutral form). 

HPLC is another convenient method for measurement of the NCE pKa values. As was shown by Melander and Horvath [13], the retention of any ionizable analyte closely resembles the curve shown in Figure 12-3. Chromatographic determination of the pKa could be accurately performed with very limited amount of sample. Fast HPLC method with optimum analyte retention is suitable for this purpose, but the influence of the organic mobile-phase modifier on the mobile phase pH and analyte Ka should be accounted for in order to provide the accurate calculation of the respective Ka value. Detailed discussion of the HPLC-based methods for the Ka determination is given in Chapter 4. 

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