Ionizable analyte retention

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.

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 ... 

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. 

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... 

Liquid chromatography mass spectrometry (LC-MS) is now routinely used in analytical laboratories. Traditional IPRs are non-volatile salts that are not compatible with MS techniques because they play a major role in source pollution that is responsible for reduced signals. Moreover the final number of charged ions that reach the detector is impaired by ion-pair formation actually IPRs added to the mobile phase to improve analytes retention exert a profound effect on analyte ionization. Chromatographers who perform IPC-MS must optimize the eluent composition based on both chromatographic separation and compatibility with online detection requirements. 

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. 

The pH dependencies of the basic analyte retention in Figure 2-20 are not classic sigmoidal, which is supposed to plateau at low pH, and no change in retention should occur for ionized bases with a further decrease in mobile-phase pH. However, as shown in Figure 2-20, a slight increase of the retention is observed with decrease of mobile-phase pH. This effect has been observed... 

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. 

The analyte nature and its appearance (e.g., ionization state) in the mobile phase are also factors that affect the retention mechanism. Eluent pH influences the analyte ionization equilibrium. Eluent type, composition, and presence of counterions affect the analyte solvation. These equilibria are also secondary processes that influence the analyte retention and selectivity and are of primary concern in the development of the separation methods for most pharmaceutical compounds. 

From a practical point of view, the concept of linearity of the logarithm of the analyte retention factor could be used only for the rough estimation of the eluent composition variation. Also, the curvature of this dependence can show further deviations from linearity if the analyte is changing its ionization state at varying organic composition. 

The impact of the pH in hydro-organic mixtures on the analyte ionization and retention will be thoroughly discussed. The impact of pH on analyte UV absorbance will be discussed in the method development chapter. Chapter 8 (Section 8-6). 

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. 

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 8-27 (k versus wpH) and Figure 8-28 (selectivity versus wpH) show the effect of pH on the retention of the para and ortho isomers at a constant mobile-phase composition of 50 5015 mM KH2PO4 acetonitrile, at 25°C over the aqueous pH range 2.0-10.7 analyzed on a Luna C18(2) (Phenomenex, Torrance, CA) column. Both of these isomeric compounds are acidic, and it is expected that an increase in the mobile-phase pH will cause a decrease in the analyte retention because these compounds are becoming progressively more ionized. At 25°C for these isomers analyzed at pH < 8 the undesired isomer, ortho isomer, is eluting after the para isomer and at pH > 9 the ortho isomer elutes before the para isomer (desired elution order). 

In step 3, for this study the upper pH for the mobile phase to be prepared was determined to be JpH 6.7 (at least two units greater than the highest spA a of the molecule). The lower pH for the mobile phase (containing 30v/v% MeCN) that should be prepared for this study should be 1.7, but this would mean that an aqueous mobile-phase wpH of 1.1 would have to be prepared to obtain a spH of 1.7 (see Chapter 4, Section 4.5 for pH shift). Remember that the pH shift of the mobile phase for a phosphate buffer is approximately 0.2 pH units in the upward direction for every 10v/v% acetonitrile. In this case, not to compromise the stability of the packing material (column chosen has recommended a lower pH limit of wpH 1.5), a pH of pH 1.6 was chosen to be prepared which correlates to a spH of 2.2 ( pH 1.6 -i- 0.6 units upward pH shift upon addition of 30v/v% acetonitrile). Most definitely the final method will not be set at this low pH, since the analyte would exist in multiple ionization states however, the experiment was performed at this low pH to elucidate the effect of the pH on the analyte retention in this low-pH region. 

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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