Ionizable analyte selectivity

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. 

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... 

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. 

Ionic interactions are exhibited between two groups (of analyte and sorbent) with opposite charge. These are the strongest interactions that can be seen between an analyte and a sorbent. Because few compounds possess either a cationic or an anionic group, the selectivity is high. Ionic interactions can only be effected in a polar environment. For an actual ionic interaction, it is necessary that the respective functional groups of sorbent and analyte are both ionized. Analytes can be eluted in two ways ... 

When combined with tandem mass spectrometry, capable of selectively detecting a few analytes from the many that could be present, this approach provides for unsurpassed analytical selectivity for difficult chemical problems such as the study of drug—receptor binding [36] or the separation of complex mixtures of proteins or peptides [37]. The detection approach can be implemented in either on- or off-line formats. Alternatively, the purified antibody can be immobilized on a matrix-assisted laser-desorption ionization probe to allow direct application and characterization of a liquid sample containing the target molecule [38]. 

Ionizable compounds may have to be dissolved in buffered solutions, to ensure that only one form of the analyte exists in solution. The pH employed is commonly at least two pH units above or below the pKa, depending on which pH yields the optimal chromophore. Just as pH equilibria can be employed, chemical complexation equilibria can also be employed to improve analytical selectivity. 

If nothing forces us to use normal-phase chromatography, we should preferentially select one of the other techniques. We can divide the analytes into three categories very polar, neutral analyte ionic or ionizable analytes and everything else. 

Wagner M, Varesio E, Hopfgartner G. Ultra-fast quantitation of saquinavir in human plasma by matrix-assisted laser desorption/ionization and selected reaction monitoring mode detection. J Chromatogr B Analyt Technol Biomed Life Sci 2008 872 (l-2) 68-76. 

Figure 9.2 Using different chemical functionalities, pSi surfaces can be tailored to selectively adsorb and efficiently ionize analytes. Left to right DIOS-MS spectra of 500fmol of BSA digest analyzed on 8-month-old TMS-derivatized DIOS chip, carbohydrate mix containing sucrose (MNa 365) and maltotriose (M Na 527) on amine-silylated pSi surface, and small-mole-...

Ion thermochemistry is of great practical importance to MS. Mass spectral patterns are intimately related to ion energy. In chemical ionization (Cl)MS, the absence of appreciably endoergic IMRs results in analytical selectivity. Schemes for analyzing and sequencing peptides and other btomolecules by MS involve site-specific protonation or cationization The site specificity is determined by the relative proton affinities or metal ion affinities of various functional groups. The dynamics of the reaetions is important too, but thermodynamics ultimately controls the favored proeesses [31]. 

Acid/base/neutral fractionation of aqueous or organic solvent-based aroma isolates are relatively simple to accomplish taking advantage of the effect of pH on the solubility of ionizable analytes. One can, by changing pH, selectively partition an aroma isolate into acid, basic, and neutral fractions. 

Figure 9 serves to demonstrate this equalizing of the stationary phases in the presence of buffers even for non-ionic analytes. In Fig. 9a, the separation of the isomers of nitroaniline on four rather different stationary phases with the help of an alkaline acetonitrile buffer is shown. Apart from small differences in the retention time, the separation of the three peaks looks rather similar on each of the four columns. Fig. 9b shows the separation of the nitroanilines on Symmetry Shield and on Zorbax Bonus in a methanol/water mixture. The chromatograms look absolutely different even an inversion of the elution order is observed. This means that to exploit the individual properties of the stationary phases in the realm of ultimate selectivity, one should dispense with buffers, which is not easy to realize in routine work, where reproducible retention times are required. Nevertheless, one should remember this in the case of orthogonal tests see below. These phenomena are observed even with simple, polar, non-ionizable analytes such as ketones (see Fig. 10). 

The fact that hydrophobic weU-covered phases do not facilitate polar interactions, which is often the case in the context of the neutralization of ionizable analytes by the pH value, can result in a good peak symmetry, but frequently also in inferior selectivity (see Fig. 19 in Chapter 1.1). The following risk thus arises in the case of well-covered, hydrophobic phases, a good peak symmetry frequently gives a false impression of similarly good selectivity. 

Cotte-Rodriguez, L Takats, Z. Talaty, N. Chen, H. Cooks, R.G. (2005). Desorption electrospray ionization of explosives on surfaces sensitivity and selectivity enhancement by reactive desorption electrospray ionization Analytical Chemistry, Vol.77, No.21, pp. 6755-6764, ISSN 1520-6882... 

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