Conductivity detection is a universal detection technique often encountered in electrophoretic separation systems, especially in modern chip-electrophoresis based devices. On the other hand, it is sparsely combined with another contemporary trend of enhancing limits of detection by means of various preconcentration strategies. This can be attributed to the fact that a preconcentration experimental setup usually brings about disturbances in a conductivity baseline. Sweeping with a neutral sweeping agent seems a good candidate for overcoming this problem. A neutral sweeping agent does not hinder the conductivity detection while a charged analyte may preconcentrate on its boundary due to a decrease in its effective mobility. This study investigates such sweeping systems theoretically, by means of computer simulations, and experimentally. A formula is provided for the reliable estimation of the preconcentration factor. Additionally, it is demonstrated that the conductivity signal can significantly benefit from slowing down the analyte and thus the overall signal enhancement can easily overweight amplification caused solely by the sweeping process. The overall enhancement factor can be deduced a priori from the linearized theory of electrophoresis implemented in the PeakMaster freeware. Sweeping by neutral cyclodextrin is demonstrated on an amplification of a conductivity signal of flurbiprofen in a real drug sample. Finally, a possible formation of unexpected system peaks in systems with a neutral sweeping agent is revealed by the computer simulation and confirmed experimentally.
Interactions among analyte forms that undergo simultaneous dissociation/protonation and complexation with multiple selectors take the shape of a highly interconnected multi-equilibrium scheme. This makes it difficult to express the effective mobility of the analyte in these systems, which are often encountered in electrophoretical separations, unless a generalized model is introduced. In the first part of this series, we presented the theory of electromigration of a multivalent weakly acidic/basic/amphoteric analyte undergoing complexation with a mixture of an arbitrary number of selectors. In this work we demonstrate the validity of this concept experimentally. The theory leads to three useful perspectives, each of which is closely related to the one originally formulated for simpler systems. If pH, IS and the selector mixture composition are all kept constant, the system is treated as if only a single analyte form interacted with a single selector. If the pH changes at constant IS and mixture composition, the already well-established models of a weakly acidic/basic analyte interacting with a single selector can be employed. Varying the mixture composition at constant IS and pH leads to a situation where virtually a single analyte form interacts with a mixture of selectors. We show how to switch between the three perspectives in practice and confirm that they can be employed interchangeably according to the specific needs by measurements performed in single- and dual-selector systems at a pH where the analyte is fully dissociated, partly dissociated or fully protonated. Weak monoprotic analyte (R-flurbiprofen) and two selectors (native β-cyclodextrin and monovalent positively charged 6-monodeoxy-6-monoamino-β-cyclodextrin) serve as a model system.
In this paper we determine acid dissociation constants, limiting ionic mobilities, complexation constants with β-cyclodextrin or heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, and mobilities of resulting complexes of profens, using capillary zone electrophoresis and affinity capillary electrophoresis. Complexation parameters are determined for both neutral and fully charged forms of profens and further corrected for actual ionic strength and variable viscosity in order to obtain thermodynamic values of complexation constants. The accuracy of obtained complexation parameters is verified by multidimensional nonlinear regression of affinity capillary electrophoretic data, which provides the acid dissociation and complexation parameters within one set of measurements, and by NMR technique. A good agreement among all discussed methods was obtained. Determined complexation parameters were used as input parameters for simulations of electrophoretic separation of profens by Simul 5 Complex. An excellent agreement of experimental and simulated results was achieved in terms of positions, shapes, and amplitudes of analyte peaks, confirming the applicability of Simul 5 Complex to complex systems, and accuracy of obtained physical-chemical constants. Simultaneously, we were able to demonstrate the influence of electromigration dispersion on the separation efficiency, which is not possible using the common theoretical approaches, and predict the electromigration order reversals of profen peaks. We have shown that determined acid dissociation and complexation parameters in combination with tool Simul 5 Complex software can be used for optimization of separation conditions in capillary electrophoresis.
- MeSH
- antiflogistika nesteroidní chemie MeSH
- beta-cyklodextriny chemie MeSH
- elektroforéza kapilární metody MeSH
- flurbiprofen chemie MeSH
- ibuprofen chemie MeSH
- ketoprofen chemie MeSH
- koncentrace vodíkových iontů MeSH
- magnetická rezonanční spektroskopie MeSH
- naproxen chemie MeSH
- osmolární koncentrace MeSH
- počítačová simulace MeSH
- software MeSH
- termodynamika MeSH
- viskozita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The complete mathematical model of electromigration dispersion in systems that contain a neutral complex forming agent and a fully charged analyte was introduced in the previous part of this series of papers (Part III - Theory). The model was implemented in the newest version of our simulation program PeakMaster 5.3 that calculates the effective mobility of the analyte and its nonlinear electromigration mobility slope, S(EMD), in the presence of a complex forming agent in the background electrolyte. The mathematical model was verified by both experiments and simulations, which were performed by our dynamic simulator Simul 5 Complex. Three separation systems differing in the chiral selector used (having different values for the complexation constant and the mobility of the complex) were chosen for the verification. The nonlinear electromigration mobility slope values were calculated from the simulations and the experiments that were performed at different complex forming agent concentrations. These data agree very well with those predicted by the mathematical model and provided the foundation for the discussion and explanation of the electromigration dispersion process that occurs in systems which contain a complex forming agent. The new version of PeakMaster 5.3 was shown to be a powerful tool for optimization of the separation conditions by minimizing electromigration dispersion which improves the symmetry of the analyte peaks and their resolution.
