Iva Zusková

Optimization of background electrolytes for capillary electrophoresis: II. Computer simulation and comparison with experiments.

A mathematical and computational model described in the previous paper (Gaš, B., Coufal, P., Jaroš, M., Muzikář, J., Jelínek, L., J. Chromatogr. A 2001, 905, 269–279) is adapted, algorithmized, and a computer program PeakMaster having a status of freeware (http://natur.cuni.cz/∼gas) is introduced. The model enables optimization of background electrolyte (BGE) systems for capillary zone electrophoresis. The model allows putting to use uni‐ or di‐ or trivalent electrolytes and allows also for modeling highly acidic or alkaline BGEs. It takes into account the dependence of ionic mobilities and dissociation of weak electrolytes on the ionic strength. The model calculates the effective mobility of analytes and predicts parameters of the system that are experimentally available, such as the transfer ratio, which is a measure of the sensitivity in the indirect UV detection or the molar conductivity detection response, which expresses the sensitivity of the conductivity detection. Further, the model enables evaluation of a tendency of the analyte to undergo electromigration dispersion or peak broadening. The suitability of the model is verified by comparison of the predicted results with experiments, even under conditions that are far from ideal (under extreme pH and a high ionic strength).

Simulation of the effects of complex‐ formation equilibria in electrophoresis: I. Mathematical model

Simul 5 Complex is a one‐dimensional dynamic simulation software designed for electrophoresis, and it is based on a numerical solution of the governing equations, which include electromigration, diffusion and acid–base equilibria. A new mathematical model has been derived and implemented that extends the simulation capabilities of the program by complexation equilibria. The simulation can be set up with any number of constituents (analytes), which are complexed by one complex‐forming agent (ligand). The complexation stoichiometry is 1:1, which is typical for systems containing cyclodextrins as the ligand. Both the analytes and the ligand can have multiple dissociation states. Simul 5 Complex with the complexation mode runs under Windows and can be freely downloaded from our web page http://natur.cuni.cz/gas. The article has two separate parts. Here, the mathematical model is derived and tested by simulating the published results obtained by several methods used for the determination of complexation equilibrium constants: affinity capillary electrophoresis, vacancy affinity capillary electrophoresis, Hummel–Dreyer method, vacancy peak method, frontal analysis, and frontal analysis continuous capillary electrophoresis. In the second part of the paper, the agreement of the simulated and the experimental data is shown and discussed.

Determination of stability constants of complexes of neutral analytes with charged cyclodextrins by affinity capillary electrophoresis.

A novel procedure for the determination of stability constants in systems with neutral analytes and charged complexation agents by affinity capillary electrophoresis was established. This procedure involves all necessary corrections to achieve precise and reliable data. Temperature, ionic strength, and viscosity corrections were applied. Based on the conductivity measurements, the average temperature of the background electrolyte in the capillary was kept at the constant value of 25°C by decreasing the temperature of the cooling medium. The viscosity correction was performed using the viscosity ratio determined by an external viscosimeter. The electrophoretical measurements were performed, at first, at constant ionic strength. In this case, the increase of ionic strength caused by increasing complexation agent concentration was compensated by changing of the running buffer concentration. Subsequently the dependence of the analyte effective mobility on the complexation agent concentration was measured without the ionic strength compensation (at variable ionic strength). The new procedure for determination of the stability constants even from such data was established. These stability constants are in a very good agreement with those obtained at the constant ionic strength. The established procedure was applied for determination of the thermodynamic stability constants of (R, R)‐(+)‐ and (S, S)‐(‐)‐hydrobenzoin and R‐ and S‐(3‐bromo‐2‐methylpropan‐1‐ol) complexing with 6‐monodeoxy‐6‐mono(3‐hydroxy)propylamino‐β‐cyclodextrin hydrochloride.

Simulated quantitative and qualitative isotachophoretic indices of 73 amino acids and peptides in the pH range 6.4–10

Qualitative and quantitative isotachophoretic indices of 73 amino acids, dipeptides and tripeptides were simulated under 24 leading electrolyte conditions covering the pH range 6.4-10. The RE values and time-based zone lengths are tabulated together with the absolute mobility (m0) and pKa values used. The leading electrolyte used was 10 mM HCl and the pH buffers were imidazole, tris(hydroxymethylamino)methane, 2-amino-2-methyl-1,3-propanediol and ethanolamine. The simulated indices will be useful in the assessment of the separability and determination of the listed and related compounds.

Enantioseparation of selected N-tert.-butyloxycarbonyl amino acids in high-performance liquid chromatography and capillary electrophoresis with a teicoplanin chiral selector

Enantioseparation of N-tert.-butyloxycarbonyl amino acids (N-t-Boc-Aas) with teicoplanin chiral selector was performed in two different separation systems: A teicoplanin-based chiral stationary phase (CSP-TE) was used in reversed-phase HPLC, and the same chiral selector (CS) was added into a background electrolyte (BGE) in HPCE. The enantioselective interaction with the same CSP/CS can be influenced by several factors, such as mobile phase/background electrolyte composition: the buffer concentration, pH, the CS concentration, the presence of organic modifiers. In addition, the charge of the chiral selector related to the charge of the analyte and to EOF are important variables in CE. The effect of these parameters on enantioselectivity and enantioseparation of selected N-t-Boc-Aas was studied. The presence of a sufficient concentration (1% solution) of a triethylamine acetate buffer in the mobile phase was shown to be essential for enantioseparation of these blocked amino acids in HPLC. A certain concentration of teicoplanin aggregates (along with teicoplanin molecules) in the BGE is required to obtain enantioseparation of N-t-Boc-Aas in HPCE.

Determination of thermodynamic values of acidic dissociation constants and complexation constants of profens and their utilization for optimization of separation conditions by Simul 5 Complex

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.

A nonlinear electrophoretic model for PeakMaster: part IV. Electromigration dispersion in systems that contain a neutral complex-forming agent and a fully charged analyte. Experimental verification.

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.

Detailed kinetic analysis of the interaction between the FOXO4–DNA-binding domain and DNA

The FOXO forkhead transcription factors are potent transcriptional activators involved in a wide range of key biological processes. In this work, the real-time kinetics of the interaction between the FOXO4-DNA binding domain (FOXO4-DBD) and the DNA was studied by using surface plasmon resonance (SPR). SPR analysis revealed that the interaction between FOXO4-DBD and the double stranded DNA containing either the insulin-responsive or the Daf-16 family member-binding element is preferably described by using a conformational change model which suggests a structural change of FOXO4-DBD upon binding to the DNA. This was further confirmed by using the time-resolved tryptophan fluorescence anisotropy decay measurements which revealed profound reduction of segmental dynamics of FOXO4-DBD upon the complex formation. Alanine scanning of amino acid residues engaged in polar contacts with the DNA showed that certain non-specific contacts with the DNA backbone are very important for both the binding affinity and the binding specificity of FOXO4-DBD.

Electromigration behavior of metal ions in the presence of complexing polymer

Abstract Electromigration behavior of metal ions in the presence of high concentrations of polyethylene glycol as a complexing agent was investigated under acidic non-buffered conditions. Simulation of the isotachophoretic steady state of the system containing the neutral polymer was performed. Both the complexation and the influence of viscosity on the electrophoretic mobility were taken into account. Good agreement between the calculated results and the experimentally obtained values was achieved. Based on the theoretical model, a new approach for the estimation of polymer–metal stability constants from experimental isotachophoretic data was proposed. Stability constants of Li+, Na+, Cs+, Mg2+, Sr2+, Mn2+, Co2+, Ni2+, Zn2+, Pb2+, Lu3+ and Y3+ ions with polyethylene glycol were determined.

Electromigration in systems with additives in background electrolytes. II: Ionic admixture

A theoretical model is presented for isotachophoretic migration in systems where both the leading and terminating electrolytes contain another common component (admixture) (e.g., a difficult to remove impurity). The effective mobility of the admixture is lower than that of the leading ion and higher than that of the terminating ion. The model system includes a further two samples. The mathematical simulation of the separation dynamics of this system was obtained by solution of basic physico-chemical laws. This facilitated a comprehensive formulation of the principles of the steady state and calculations of the parameters of all zones. Based on the theoretical model, a method for the determination of admixture concentrations in the leading and terminating electrolytes from isotachophoretic measurements and accurate determination of the limiting mobilities of the separated compounds from experimental data affected by the presence of the admixture are described.