Hans F. M. Boelens

Comparison of five methods for the study of drug–protein binding in affinity capillary electrophoresis

The qualitative and quantitative aspects of capillary electrophoretic methods used to study drug-protein interactions, viz. the affinity capillary electrophoresis (ACE). Hummel-Dreyer (HD), frontal analysis (FA), vacancy peak (VP) and vacancy affinity capillary electrophoresis (VACE) methods have been investigated. In the ACE and the VACE methods the binding parameters can be calculated from the change in the electrophoretic mobility of the drug on complexation with a protein. In the frontal analysis and the vacancy peak method the free drug concentration is measured with UV detection. In the Hummel-Dreyer method the amount of drug bound is measured with UV detection. For the comparison of these five methods the warfarin-bovine serum albumin (BSA) system was used. Several factors that might influence the determination of association parameters were examined. With the FA, VP, HD and VACE methods the absolute numbers of the different binding sites involved in the complex formation can be determined, a major advantage in drug-binding studies.

Quantitative Aspects of Comprehensive Two‐Dimensional Gas Chromatography (GC×GC)

A software program was developed to enable the quantification of the complex 3D-data sets as produced by GC×GC. Using this software, it was demonstrated that the detectability limit of GC×GC in our study is 18 times better than that of ‘normal’ capillary gas chromatography (CGC). This enhancement is due to the signal increase produced by the thermal modulation effect. The relative standard deviation of 0.9% as measured on a test mixture was excellent. Furthermore, a comparison was made for the group-type separation of heavy gas oils between the hyphenation of LC and GC (LC-GC) and GC×GC. Although these separations are different in nature, the agreement of the results of both methods was very good. The results of GC×GC may even be more accurate, since, different from CGC, even in the most complex chromatograms the baseline in the second dimension chromatograms is always present.

Analysis of longitudinal metabolomics data

MOTIVATION Metabolomics datasets are generally large and complex. Using principal component analysis (PCA), a simplified view of the variation in the data is obtained. The PCA model can be interpreted and the processes underlying the variation in the data can be analysed. In metabolomics, often a priori information is present about the data. Various forms of this information can be used in an unsupervised data analysis with weighted PCA (WPCA). A WPCA model will give a view on the data that is different from the view obtained using PCA, and it will add to the interpretation of the information in a metabolomics dataset. RESULTS A method is presented to translate spectra of repeated measurements into weights describing the experimental error. These weights are used in the data analysis with WPCA. The WPCA model will give a view on the data where the non-uniform experimental error is accounted for. Therefore, the WPCA model will focus more on the natural variation in the data. AVAILABILITY M-files for MATLAB for the algorithm used in this research are available at http://www-its.chem.uva.nl/research/pac/Software/pcaw.zip.

Selection of optimal sensor position in a tubular reactor using robust degree of observability criteria

Robust selection criteria for the optimal location for in-process concentration or temperature sensors along the length of a tubular reactor for the partial oxidation of Benzene to Maleic Anhydride are developed. A model of the reactor is constructed by rewriting the Pdes describing the mass and heat balances into a set of Odes through the method of lines on a grid defined over the reactor length. The linearized model is described as a continuous, time-invariant state-space model where the state is formed by temperature and concentration profiles on the grid points. The best sensor location for the reactor is found by specifying scalar measures on the observability Gramian integral from the linear least-squares state estimation problem. New robust criteria for a degree of observability are specified. The scores on these criteria are determined by the amount of signal received by a sensor for a specific system configuration. These new selection criteria are compared with known measures for degree of observability for the optimal sensor location problem from the literature.

Vacancy affinity capillary electrophoresis, a new method for measuring association constants

A new method to determine association constants, the vacancy affinity capillary electrophoretic (VACE) method is described. The capillary is filled with a mixture of the interacting components and neat buffer is injected. The concentration of one of the components is fixed, and the concentration of the other one is varied during a series of experiments. The presence of both species in the capillary causes a large background signal. Two negative peaks will appear in the electropherogram. The migration times of the negative peaks reflect the fractions of the free and bound components in the mixture, and depends on the concentration in the buffer. This shift can be used to construct a binding curve from which the association parameters can be estimated. Nearly the whole system is saturated with the interacting components, so that losses due to wall adsorption are less detrimental.

Estimating reaction rate constants: comparison between traditional curve fitting and curve resolution

Abstract A traditional curve fitting (TCF) algorithm is compared with a classical curve resolution (CCR) approach for estimating reaction rate constants from spectral data obtained in time of a chemical reaction. In the TCF algorithm, reaction rate constants are estimated from the absorbance versus time data obtained from selective wavelengths. In this case, wavelengths are selected at which mainly one species is absorbing in time. In CCR, pure spectra of reacting absorbing species and the reaction rate constants are estimated simultaneously. Both TCF and CCR have been applied to experimental data. The reaction rate constants and the individual pure spectra of the reacting absorbing species were estimated simultaneously from the UV–VIS spectra taken in time of the two-step biochemical consecutive reaction of 3-chlorophenylhydrazonopropane dinitrile with 2-mercaptoethanol. This reaction was performed under second order and pseudo-first order conditions. For both conditions, the signal-to-noise ratio was approximately the same. However, if second order conditions are chosen, the first reaction step is very slow, which results in small absorbance differences in time. For the pseudo-first order dataset, the best precision of the reaction rate constant estimates has been obtained with TCF. For the second order dataset, CCR performed the best with respect to the precision of the reaction rate constant estimates.

Critical evaluation of the applicability of capillary zone electrophoresis for the study of hapten-antibody complex formation

The applicability of capillary zone electrophoresis (CZE) for the determination of association constants of an hapten-antibody complex with values as high as 10(7) (mol/I)-1 was investigated (dissociation constant in the nmol range). As a reference method the well known, enzyme-linked immunosorbent assay (ELISA) was selected. The study describes the optimisation of the experimental conditions of the CZE technique. The CZE measurements were optimised according to an experimental design. The results of the CZE and ELISA methods are compared giving consideration to the reproducibility (repeatability) of the two methods.

Determination of Rate Constants in Second-Order Kinetics Using UV-Visible Spectroscopy

A general method for estimating reaction rate constants of chemical reactions using ultraviolet-visible (UV-vis) spectroscopy is presented. The only requirement is that some of the chemical components involved be spectroscopically active. The method uses the combination of spectroscopic measurements and techniques from numerical mathematics and chemometrics. Therefore, the method can be used in cases where a large spectral overlap of the individual reacting absorbing species is present. No knowledge about molar absorbances of individual reacting absorbing species is required for quantification. The reaction rate constants and the individual spectra of the reacting absorbing species of the two-step consecutive reaction of 3-chlorophenylhydrazonopropane dinitrile with 2-mercaptoethanol were estimated simultaneously from UV-vis recorded spectra in time. The results obtained were excellent.

Origin and correction of bias caused by sample injection and detection in capillary zone electrophoresis

It is well known that electrokinetic sample introduction leads to a bias in CZE due to differences in the electrophoretic mobility of the substances. Therefore, a correction by multiplying the peak area with the migration time is often deemed necessary. With on-column detection, the area of a peak in the electropherogram depends on the velocity of the zone. In CZE every component has a different velocity, leading to a bias in detection. For that reason another correction by dividing the peak area by the migration time can be used. It will be shown that the injection and detection bias are inversely proportional to each other. This means that no correction of peak area is necessary for these biases in the case of electrokinetic injection being used in combination with UV detection, in contrast with hydrodynamic injection.

Applicability of capillary zone electrophoresis to study metal complexation in solution

Abstract The suitability of capillary zone electrophoresis for the determination of stability constants of metal complexes was investigated. Three variants were tested viz., the frontal analysis, the Hummel-Dreyer method and the vacancy peak method, in the step-wise complexation between copper(II) and 1,10-phenanthroline as well as 2,2′-bipyridyl. The investigation reveals that CZE has attractive features for the quantitative study of complex formation with neutral ligands. However, the full exploitation requires more sensitive detection systems and application of an automated CZE system.