Miroslav Macka

The use of the Box–Behnken experimental design in the optimisation and robustness testing of a capillary electrophoresis method for the analysis of ethambutol hydrochloride in a pharmaceutical formulation

Box-Behnken experimental designs do not appear to be extensively used in optimisation of analytical methods using capillary electrophoresis (CE). This paper describes the use of the Box-Behnken experimental design to optimise the factors affecting the separation of ethambutol hydrochloride (EB), its impurity 2-amino-1-butanol and the internal standard (phenylephrine hydrochloride) in a CE method for a pharmaceutical tablet assay. The three factors studied simultaneously were: buffer pH, buffer concentration and applied electric field, each at three levels. The method was optimised with respect to three responses: resolution between peaks, theoretical plate count and the migration time of the EB peak. A statistical programme, which applies a multiple response optimisation algorithm, was used to calculate and optimise the three responses simultaneously. The optimum conditions were established to be 58.0 mM sodium borate buffer at pH 9.50 and an applied electric field of 412 V/cm. The robustness of the method was also determined and confirmed using a second Box-Behnken design, as part of the validation exercise. System suitability values for the method were derived from the regression surface analysis. The CE method for a pharmaceutical tablet formulation was further validated according to current regulatory requirements, with respect to linearity and range, precision, specificity, accuracy and limit of quantitation. The optimised method gives a fast and efficient separation under 4 min, with complete resolution between the three peaks, and represents an improvement over the existing USP method. It can be concluded that the Box-Behnken experimental design provides a suitable means of optimising and testing the robustness of a CE pharmaceutical method.

Developments in sample preparation and separation techniques for the determination of inorganic ions by ion chromatography and capillary electrophoresis

A review is presented of sample preparation and separation techniques for the determination of inorganic ions by ion chromatography (IC) and capillary electrophoresis (CE). Emphasis has been placed on those sample treatment methods which are specific to inorganic analysis, and the developments in separation methods which are discussed are those which enhance the capabilities of IC and CE to handle complex sample matrices. Topics discussed include solid-phase extraction for sample clean-up and preconcentration, dialytic methods, combustion methods, matrix-elimination IC, electrostatic IC, electrically polarised ion-exchange resins, electromigration sample preparation in CE, chromatographic sample preparation for CE, use of high-ionic strength background electrolytes, buffering of background electrolytes in CE, use of capillary electrochromatography for inorganic determinations, and methods for the manipulation of separation selectivity in both IC and CE. Finally, some possible future trends are discussed.

Solid-phase trapping of solutes for further chromatographic or electrophoretic analysis

Because of its simplicity, speed and effectiveness, solid-phase extraction (SPE) has become the preferred technique for concentration of selected analytes prior to chromatographic or electrophoretic analysis. In this review the historical development of SPE is briefly traced. Then the principles of SPE are reviewed in some detail. Numerous references are given on the format, sorbents, elution conditions, online techniques and automation with special emphasis on relatively recent developments. The principles and recent advances in solid-phase microextraction (SPME) are also reviewed. The final section on selected recent applications includes an extensive list of references to work published within the last three years. Future trends and developments are discussed briefly.

Separation of uranium(VI) and lanthanides by capillary electrophoresis using on-capillary complexation with arsenazo III

The viability of the separation of lanthanides and uranium(VI) in the form of strongly absorbing complexes with arsenazo III (AIII) was studied with the aim to increase the sensitivity of absorbance detection in determination of these metals by capillary electrophoresis (CE). Special attention was paid to the complexation equilibria in the background electrolyte (BGE). On-capillary complexation provided better peak shapes for lanthanides compared to pre-column complexation. While the BGE composition had very little effect on the peak shape of the kinetically inert uranium(VI) complex, it played a crucial role in the peak shapes of the kinetically labile lanthanide complexes. Addition of a second ligand competing with the metallochromic ligand AIII for the metal ions was found to be critical to achieve good peak shape. The nature and concentration of the competing complexing ligand added to the BGE, the pH, and the concentration of AIII were found to exert a strong influence on the separation selectivity, peak shapes and the detection sensitivity. Several carboxylic acids were compared as BGE competing ligands and citrate provided best selectivity and peak shapes. A citrate BGE at pH 4.7 and containing 0.1 mM AIII was used for the separation of uranium(VI) (350 000 theoretical plates) and LaIII (63 000 theoretical plates) while, to separate most lanthanides and uranium(VI), a similar BGE with a lower (0.03 mM) AIII concentration was used. Using hydrostatic sampling (100 mm for 10 s) detection limits of 0.35 μM (49 ppb) LaIII and 25 μM (60 ppb) UO2 were obtained. Using on-capillary complexation, sample stacking was retained for injection times of up to at least 100 s (ca. 30-mm sample plug) without loss of peak shapes for lanthanides or recovery for LaIII. When this process was used, the detection limit for LaIII was reduced to about 5 ppb. Optimal properties of metallochromic ligands for separation and detection of metals by CE are discussed.

Anion-exchange capillary electrochromatography with indirect UV and direct contactless conductivity detection

Conductivity detection is applied to ion‐exchange capillary electrochromatography (IE‐CEC) with a packed stationary phase, using a capacitively coupled contactless conductivity detector with detection occurring through the packed bed. Columns were packed with a polymeric latex‐agglomerate anion‐exchanger (Dionex AS9‐SC). A systematic approach was used to determine suitable eluants for IE‐CEC separations using simultaneous indirect UV and direct conductivity detection. Salicylate and p‐toluenesulfonate were identified as potential eluant competing anions having sufficient eluotropic strength to induce changes in separation selectivity, but salicylate was found to be unsuitable with regard to baseline stability. It was also found for both indirect UV and direct conductivity detection that homogenous column packing was imperative, and monitoring of the baseline could be used to assess the homogeneity of the packed bed. Using a p‐toluenesulfonate eluant, the separation of eight common anions was achieved in 2.5 min. Direct conductivity detection was found to be superior to indirect UV detection with regard to both baseline stability and detection sensitivity with detection limits of 4–25 μg/L being obtained. However, the calibration for each anion was not linear over more than one order of magnitude. When using conductivity detection, the concentration of the eluant could be varied over a wider range (2.5–50 mM p‐toluenesulfonate) than was the case with indirect UV detection (2.5–10 mM), thereby allowing greater changes in separation selectivity to be achieved. By varying the concentration of p‐toluenesulfonate in the eluant, the separation selectivity could be manipulated from being predominantly ion‐exchange in nature (2.5 mM) to predominantly electrophoretic in nature (50 mM).

Design of background electrolytes for indirect detection of anions by capillary electrophoresis

In capillary electrophoresis of inorganic and other low molecular weight anions using indirect photometric detection, the correct design of the background electrolyte can considerably reduce the time needed for method development and can increase the quality of the separation achieved. This article discusses the basic steps for development of suitable electrolytes, based on fundamental knowledge of the nature of the analytes and the proposed background electrolyte. Consideration is given to the choice of the absorbing probe used for indirect detection, based on the relative mobilities of the probe and the analytes, and the desired limits of detection. Choice of the electro-osmotic flow modifier and correct buffering protocols are also discussed. Finally, strategies to increase the useful mobility range of the background electrolyte are given, based on matching the mobilities of multiple electrolyte probes with the mobilities of the analytes.

Use of dyes as indirect detection probes for the high-sensitivity determination of anions by capillary electrophoresis

High sensitivity for indirect detection was achieved by utilising highly absorbing species as the displaced co-ion (or probe). Two highly absorbing dyes, bromocresol green and indigo-tetrasulfonate, were investigated as potential probes in the determination of small organic and inorganic anions. The concentration of these probes was kept as low as possible to ensure the signal-to-noise ratios were reasonable and the background absorbance was within the linear range of the detector. Four different protocols for buffering the electrolyte with such low probe concentrations were investigated. Buffering with agents that introduce co-anions [acetate or 2-(cyclohexylamino)ethanesulfonic acid (CHES) buffers] proved unsuitable as detection sensitivities were diminished due to competitive displacement by the analytes and system peaks were also induced. Buffering without introduction of co-anions was achieved using the buffering base, diethanolamine, or the use of ampholytes, lysine and glutamic acid. For separations performed with these two buffering approaches, migration time reproducibilities were less than 1% R.S.D. for most analytes. Minimal detectable amounts were in the low attomol region (1·10−18 mol), corresponding to sub-μM vacuum injected solution concentrations. These were an order of magnitude lower than the general detection limit reported for indirect photometric detection, and were comparable with detection limits achieved with indirect fluorescence detection. Finally, the detection limits were further improved by approximately three times for anions analysed with indigo-tetrasulfonate as the probe when a Z cell was employed as the detection cell.

Performance of a simple UV LED light source in the capillary electrophoresis of inorganic anions with indirect detection using a chromate background electrolyte

Light emitting diodes (LEDs) are known to be excellent light sources for detectors in liquid chromatography and capillary electromigration separation techniques, but to date only LEDs emitting in the visible range have been used. In this work, a UV LED was investigated as a simple alternative light source to standard mercury or deuterium lamps for use in indirect photometric detection of inorganic anions using capillary electrophoresis with a chromate background electrolyte (BGE). The UV LED used had an emission maximum at 379.5 nm, a wavelength at which chromate absorbs strongly and exhibits a 47% higher molar absorptivity than at 254 nm when using a standard mercury light source. The noise, sensitivity and linearity of the LED detector were evaluated and all exhibited superior performance to the mercury light source (up to 70% decrease in noise, up to 26.2% increase in sensitivity, and over 100% increase in linear range). Using the LED detector with a simple chromate-diethanolamine background electrolyte, limits of detection for the common inorganic anions, Cl-, NO3-, SO4(2-), F- and PO4(3-) ranged from 3 to 14 microg L(-1), using electrostatic injection at -5 kV for 5 s.

Artificial neural networks for computer-aided modelling and optimisation in micellar electrokinetic chromatography.

The separation process in capillary micellar electrochromatography (MEKC) can be modelled using artificial neural networks (ANNs) and optimisation of MEKC methods can be facilitated by combining ANNs with experimental design. ANNs have shown attractive possibilities for non-linear modelling of response surfaces in MEKC and it was demonstrated that by combining ANN modelling with experimental design, the number of experiments necessary to search and find optimal separation conditions can be reduced significantly. A new general approach for computer-aided optimisation in MEKC has been proposed which, because of its general validity, can also be applied in other separation techniques.

Separation of metal ions and metal-containing species by micellar electrokinetic capillary chromatography, including utilisation of metal ions in separations of other species

The use of micellar electrokinetic capillary chromatography (MEKC) for the separation of metal ions and metal-containing species is reviewed, together with the use of metal ions as a means to separate other species. Topics covered include the manipulation of separation selectivity through the use of complexation reactions induced by addition of a metal ion to the background electrolyte, enantiomeric separations facilitated through metal-analyte interactions, separation of organometallic species, separation of stable metal complexes in which the entire complex is the analyte and the separation of metal ions as analytes using pre-capillary or on-capillary complexation reactions with a suitable ligand.