David M. Goodall

Differential binding of tioconazole enantiomers to hydroxypropyl-β-cyclodextrin studied by capillary electrophoresis

Capillary electrophoresis has been used to determine binding constants of tioconazole enantiomers with hydroxypropyl-beta-cyclodextrin (HP-beta-CD), after correcting for changes in mobility with increasing viscosity. The predicted and observed values of enantiomeric mobility difference were found to be maximum at a concentration of HP-beta-CD equal to the reciprocal of the average binding constant.

Chiral separation of β-amino alcohols by capillary electrophoresis using cyclodextrins as buffer additives. I. Effect of varying operating parameters

SummaryCapillary electrophoresis has been used for the chiral analysis of two β-amino alcohol pharmaceutical compounds. Capillary zone electrophoresis conditions were used with β-cyclodextrin as a chiral mobile phase additive. The effects of variation of β-cyclodextrin concentration, temperature, pH, background electrolyte composition and concentration have been investigated. Optimum separations were achieved for clenbuterol using β-cyclodextrin at its solubility limit (16mM), the lowest practicable temperature (19°C), pH 4.0 and an electrolyte solution with a high ionic strength prepared from 0.1 M citric acid and 0.3 M Na2HPO4. For the development compound picumeterol and its (S)-enationmer, the optimum pH 4.0 buffer was prepared from 0.1 M citric acid and 0.2 M sodium acetate. Baseline separation with resolution greater than 2 was achieved for both compounds.

Analysis of glyphosate and glufosinate by capillary electrophoresis-mass spectrometry utilising a sheathless microelectrospray interface

The potential of capillary electrophoresis combined with mass spectrometry for the simultaneous determination of two herbicides (glyphosate and glufosinate) and their metabolites (aminomethylphosphonic acid and methylphosphinicopropionic acid) as the native species is demonstrated utilising a simple microelectrospray interface. The interface uses the voltage applied to the CE capillary to drive separation and generate the electrospray, avoiding sample dilution associated with the use of a sheath liquid interface. The chemistry of the internal walls of the capillary has a marked influence on peak shape, and appropriate choice is essential to successful operation of the interface. A linear polyacrylamide coated capillary, which has no electroosmotic flow, gave best reproducibility, with precision of migration time and peak area in the range 1-2 and 7-12% RSD, respectively, for the four analytes. Limits of detection, low-pg on-column, are substantially better than for previous methods and calibration curves over the range 1-100 microM have R2 values greater than 0.97. The observed concentration limit of detection for glyphosate in water is 1 microM and for a water-acetone extract of wheat is 2.5 microM, allowing the underivatised herbicide to be detected at 10% of the maximum residue limit in wheat.

Analysis of glyphosate using capillary electrophoresis with indirect detection

Abstract A method has been developed for the analysis of glyphosate and aminomethylphosphonic acid (AMPA) in water. Using a pH 7.5 10 mM phthalate background electrolyte containing of 0.5 mM tetradecyltrimethylammonium bromide (TTAB) as an electro-osmotic flow modifier, the analytes were separated in less than 4 min under reverse polarity conditions and detected indirectly. Response was shown to be linear for over two orders of magnitude, calibration in the range 0.01–1.0 mM (1.7–170 μg ml−1 glyphosate, 1.1–110 μg ml−1 AMPA) giving least squares correlation coefficients of 0.9998 and 0.9999. Precisions of migration times and normalised peak areas were typically less than 0.7 and 2.0% respectively. Calibration slopes gave transfer ratios for glyphosate (0.98) and AMPA (0.75) in good agreement with values computed from theory using coupled transport equations. With stacking from water, the limit of detection for glyphosate was 5 μM, i.e., 0.8 μg ml−1 (twice peak-to-peak noise) for a 6.7 nl injection, calculated from a 0.05 mM standard solution. Field-amplified sample injection has enabled 0.01 μM (2 ng ml−1) to be detected, a factor of ∼ 1000 in signal enhancement being obtained over conventional hydrodynamic injection with no stacking.

Plackett-Burman experimental design in chiral analysis using capillary electrophoresis

SummaryPlackett-Burman experimental design has been studied in the chiral separation of clenbuterol using capillary electrophoresis (CE). This saturated fractional design approach was used to simultaneously investigate the variables pH, cyclodextrin concentration, electrolyte ionic strength, methanol concentration, and injection time, each at three levels. Percentage main effects of each variable on resolution, retention time and peak efficiency were calculated. Ionic strength, pH and β-cyclodextrin concentration were found to exert the greatest effect on resolution, pH on retention time and pH and ionic strength on peak efficiency. Results are in agreement with those from a previous univariate study. It is concluded that Plackett-Burman experimental design offers of rapid means for testing robustness of chiral CE methods or for the simultaneous screening of experimental variables.

Calorimetric and chiroptical evidence of aggregate-driven helix formation in carrageenan systems

Abstract Thermally induced, order-disorder transitions of iota- and kappa-carrageenan have been monitored by optical rotation and differential-scanning calorimetry in various ionic environments. Conformational ordering in kappa-carrageenan is observed only in the presence of cations that have been shown previously to promote helix-helix aggregation, and shows marked hysteresis between heating and cooling. Iota-carrageenan, by contrast, shows an order-disorder transition in the non-aggregating, tetramethylammonium salt form, at substantially lower temperature than for kappa-carrageenan, and without hysteresis. In the presence of potassium ions, which are known to promote aggregation, iota-carrageenan shows two distinct thermal-transitions, one without hysteresis at the same temperature as observed under non-aggregating conditions, and one with significant hysteresis close to the temperature of the kappa-carrageenan transition. We interpret these transitions as helix-to-coil and aggregated helix-to-coil, respectively. This interpretation is supported by measurements of the enthalpy changes of the transitions; ΔH values show a systematic increase with increasing aggregation and hysteresis. We conclude that the double helix of iota-carrageenan can exist as a stable entity in isolation, but may be further stabilised by aggregation, whereas the kappa-carrageenan helix is stable only when aggregated.

Quantitative aspects of capillary electrophoresis

Abstract Whilst rapid, high efficiency separations by capillary electrophoresis were achieved a decade ago, questions concerning the quantitative abilities of capillary electrophoresis techniques have been answered more slowly. Today, with improvements in instrumentation and capillary technology it is apparent that capillary electrophoresis can provide quantitative analyses which rival high-performance liquid chromatography and conventional electrophoresis in many applications.

Systematic approach to treatment of enantiomeric separations in capillary electrophoresis and liquid chromatography II. A study of the enantiomeric separation of fluoxetine and norfluoxetine

Abstract A systematic approach to enantiomeric separations in capillary electrophoresis (CE) and liquid chromatography (LC) with chiral mobile phase additives (MPA) or a chiral stationary phase (CSP) is used in the study of fluoxetine and norfluoxetine with cyclodextrins as chiral selectors. Binding constants and selectivities are determined under the same experimental conditions (mobile phase, buffer composition). Good agreement is found between results from the three techniques. The role of the buffer salt is investigated by comparison of binding constants obtained with triethylammonium and sodium acetate buffers. Investigation of the effects of derivatisation of the selector in CE and LC with MPA demonstrates the appropriate choice of cyclodextrin type for use in LC. By studying the influence of organic modifier content on separation parameters, CE can predict a useful solvent working range for a CSP.

Self-consistent framework for standardising mobilities in free solution capillary electrophoresis : applications to oligoglycines and oligoalanines

A theoretical analysis of deviations from ideality in ionic transport is presented to correct mobilities, mu, measured in free solution capillary electrophoresis (CE) to mobility at infinite dilution, mu degree (limiting mobility). Non-ideality is treated at the same level of approximation as in equilibrium, using a correction factor for the sum of the analyte and counter-ion radius originally suggested by Robinson and Stokes (Electrolyte Solutions, 1961). Unlike previous corrections using Debye-Hückel-Onsager theory, which are strictly applicable only at very low ionic strengths, this treatment is expected to be valid for univalent ions migrating in a uni-univalent background electrolyte for ionic strengths up to 0.075 mol kg-1, a range typical of CE experiments. The analysis is applied to the determination of mu degree in acidic and basic buffers for oligoalanines and oligoglycines with degree of polymerisation 2 to 6. Limiting mobilities for the fully protonated and deprotonated peptides are found to be numerically equal but opposite in sign, consistent with a change in charge from +1 to -1. In all uni-univalent buffers studied (borate, citrate, low pH lithium phosphate and sodium phosphate) mu degree values established using data over a range of pH and ionic strength are found to be identical and in excellent agreement with previous values from isotachophoresis. Values of mu degree in high pH sodium phosphate buffer are systematically 0.2.10(-8) m2 V-1 s-1 higher than those in other buffers; this may be attributed to limitations of the model for a buffer with 1+:2- and 1+:3- ions. This self-consistent framework for standardising mobilities in free solution CE is expected to be widely applicable to univalent analytes migrating in a 1:1 background electrolyte.

Role of cations in the conformation of iota and kappa carrageenan

Cation binding to iota and kappa carrageenan has been investigated by i.r. spectroscopy, differential scanning calorimetry, optical rotation, intrinsic viscosity and light scattering. From measurements of the salt dependence of the midpoint of the thermally induced order–disorder transition the increase in the number of cations associated with each disaccharide unit was estimated. I.r. studies of sulphate stretching frequencies for the ordered conformation indicate that association of tetramethylammonium ions to carrageenan is by simple atmospheric binding. With K+, by contrast, there is evidence of ion-pair formation, with stronger binding to kappa carrageenan than to iota. Enthalpy changes for the order–disorder transition support these conclusions. Comparison of intrinsic viscosity at high temperature in sodium or potassium chloride solution with values obtained in tetramethylammonium chloride solution indicate a less expanded conformation, consistent with a reduction in charge density by site binding of Na+ and K+, but not Me4N+. Binding constants were estimated from the variation in optical rotation of the disordered form with [Na+] and [K+]. Light-scattering studies for segmented iota carrageenan in the high-temperature (disordered) conformation give radii of gyration for the Na+ and K+ salt forms which are close to the value calculated for an unperturbed random coil, while in tetramethylammonium chloride the radius of gyration is appreciably higher.