Edmund T. Bergström

Systematic approach to treatment of enantiomeric separations in capillary electrophoresis and liquid chromatography.: I. Initial evaluation using propranolol and dansylated amino acids

Abstract A systematic approach is outlined for treatment of enantiomeric separations in capillary electrophoresis (CE) and liquid chromatography (LC) using chiral mobile phase additives. General equations and data analysis methods are presented to relate mobilities or capacity factors to equilibrium constants in binding equilibria, and to maximise mobility or retention time differences as a function of selector concentration. The use of cyclohexanol as a competitor is shown to be beneficial in optimising chiral separations of species which bind strongly to β-cyclodextrins. This general treatment has been applied with the test systems 1: propranolol and β-cyclodextrin and 2: dansylated amino acids and β-cyclodextrin. Chiral separations and binding constants, determined using LC with β-cyclodextrin as a mobile phase additive or a chiral stationary phase, are compared with results using the same selector in CE for system 2. Mobile phase equilibria defined by CE reveal more complex stationary phase binding equilibria in LC. Our studies make a link between LC and CE which may allow rational separation strategies to be transferred between the two fields.

Electromigration dispersion in capillary zone electrophoresis. Experimental validation of use of the Haarhoff-Van der Linde function.

This paper provides experimental validation of the use of the Haarhoff-Van der Linde (HVL) peak fitting function to fit experimental capillary zone electrophoresis (CZE) electropherograms. The test mixtures were composed of paraquat over a five order of magnitude concentration range (1.2 microM to 120 mM) and 4-aminopyridine at constant concentration (0.53 mM) as internal standard. Peak descriptors and electrophoresis parameters were extracted reliably by a Gaussian function from 4 to 40 microM; by the HVL function from 120 microM to 4 mM; and by a triangular function from 4 to 120 mM. The HVL function can be used where there is significant peak asymmetry due to electromigration distortion (EMD) and the Gaussian contribution toward the peak variance is greater than 25%. The peak centre (a1) and the Gaussian variance (a2) of the paraquat peak are shown to be independent of concentration. Diffusion coefficients obtained from a2 for both analytes were found to be in good agreement with their theoretical values. For all peaks where the distortion coefficient (a3) can be extracted, this parameter is shown to be directly proportional to the sample loading, as predicted by EMD theory. For the 4-aminopyridinium ion, mobilities calculated from a3 and measured independently are in excellent agreement. These results show that the HVL function accurately describes the two major processes, diffusion and EMD. contributing to the variance during a CZE separation.

A charge coupled device array detector for single-wavelength and multiwavelength ultraviolet absorbance in capillary electrophoresis.

A fundamental limitation to the use of single-point absorbance detection for capillary electrophoresis is irradiance, since it is not possible to create an image at the detection point on capillary that is brighter than the light source. This limitation may be overcome by illuminating a length of the capillary using a fiber-optic bundle and using a charge coupled device (CCD) camera that can image the full length of the illuminated zone. The present paper describes design and development of a CCD detector for UV absorbance that can be used in both multiwavelength and single-wavelength modes. The CCD camera images analyte peaks in the capillary dimension, together with wavelength-resolved absorbance in the dimension perpendicular to the capillary. Successive snapshots of the peaks are added together, after appropriate correction for time-dependent peak displacement, without sacrificing spatial resolution. Measured baseline rms noise values at 200 nm are 34 μAU using a holographic grating in multiwavelength mode and 8 μAU with the addition of a band-pass filter. Both values are in excellent agreement with calculations of limiting shot noise. Performance in multiwavelength mode is constrained by the 470-ms readout time of the CCD used, which sets a maximum duty cycle of 2.3%. Noise contributions from source intensity fluctuations are reduced by using a portion of the CCD image to provide a baseline reference signal. With 4-hydroxybenzoate as test analyte, the linear dynamic range in multiwavelength mode is shown to be between 3 and 4 orders of magnitude. High-quality spectra of 2-, 3-, and 4-methylbenzoates are obtained on capillary and used in deconvolution of closely migrating peaks of the 2- and 3-isomers.

Diode laser-based indirect absorbance detector for capillary electrophoresis

A near-infrared detector has been developed for use in capillary electrophoresis (CE). The detector has a double beam arrangement with signal- to-reference ratioing and operates with a 670-nm diode laser as the light source. The laser beam can be modulated to allow a.c. signal recovery using lock-in amplification. Near-infrared laser dyes have been investigated as background absorbers, and the singly-charged cationic dye rhodamine 700 found to exhibit suitable characteristics for use in indirect absorbance detection in methanol-water mixtures. Separations of a series of tetraalkylammonium compounds have been performed on both a commercial CE apparatus with indirect UV detection and on a home-made instrument incorporating the diode laser detector. The limit of detection (2 × peak-to-peak noise) for the tetrabutylammonium ion with diode laser-based indirect absorbance is 2 · 10−5 M. Both positive and negative peaks are found for these positively- charged analytes displacing a positively charged background absorber. Transfer ratios for all peaks are quantitated and the results compared with theoretical treatments of displacement in indirect detection. For analytes with mobility less than the indirect absorbing ion, measurement of peak area leads directly to the quantity of analyte present in the sample solution.

Ultrastructural evidence for intramolecular double stranding in iota-carrageenan

Kinetic studies of primary processes of conformational ordering in gel-forming biopolymers have suggested that a change in mechanism from intermolecular to intramolecular multistrand formation occurs on lowering the concentration of biopolymer. We report here ultrastructural observations consistent with intramolecular double stranding in a carbohydrate polymer, iota-carrageenan, by arresting this process of primary conformational ordering by an ultra-rapid freeze fixation technique. High-resolution transmission electron microscopy (TEM) revealed isolated iota-carrageenan chains showing a range of morphologies (linear, circular, and hairpin) consistent with intramolecular stranding. Control experiments in which iota-carrageenan was frozen in the disordered form revealed longer and thinner strands.

Multi-compound electrophoretic assays for tyramine oxidase with a UV area detector imaging multiple windows on a looped capillary

Active pixel sensor UV area imaging and capacitively coupled contactless conductivity detection have been applied in an electrophoretically mediated microanalysis (EMMA) assay for substrate specificity of tyramine oxidase (Arthrobacter sp.). Use of the UV area imaging detector to monitor four windows in a capillary with three loops provided intrinsic self-referencing for all species and identified tyramine and 2-phenethylamine as the only reactive components in a multi-compound mixture. Continuous engagement EMMA experiments showed significant benefits by comparison with plug-plug EMMA, improving sensitivity by extending enzyme-substrate interaction times and allowing measurement of time-dependent reaction in the substrate zones passing the four windows.

Dynamic analysis of capillary electrophoresis data using real-time neural networks

A method for real-time processing of snapshot data obtained from a capillary electrophoresis instrument using laser-induced fluorescence detection is described. The detector is based on a charge coupled device imaging 25 mm of the capillary length. Output data from the camera consists of a series of snapshot images of the analyte bands moving across the capillary window. The snapshot images are first pre-processed in order to obtain spatial profiles of fluorescence intensity. Each profile from successive snapshot images is processed on-line using a neural network, allowing the real-time measurement of separation parameters such as the velocity and width for each peak. This approach enables dynamic information to be extracted during the separation and the reduction of storage requirements, since the profiles where there are no peaks need not be recorded. The signal processing is based on profile approximation using a radial basis neural network which provides accurate and sensitive peak determination for each camera snapshot. The high spatial resolution of the system and accurate fluorescence profile approximation leads to good peak shape description and therefore to improved deconvolution of overlapping peaks. Examples of real-time analysis of complex peak patterns in separations of sulfonated aluminium phthalocyanines are given.

Capillary action liquid chromatography.

Capillary action LC (caLC) is introduced as a technique using capillary action as the driving force to perform LC in capillary columns packed with HPLC type microparticulate materials. A dry packing method with centrifugal force was developed to prepare capillary columns in parallel (10 columns per 3 min) to support their disposable use in caLC. Using a digital microscope for real-time imaging and recording separations of components in a dye mixture, caLC was found to have flow characteristics similar to TLC. Based on the investigation of microparticulate HPLC silica gels of different size (1.5-10 microm) and a typical TLC grade irregular medium, Merck 60G silica, the van Deemter curves suggested molecular diffusion as the major contribution to band broadening in caLC. With Waters Xbridge 2.6 microm silica, plate heights down to 8.8 microm were obtained, comparable to those achievable in HPLC. Assisted by an image-processing method, the visual caLC separation was converted to a classical chromatogram for further data analysis and such a facility confirmed the observation of highly efficient bands.

High-performance liquid chromatography applications of optical rotation detection with compensation for scattering and absorbance at the laser wavelength

Use of instrumentation developed to enable simultaneous monitoring of optical rotation (OR) and transmittance allows OR measurements to be made in the presence of high levels of absorbance, scattering or other effects that change the intensity of the plane-polarised light at the photodiode detector. This extends the application of OR detection to areas where it was previously difficult. Examples of the application of high-performance liquid chromatography (HPLC) with the improved OR detector include (i) the analytical scale separation of fructose and sucrose and (ii) the semi-preparative separation of enantiomers of warfarin and Trögers base. A signal-to-noise improvement of up to 150% is found when comparing signals with and without correction for transmittance changes. The improved OR detector has been used in series with a UV detector and the system shown to be suitable for on-line measurement of peak purity in separations using a chiral column under overload conditions.

Electrically assisted capillary liquid chromatography using a silica monolithic column.

A silica monolithic capillary column was linked to an open capillary of the same internal diameter via a Teflon sleeve to form a duplex column to investigate the combination of chromatography and electrophoresis in the mode of electrically assisted capillary liquid chromatography (eCLC). Using a commercial CE instrument with an 8.5 cm long, 100 microm i.d. reversed phase silica monolithic section and a window 1.5 cm beyond the end of this in a 21.5 cm open section, a minimum plate height of 9 microm was obtained in capillary liquid chromatography (CLC) mode at a low driving pressure of 50 psi. In eCLC mode, high speed and high resolution separations of acidic and basic compounds were achieved with selectivity tuning based on the flexible combination of pressure (0-100 psi) and voltage. Taking advantage of the excellent permeability of silica monolithic columns, use of a step flow gradient enabled elution of compounds with different charge state.