Predicting peak shape in capillary zone electrophoresis: a generic approach to parametrizing peaks using the Haarhoff-Van der Linde (HVL) function.

We have found that the Haarhoff-Van der Linde (HVL) peak function provides excellent fitting to the shapes of CZE peaks. Initially designed for overloaded peaks in gas chromatography, this function describes a Gaussian peak when there is no peak distortion, and a triangular peak when there is no diffusional peak broadening. As such, it is ideal for CZE peaks distorted by electromigration dispersion (EMD). Fitting peaks with this function gives four parameters: three of them can be related to the Gaussian peak that would have been obtained in case of no EMD; the last one is a measure of the peak distortion. Using moving boundary theory, this peak distortion parameter may readily be expressed in terms of analyte and background electrolyte mobilities and concentrations, electric field, and sample injection length. The variance of an HVL peak is shown to be described by a universal function, and a master equation is presented. The region where EMD adds less than 10% to the Gaussian variance is shown to be very narrowly spread around the mobility matching condition. Under typical CZE operating conditions with an analyte at 1% of the BGE concentration, significant peak distortion is always present. Because the total peak variance is not an addition of the Gaussian and triangular contributions, the HVL model and the methodology introduced here should always be used to correctly combine variances.

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.

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.

Capillary electrophoresis with chiral selectors: optimization of separation and determination of thermodynamic parameters for binding of tioconazole enantiomers to cyclodextrins.

A systematic approach is outlined for optimization of enantiomeric separations in free solution capillary electrophoresis using chiral mobile-phase additives. Maximum electrophoretic mobility difference between the enantiomers occurs when the concentration of free selector is equal to the reciprocal of the average binding constant. General equations and data analysis methods are presented to relate mobilities to equilibrium constants in simple and competitive binding equilibria and used to determine thermodynamic parameters for host-guest complexation of tioconazole enantiomers with a range of cyclodextrin selectors. Selectivities are found to be in the reverse order of binding constants in the series dimethyl-beta-cyclodextrin (K1 = 6.9 x 10(3) M-1, alpha = 1.10) to hydroxypropyl-beta-cyclodextrin (K1 = 0.72 x 10(3) M-1, alpha = 1.29). For beta-cyclodextrin (K1 = 1.32 x 10(3)M-1, alpha = 1.20), delta H zero provides the dominant contribution to binding but delta delta H zero and T delta delta S zero terms give comparable contributions to the selectivity. Addition of alcohol does not affect the selectivity, but allows displacement of the optimum separation conditions to higher cyclodextrin concentration through either competitive binding (with cyclohexanol) or preferential solvation of reactants (with methanol).

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.