David K. Lloyd

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.

Airway surface fluid composition in the rat determined by capillary electrophoresis

The apical surface of respiratory epithelial cells is covered by a thin layer of low-viscosity fluid termed airway surface fluid (ASF), about which relatively little is known. We collected samples of ASF from anesthetized rats, which were then analyzed using capillary electrophoresis, a method that enables extremely small quantities of fluid to be analyzed. We found values for Na+ (40.57 ± 3.08 mM), K+ (1.74 ± 0.36 mM), and Cl- (45.16 ± 1.81 mM), indicating that this fluid is hypotonic compared with rat plasma. In contrast, the concentrations of nitrite and nitrate within ASF were higher than reported plasma values. Additionally, intravenous administration of the cholinergic agonist methacholine (MCh) resulted in a dose-dependent increase in the concentration of Na+ and Cl- within the ASF. This increase is ∼50% in these ions after a dose of 100 ng MCh/g body wt. This animal model, together with this microanalytical technique, may be useful for investigating the in vivo regulation of ASF composition.The apical surface of respiratory epithelial cells is covered by a thin layer of low-viscosity fluid termed airway surface fluid (ASF), about which relatively little is known. We collected samples of ASF from anesthetized rats, which were then analyzed using capillary electrophoresis, a method that enables extremely small quantities of fluid to be analyzed. We found values for Na+ (40.57 +/- 3.08 mM), K+ (1.74 +/- 0.36 mM), and Cl- (45.16 +/- 1.81 mM), indicating that this fluid is hypotonic compared with rat plasma. In contrast, the concentrations of nitrite and nitrate within ASF were higher than reported plasma values. Additionally, intravenous administration of the cholinergic agonist methacholine (MCh) resulted in a dose-dependent increase in the concentration of Na+ and Cl- within the ASF. This increase is approximately 50% in these ions after a dose of 100 ng MCh/g body wt. This animal model, together with this microanalytical technique, may be useful for investigating the in vivo regulation of ASF composition.

Determination of enantiomeric purity of ephedrine and pseudoephedrine by high-performance liquid chromatography with dual optical rotation/UV absorbance detection

A reversed-phase high-performance liquid chromatography (HPLC) method with dual optical rotation/UV absorbance detection has been developed for the determination of enantiomeric purity of ephedrine hydrochloride and pseudoephedrine hydrochloride using an achiral column. The method gave a correlation coefficient of 0.9997 for the plot of log(optical rotation response) versus log (concentration) over the range of 0.06-10 mg ml(-1) of (+)-ephedrine hydrochloride (20 microliters injection). The limit of detection was 1.0 micrograms. Enantiomeric purity is shown to be most readily determined by measuring optical rotation, alpha, and absorbance, A, responses for standard and unknown samples, and using the equation (alpha/A)u/(alpha/A)s = (2xu - 1)/(2xs - 1), where x is the mole fraction of one of the enantiomers and subscripts s and u refer to standard and unknown, respectively. In blind trials using unknown mixtures of (+)- and (+/-)-ephedrine hydrochloride and a (+)-ephedrine hydrochloride standard, enantiomeric purities were determined to +/- 0.4% (95% confidence level) with five or six replicate 50 micrograms injections. The method has also been applied to the determination of the enantiomer mole fraction of (+)-pseudoephedrine hydrochloride in a cough linctus, giving xu = 0.99 +/- 0.01 with seven replicate injections of 20-fold diluted linctus samples containing 7.5 micrograms of the chiral compound being assayed. Unlike conventional polarimetry, the method does not require chemically-pure samples and can be orders of magnitude more economical in material.

Determination of enantiomeric purity of paclobutrazol and fluazifop-P-butyl using a diode-laser-based polaimetric high-performance liquid chromatography detector

Enantiomeric purities of resolved enantiomers of the agrochemicals paclobutrazol (I) and fluazifop-P-butyl (II) have been determined using a diode-laser-based polarimetric high-performance liquid chromatography detector. Reversed-phase achiral chromatography was used with polarimetric and absorbance detectors in series to measure optical rotation, α, and absorbance, A. In blind trials enantiomer mole fractions in unknowns, xu, were calculated from a standard, xs, and α/A values using the equation (α/A)u/(α/A)s = (2xu — 1)/(2xs — 1). The method always gave xu within 1% of actual values. 95% confidence limits were roughly twice those from chiral chromatography and better than conventional polarimetry. The linear range of the polarimetric detector was 0.02 – 10 mg ml−1 for I and 0.1 – 10 mg ml−1 for II (20 μl injection). Limits of detection for I and II of 0.19 and 1.0 μg correlate with specific rotations of the compounds.

A Diode Laser Polarimetric High-Performance Liquid Chromatography Detector and Applications to Enantiomeric Purity Determinations and Enantioselective Reactions

The principles of operation of a polarimetric high-performance liquid chromatography (HPLC) detector based on an 820 nm diode laser are discussed. Root-mean-square noise is 4 µ° (1 sec time constant), and detection limits are found in the range 0.10–2 µg dependent on specific rotation and chromatographic peak width. Determinations of enantiomeric purity have been carried out using achiral chromatography with dual polarimetric/absorbance detection. Applications to the pharmaceuticals ephedrine hydrochloride, pseudoephedrine hydrochloride (in a cough linctus) and the agrochemicals paclobutrazol and fluazifop-P-butyl demonstrate the accuracy and precision of the technique and its potential in quality control. Determinations of enantiospecificity in enzyme and free radical reactions are reviewed. Polarimetric detection with chiral chromatography of complex reaction mixtures has allowed identification of enantiomers, and with achiral chromatography of near racemates has given enantiomeric purities to 0.1%.