Determination of enantiomeric excess by chiral liquid chromatography without enantiomerically pure starting standards.

A facile approach for the enantiomeric excess determination of enantiomeric mixtures without the necessity of pure enantiomer standards is presented. Promethazine and trimeprazine commercial nonracemic mixtures were used as cases study to probe the validity of the method. Chromatographic resolutions obtained with a chiral column AGP in reverse phase mode were 1.32-1.16 (promethazine) and 1.20-0.93 (trimeprazine) for the three detectors (circular dichroism, photometric and fluorimetric) in series. Results obtained showed that enantiomeric excess was 10.4, 8.71 and 8.58% for promethazine and 1.60, 1.23 and 1.80% for trimeprazine (medium values of 9.23 ± 1.01% and 1.54 ± 0.29%, respectively). Recovery assay over human serum samples, at three concentration levels, spiked with prometazine and submitted to solid-phase extraction, gave values of 99.09-93.48% [S-(-) enantiomer] and 98.51-91.89% [R-(+)-enantiomer]. Detection limits of promethazine enantiomers were between 0.02 µg (fluorimetric) and 1 µg (circular dichroism), and 0.02-1.1 µg for trimeprazine.

Resolution of (+)-cinchonine and (-)-cinchonidine by phase-modulation fluorescence spectroscopy.

The different fluorescence lifetime profiles of the optical isomers (+)-cinchonine (CN) and (-)-cinchonidine (CD) have been used to resolve their mixtures, on the basis of phase and modulation fluorescence lifetime measurements. To resolve mixtures of these compounds two distinct approaches have been used: simultaneous equations from phase-resolved and modulation-based fluorescence lifetime measurements. Results indicate that frequency domain fluorimetry is useful to resolve stereomeric compounds for determination without physical separation. The precision of the measurements gives RSD values of 5.2% and 4.0% for CD and CN, respectively. Detection limits with the modulation fluorescence lifetime method were 11.3 and 17.2 microM for CD and CN, respectively.

Determination of asulam by fast stopped-flow chemiluminescence inhibition of luminol/peroxidase.

An efficient, sensitive and fast stopped-flow method has been developed to determine asulam in water, based on its inhibition effect on the horseradish peroxidase-luminol-hydrogen peroxide chemiluminescence reaction, (HRP-luminol-H(2)O(2)). Ultra fast data acquisition (0.20s) facilitates excellent selectivity because no interferences from concomitants in the matrix act in such short time scale. The precision as repeatability (expressed as relative standard deviation, n=10) was 0.4% at a 40 pM level. The detection limit was 1.5 pM (0.35 ng/L) and 7.15 pM in pure and raw water, respectively. The calibration data over the range 5-60 pM present a correlation coefficient of r=0.9993. The proposed method has been applied to determine asulam in water samples by using solid-phase extraction (SPE). Mean recovery value was 98.1+/-2% at 50 pM level.

Simultaneous determination of glycols based on fluorescence anisotropy.

Simultaneous determination of non-fluorescent glycols in mixtures without separation or chemical transformation steps is described. Two methods based in the measure of fluorescence anisotropy of a probe such as fluorescein dissolved in the analyte or analyte mixtures are described. In the first method, the anisotropy spectra of pure and mixtures of analytes are used to quantitative determination (if the fluorophor concentration is in a range where fluorescence intensity is proportional to concentration). In the second method, a calibration curve anisotropy-concentration based on the application of the Perrin equation is established. The methods presented here are capable of directly resolving binary mixtures of non-fluorescent glycols on the basis of differences on the fluorescence anisotropy of a fluorescence tracer. Best analytical performances were obtained by application of the method based on Perrin equation. This method is simple, rapid and allows the determination of mixtures of glycols with reasonable accuracy and precision. Detection limits are limited by the quantum yield and anisotropy values of the tracer in the solvents. Recovery values are related to the differences in anisotropy values of the tracer in the pure solvents. Mixtures of glycerine/ethylene glycol (GL/EG), ethylene glycol/1,2-propane diol (EG/1,2-PPD) and polyethylene glycol 400/1,2-propane diol (PEG 400/1,2-PPD) were analysed and recovery values are within 95-120% in the Perrin method. Relative standard deviation are in the range 1.3-2.9% and detection limits in the range 3.9-8.9%.

Phenol derivatives as enhancers and inhibitors of luminol-H2O2-horseradish peroxidase chemiluminescence.

Systematic studies on phenol derivatives facilitates an explanation of the enhancement or inhibition of the luminol-H2O2-horseradish peroxidase system chemiluminescence. Factors that govern the enhancement are the one-electron reduction potentials of the phenoxy radicals (PhO./PhOH) vs. luminol radicals (L./LH-) and the reaction rates of the phenol derivatives with the compounds of horseradish peroxidase (HRP-I and HRP-II). Only compounds with radicals with a similar or greater reduction potential than luminol at pH 8.5 (0.8V) can act as enhancers. Radicals with reduction potentials lower than luminol behave in a different way, because they destroy luminol radicals and inhibit chemiluminescence. The relations between the reduction potential, reaction rates and the Hammett constant of the substituent in a phenol suggest that 4-substituted phenols with Hammett constants (sigma) for their substituents similar or greater than 0.20 are enhancers of the luminol-H2O2-horseradish peroxidase chemiluminescence. In contrast, those phenols substituted in position 4 for substituents with Hammett constants (sigma) lower than 0.20 are inhibitors of chemiluminescence. On the basis of these studies, the structure of possible new enhancers was predicted.

Enhancer effect of fluorescein on the luminol-H2O2-horseradish peroxidase chemiluminescence: energy transfer process.

The chemiluminescence of the luminol-H2O2-horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intensity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects.

Normal-phase liquid chromatography of plant hormones using reversed cholic acid micelles as the mobile phase.

When several plant hormones with similar structures are present in a sample, conventional analytical methods are not sufficiently selective to effectively differentiate them. However, the excellent selectivity of micellar liquid chromatography allows similarly structured compounds to be easily differentiated. We used reversed cholic acid micelles dissolved in tetrahydrofuran as the mobile phase in normal-phase chromatography of seven plant hormones. Our results conformed closely to those predicted by the pseudophase micellar liquid chromatography theory. The chromatographic efficiencies were calculated from the peak width and the retention times, and we compared these values with those obtained by reversed-phase chromatography using sodium dodecyl sulfate mobile phase in water. To test the efficiency of this selective method for the analysis of plant tissues that may also contain contaminants, we applied it to homogeneous suspensions of spiked maize roots. Recoveries ranged from 91 to 110%, the relative standard deviations were between 0.46 and 10.03%, and the detection limits were between 0.08 and 0.80 micrograms g-1.

Variable-angle synchronous fluorescence spectrometry and rank annihilation methods for mixture resolution.

The potential of variable angle synchronous spectroscopy (VASS) for fluorescent mixtures resolution was assessed and compared with the rank annihilation method (RAM). For this purpose, a set of excitation-emission matrices from three standard cyclodextrin fluorescence-enhanced solutions of the pesticides aminocarb, carbendazim and coumatetralyl and a mixture of them was obtained. Careful selection of the spectral routes to be scanned provides analyte signals that are free of interferences. Application of the rank annihilation method to excitation-emission matrices (EEMs) obtained by conventional scanning spectrofluorimetry gives quantitative results that show poor precision and accuracy when compared to those of VASS. The recoveries from ternary mixtures by VASS are within 99-104% and by RAM within 84-130%.

Assay of cholinesterase using a pro-enhancer of the luminol-H2O2-horseradish peroxidase reaction.

2-Naphthyl acetate acts as a pro-enhancer of the luminol-H2O2-horseradish peroxidase reaction. Cholinesterase hydrolyses the bound acetyl group and produces 2-naphthol, and this compound is an enhancer of the chemiluminescent reaction. We studied the kinetics of chemiluminescent emission and the influence of 2-naphthyl acetate and cholinesterase enzyme concentrations. The cholinesterase concentration versus chemiluminescence intensity maximum was linear for cholinesterase between 0 and 181 microU/mL, with a detection limit of 8 microU/mL and a relative standard deviation of 9.5% (n = 3), for a sample containing 90.67 microU/mL of cholinesterase.

Enhancement and inhibition of luminol chemiluminescence by phenolic acids.

We explored the behaviour of a series of phenolic acids used as enhancers or inhibitors of luminol chemiluminescence by three different methods to determine if behaviour was associated with phenolic acid structure and redox character. All the phenolic acids inhibited chemiluminescence when hexacyanoferrate (III) was reacted with the phenolic acids before adding luminol. The redox character of these compounds was clearly related to structure. When hexacyanoferrate(III)-luminol-O2 chemiluminescence was initiated by phenolic acid-luminol mixtures some phenolic acids behaved as enhancers of chemiluminescence, and others as inhibitors. We propose a mechanism to explain these findings. We found direct relationships between the redox character of the phenolic acids and the enhancement or inhibition of the chemiluminescence of the luminol-H2O2-peroxidase system and we propose mechanism to explain these phenomena.

Mineralization procedure for determination of copper in aerosols using photometric method based on copper-BPKQH complex.

A mineralization procedure is proposed for copper aerosols in which samples are mineralized by wet-ashing with HNO3 + HCIO4 mixture. Copper content is determined by a photometric method based on formation of a complex between copper and benzyl 2-pyridyl ketone 2-quinolylhydrazone (BPKQH). The influence of pH, ethanol content, and reagent concentration is studied. Copper determination at pH = 7.3, in the range 0.05 to 3 ppm is proposed. Mineralization with HNO3 is carried out in different conditions of evaporation (with or without dryness) and manipulation. Analysis of variance by the (ANOVA) single factor method shows that digestion conditions significantly increase the variability of results. Repeatability assays suggest that nitric acid alone gives low precision. Recovery assays reveal that the HNO3 digestion process causes copper loss by volatilization. Mineralization with HNO3 + HCIO4 is optimized on the basis of the accuracy and precision obtained. The ANOVA method suggests that mineralization conditions significantly increase total variability of results and also that results of the photometric method are not significantly different from those of the AAS method. The optimum mineralization procedure gave an RSD of 0.63% and a copper recovery of 98.4%. The combined digestion and quantification method was applied to determination of copper in aerosol samples obtained during leaf spraying operations of olive trees in the field.