Toxicological evaluation of harmful substances by in situ enzymatic and biological detection in high-performance thin-layer chromatography

The efficiency of a chromatographic analysis method is determined by the selectivity of the chromatographic separation and the specificity of the detection method. In the case of high-performance thin-layer chromatography (HPTLC) the separated components can be detected and quantified directly on the chromatogram by physical and chemical methods. By coupling high-performance thin-layer chromatography with biological or biochemical inhibition tests it was possible to detect toxicologically active substances in situ. A linear relationship was shown between the signal of the inhibition of cholinesterase and the concentration of the inhibitor using a constant enzyme concentration and a constant incubation time. The graph of the inhibition of the luminescence of Photobacterium vibrio fisheri in relation to the concentration of pentachlorophenol (range 20–80 ng) is nearly linear. Measurements were done by using a densitometer or a videodensitometric scanner.     

Coal-tar pitch characterization by thin-layer chromatography with flame ionization detection

Summary A rapid and quantitative method for compound class characterization of coal-tar pitches without previous fractionation, using an improved TLC method with an FID system, has been developed. Results show adequate accuracy and precision, including the sampling step.A fast calibration method, based on a variation of the internal normalization procedure, can be used for up to 18 g of whole sample application, avoiding the usually tedious absolute calibration in such analyses. This range is more than sufficient in view of the small amounts usually spotted in this technique.Speed of sample application by autospotter influences the shape of the peak nearest the point of application. For the coal tar pitch studied (9 wt.% non-eluted), slow application (0.5 L min^–1) gives a non-eluted Gaussianintegratable peak. A faster speed (10 L min^–1) is usable for analysing fossil products with lower non-eluted content. Total analysis time is less than 2 h, a considerably improvement on current methods.     

Bioautography detection in thin-layer chromatography

Bioautography is a microbial detection method hyphenated with planar chromatography techniques. It is based mainly on antimicrobial or antifungal properties of analyzed substances. The review discusses three versions of bioautography, i.e. contact, immersion and direct bioautography. The more concern is given to the last one. Many applications are quoted, not only for testing various groups of compounds, but also for investigating biochemical processes and factors influencing bacterial growth. Additionally, related methods, which can be included into direct bioautography, are discussed. The most promising among them seems to be TLC-bioluminescence screening.     

Evaluation of kinetic parameters for enzymatic interesterification synthesis of L-ascorbyl lactate by response surface methodology

The kinetics of lipase-catalyzed interesterification synthesis of L-ascorbyl lactate was studied. To determine the enzyme kinetic constants of the interesterification, a three-factor and five-level central composite design was used. The factors studied were ethyl lactate concentration, reaction temperature (T), and water content (w). Moreover, a statistical approach called the response surface method (RSM) was used to predict the kinetic constants. Finally, the relationships between the kinetic constants (Vm and Km) and the reaction parameters (T and w) were obtained. To assess the accuracy of the RSM approach for determining Vm and Km, detailed validation experiments were carried out by the conventional approach at four different reaction parameters(35 degrees C, 10 microL; 45 degrees C, 20 microL; 55 degrees C, 15 microL; 65 degrees C, 18 microL). The results indicated that the RSM approach gave reasonable results for the determination of Vm and Km in the range of tested parameters.     

Kinetic detection method for thin-layer chromatography

The feasibility of using kinetic detection and simultaneous kinetic methods for the determination of species separated on thin-layer chromatographic plates has been explored. The proposed method was tested by monitoring the reaction of ninhydrin with leucine, isoleucine, and phenylalanine. The results obtained using three instruments (two scanning instruments and an imaging instrument) were compared. Although none of the instruments provided ideal results, the premise of the kinetic approach for the in situ quantification of analytes was shown to be valid. The possibility of using kinetic methods to resolve responses from overlapped species was also investigated, and shows promise for improving the overall resolution of thin-layer chromatographic methods. A discussion of the instrumental requirements for successful utilization of the kinetic detection method is also given.     

Spectral detection in thin-layer chromatography by linear photodiode array spectrometry

Summary A new method of spectral detection in thin-layer chromatography (TLC) is given. Spectral reflectance and fluorescence can be measured by use of fiber optics and a photodiode array. The possibilities of the method with respect to quantitative evaluation, background subtraction, and recognition of overlapped spots are demonstrated at examinations of dyes in comparison to conventional single wavelength detection. The apparatus, the experimental limitations, and future developments are discussed.

---

Spektrale Detektion in der Dünnschicht-Chromatographie durch lineare Photodiodenarray-Spektrometrie

     

Multidimensional detection and analysis in thin-layer chromatography

Thin-layer chromatography has become very important in the routine separation of complex mixtures. To allow rapid data acquisition from thin-layer plates, multichannel detectors have recently been used. In this article we will review how these devices can make possible the spectral identification of the chromatographed spots in a 'hyphenated' method.     

A new spray reagent for selective detection of dichlorvos by thin-layer chromatography

The misuse of dichlorvos (DDVP), an organophosphorus insecticide, results in many instances of poisoning. This paper describes a new spray reagent for selective detection of dichlorvos in biological materials by thin-layer chromatography. Dichlorvos in presence of moisture breaks down to dichloroacetaldehyde which in turn reacts with phenylhydrazine hydrochloride to give a yellowish red colour. In acidic media the colour is intensified and consequently the sensitivity of detection increases. The reagent is selective for dichlorvos, other organophosphorus insecticides failed to give a coloured spot. Moreover organochlorine, carbamate and synthetic pyrethroid insecticides or even constituents of visceral extracts (amino acids, peptides, proteins etc.) do not interfere. The limit of detection is ca 10 mug.