Metallofluorescent indicators as spray reagents for the in situ determination of organophosphorus pesticides on thin-layer chromatograms

Fluorescence-quenched solutions of palladium(II)-calcein and palladium(II)-calcein blue are shown to be sensitive spray reagents for the detection and in situ determination of organothiophosphorus insecticides on thin-layer chromatograms. The palladium is displaced from its non-fluorescent indicator complex by the pesticide producing fluorescent spots on the plate. Visual detection limits for 16 insecticides are given. As little as 10–50 ng of phosphorodithioate pesticides can be detected within l h after spraying and drying the plate, while the detection limits for phosphorothioates are somewhat higher (ca. 50–100 ng). Quantitative measurements may be conveniently made 18–24 h after spraying and drying the plate. Plots of fluorescence (as peak area) versus quantity of pesticide are linear over a 10–15 fold range. The relative standard deviation of replicate spots of Guthion (50–400 ng) and Cygon (50–100 ng) is 4–9 % and less than 15 % for 10 ng of Cygon or 20 ng of Guthion. The method was applied to the analysis of lake water spiked with Cygon. The recovery of 2–20 μg l^-1 of the pesticide was 87–113% with no clean-up other than t.l.c. required.     

Quantitative evaluation of thin-layer chromatograms

A commercially available scanning photometer, designed to scan paper electrophoresis strips, has been used to evaluate thinlayer chromatograms. The chromatogram may be lifted off the glass plate with cellophane tape which is then cut into strips and scanned, or the thin-layer chromatogram may be photographed and the photograph scanned. Newly developed cellulose-backed thin layers of silica or alumina may also be cut into strips and scanned. The instrument has also been modified to scan glass plates. A correction is applied for non-uniformity of the thin layer. Zones containing colourless components can be located by means of an auxiliary chromatogram, and the components present determined by carbon analysis.     

Fluorescence analysis of porphyrins in thin-layer chromatograms

Summary A fluorometric method is described for the direct evaluation of porphyrins on thin-layer chromatograms. The porphyrins (in analytical amounts, i.e., 0.05–10 g), in form of their methyl esters, are separated on commercial silica gel cards which have aluminium foil as a base. The separation is carried out in the solvent system benzol-ethyl acetate-methanol (85+13.5+1.5, v+v), and proceeds according to the number of methyl ester groups in the porphyrin molecules. The intensity of fluorescence of the porphyrin zones in the chromatograms was continuously registered on the Camag-Turner TLC-Scanner (primary filter Kodak-Wratten 47-B, secondary filter Kodak-Wratten 25; photomultiplier HTV-R136). On the basis of subsequent spectrophotometric analyses of the same material, factors were determined for the quantitative evaluation of the fluorescence peaks for two or more porphyrins in each chromatogram, thus permitting correct determination of the percent distribution of the porphyrins of a separated mixture. The sensitivity of the method is such that amounts of less than 1 ng of porphyrin methyl ester/mm² of silica gel surface (layer thickness 0.2 mm) are detected. The reproducibility depends mainly upon the sharpness of separation of the porphyrin methyl esters in the chromatogram. Under identical experimental conditions the coefficient of variability amounts to from 1 to 5% in groups of five similar chromatograms.Spotting the porphyrin methyl esters using automatic micropipettes and parallel application of fixed amounts of reference substances makes possible determination of the concentrations on the basis of geometric measurements of peak areas following development of the chromatogram. The diameter of the porphyrin zones should not exceed 13 mm if they are to fall fully within the fixed height of the excitation slit.Direct evaluation of porphyrins using fluorescence scanning of thin-layer chromatograms is best suited for serial determinations, and has found application in the biochemical diagnosis of hepatic porphyrias.

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Fluorescenzanalyse von Porphyrinen in Dünnschicht-Chromatogrammen

Zusammenfassung Es wird eine fluorimetrische Methode zur direkten Bestimmung der Porphyrine im Dünnschicht-Chromatogramm beschrieben. Porphyrine werden als Methylester im analytischen Maßstab (0,05–10 g) auf fertigen Kieselgel-Karten mit Aluminiumfolie als Trägermaterial getrennt. Die Trennung erfolgt im Lösungsmittelsystem Benzol-Essigsäureäthylester-Methanol (85+13,5+1,5, v+v) nach Anzahl der Methylestergruppen im Porphyrinmolekül.Die Fluorescenzintensität der Porphyrinzonen im Chromatogramm wird mit dem Camag-Turner DC-Auswertegerät (Primärfilter Kodak-Wratten 47-B, Sekundärfilter Kodak-Wratten 25; Photomultiplier HTV-R136) fortlaufend aufgezeichnet. Basierend auf durchgeführten spektrophotometrischen Analysen wurden Faktoren zur quantitativen Auswertung der Fluorescenzpeaks für 2 oder mehrere Porphyrine im Chromatogramm ermittelt, welche die richtige Bestimmung der prozentualen Verteilung der Porphyrine eines getrennten Gemisches gestatten. Die Empfindlichkeit der Analytik liegt unter 1 ng Porphyrinmethylester/mm² Kieselgelfläche (Schichtdicke 0,2 mm). Die Reproduzierbarkeit hängt in erster Linie von der Trennschärfe der Porphyrinmethylester im Chromatogramm ab. Unter identischen experimentellen Bedingungen liegt der Variationskoeffizient zwischen 1 und 5% in Gruppen von 5 gleichartigen Chromatogrammen.Punktförmige Applikation der Porphyrinmethylester mit automatischen Mikropipetten und paralleles Auftragen definierter Mengen von Vergleichssubstanzen erlauben nach der Entwicklung des Chromatogramms Konzentrationsbestimmungen aus der Berechnung der Peakflächen, wenn der Durchmesser der Porphyrinzonen unter Berücksichtigung der fixierten Spalthöhe des Scanners etwa 13 mm nicht überschreitet.Die Methode der fluorimetrischen Direktauswertung von Porphyrinen im Dünnschicht-Chromatogramm eignet sich vorwiegend für Serienbestimmungen und wurde in der biochemischen Diagnostik hepatischer Porphyrien eingesetzt.

     

Bioautographic detection of mycotoxins on thin-layer chromatograms.

A method for the bioautographic detection of mycotoxins on thin-layer chromatograms by using Artemia salina larvae is described. The method was tested on standard samples of mycotoxins (aflatoxin B1 kojic acid and sterigmatocystin) and on the extracts from toxicogenic fungi isolated from different sources.