H. Jork

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 toxcologically 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.

Vergleichende chromatographische untersuchungen bei s-triazinen☆

Abstract The applicabilities of gas chromatography (GC), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) for residue analysis of s-triazine herbicides are compared. With the conditions discussed the detection limit for the 14 s-triazines was determined as follows: 0.02–0.03 ng in GC alkali flame-ionization detection, 0.8 ng in GC flame-ionization detection, 1 ng in HPLC (UV detection), 3–5 ng in HPTLC (UV detection) and 8–13 ng in TLC (UV detection). The linear range in GC (AFID) was 104 whereas in the three liquid chromatographic methods it was 102. The reproducibility with a confidence limit of P = 95% has a coefficient of variation of ±5%. In routine analysis requiring a large number of separations HPTLC has advantages, because the time for a single separation is only 40 sec.

Möglichkeiten der quantitativen auswertung von papierund dünnschicht-chromatogrammen : Einführungsreferat

Abstract The possibilities of quantitative evaluation of paper- and thin-layer chromatograms are briefly reviewed. It is shown that most of the methods of determination used hitherto require greater amounts of substances than are present in a single spot on a chromatogram. Only direct estimation methods are suitable in the analytical range of application of thin-layer chromatography. Disregarding the visual procedure, these methods can be divided into densitometric, spectrophotometric and fluorimetric procedures from the technical point of view, and distinguished physically from the determination fo radioactivity. The advantages and disadvantages of the different methods of determination are discussed. In particular the universal possibility of using the direct spectrophotometric estimation is pointed out. These measurements provide unlimited applications in the wavelength range λ = 200 to 2500 nm with a relative standard deviation of ±3–4%. Thus they embrace the ultraviolet and visual spectral range, and the staining of chromatogram zones can mostly be dispensed with. Also in the qualitative evaluation of chromatographically separated compounds the non-destructive direct determination of spectra (absorption and fluorescence spectra) offers many advantages in the characterization and identification of single substances.

Beeinflussung der dünnschichtchromatographischen remissionsmessung durch arbeitstechnische faktoren : III. Simultanmessung und ihre einflussfaktoren

Abstract The influence on reflectance measurements of thin-layer chromatograms by technical factors. III. Factors influencing simultaneous measurements Spectrophotometric reflectance measurements are often used for the direct determination of thin-layer chromatograms. Since the scattering behaviour of the stationary phase varies as a result of different layer thickness, particle size and packing density, disturbances occur in the baseline pattern in the visible range of the spectrum. Smoothing of the background is possible when the influencing factors are known. This is achieved by simultaneous detection of the reflected and transmitted radiation and by compensation corrections of the reflectance curve. As a result of this simultaneous measurement the detection limit will be improved by a factor of 10 to 100, so that nanogram-amounts can also be unambiguously detected both qualitatively and quantitatively. The reproducibility of the total analysis is in the nanogram-range about s = 5–7 %. Calibration curves through the zero point are obtained.

Dünnschichtchromatographische bestimmung von testosteron und Δ4-androstendion(3,17) aus testesgewebe von rinderföten

Abstract Thin-layer chromatographic determination of testosterone and Δ4-androstene-3,17-dione from bovine foetal testicular tissue A procedure for the extraction of androgens from a few grams of bovine testes tissue is described. Δ4-Androstene-3,17-dione and testosterone were identified on the two-dimensional thin-layer chromatogram by means of their RF-values, the K onig -O ertel -reaction, and their U.V.-spectra. Quantitative determination of these compounds was carried out directly on the thin-layer plate with the Zeiss chromatogram-spectrophotometer principally based on reflectance measurements. With this method 0.2–0.5 μg of testosterone or Δ4-andorstene,3,17-dione can be estimated with a standard deviation of ±10%.

PrÄchromatographische in situ-Derivatisierung von FettsÄuren im Picomol-Bereich

SummaryA simple and rapid derivatization method for C24-C6 fatty acids on HPTLC RP-18 phases is described. The prechromatographic reaction with monodansylpiperazine and monodansylcadaverine takes place at the start. Linear calibration curves are obtained after chromatography (e.g. dansyl palmityl piperazide: r=0.9998), which can be employed for quantitative analysis in the lower nanogram range (determination limit <10 ng). All 10 fatty acids can be separated excellently (R usually 1.5). Stepwise and gradient developments are employed (AMD system).This is the first time that in situ derivatization techniques have been employed for fatty acids leading to fluorescent products in the picomole range and a gradient method has been described which also leads to precise separations on RP phases.

Terpene Derivatives, Essential Oils, Balsams, and Resins

This chapter contains a discussion of possible ways of separating lipophilic mixtures of natural products, the components of which are built up from isoprene units in accordance with Ruzicka’s isoprene rule [219].

Isolation and identification of decahydrotetramethylcyclopropazulenol in the essential oil of Murraya koenigii (L.) Sprengor

SummaryThe essential oil of curry leaves contains numerous terpene and sesquiterpene hydrocarbons as well as polar compounds from which decahydrotetramethylcyclopropazulenol, selin-11-en-4α-ol and caryophyllene expoxide have been isolated and identified for the first time. This has been done with the aid both of known classical methods and of modern NMR spectroscopic techniques, such as homo- and heteronuclear COSY, COSY spectra for long-range coupling and inverse TOCSY spectra. The results are summarized and discussed.

Determination of digitalis-glycosides by HPLC and reaction-detection

ZusammenfassungEine Referenzmethode zur Bestimmung von Digoxin in Humanserum wird vorgestellt. Die Einzelschritte bestehen aus einer RP-HPLC, dem Einsatz eines Reaktionsdetektors und der anschließenden Fluorescenzdetektion. Verfahrenscharakteristica für die Analyse von Serumproben sind: Nachweisgrenze von 100 pg/ml; Reproduzierbarkeit von VK=±2,9% und Linearität bis 5 ng.Die Probenvorbereitung besteht lediglich aus der Seruminjektion auf eine Injektionskartusche und Spülinjektionen.SummaryA reference method for the determination of digoxin in serum has been developed. The method consists of RP-HPLC, reaction detection and fluorimetric measurement. Characteristics of the method for the analysis in serum are: detection limit of 100 pg/ml; reproducibility of VC= ±2.9% and linearity up to 5 ng.The sample preparation consists of serum injection on a clean-up column and purge injections.

Quantitative fluorometric in-situ determination of netilmicin and 4 gentamicins on TLC-RP-layers

SummaryThis investigation describes a thin-layer chromatographic method for the quantitative determination of netilmicin and gentamicins C1, C1a, C2 and C2a in pharmaceutical preparations. The individual components are separated on C8 or C18 reversed phase layers with a mobile phase consisting of methanol and ammonia with added lithium chloride. After optimization of the post-chromatographic derivatization with 2,2-diphenyl-1-oxa-3-oxonia-2-boratanaphthalene (DOOB) quantitative determination takes place directly on the layer. The calibration curves are linear for netilmicin in the range 50–250 ng/zone and for the gentamicin components in the range 30–140 ng/zone (this corresponds to 100 to 400 ng/zone total complex). The determination limits per zone are 50 ng total gentamicin complex and 10 ng netilmicin per zone. No clean-up is required for analysis because of the great specificity of the derivatization reaction. The reproducibility of the method for independently carried out measurement series can be described by coefficients of variation between CV = ±1.7%–4.2%.