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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.

A method is described for the determination of fifteen active ingredients of plant protecting products in ground-, raw, and drinking water. After extraction and enrichment of the pesticides from the water sample with solid-phase extraction, the extract is fractionated on silica-coated TLC plates, first in a so-called screening gradient by AMD (automated multiple development). The detection of the pesticides is performed by UV multidetection. Positive results from the first separation are confirmed by a second separation on silica in an AMD gradient of distinctly different selectivity, compared to the first separation. Following this strategy, it is possible to distinguish nearly all nonvolatile, from water extractable, active ingredients of plant protecting products in the market. UV spectra are taken for further confirmation of positive results. In most cases, this is possible even at the limit of determination. All chromatograms and spectra shown are raw data, resulting from the confirmatory test DIN 38407 part 11, in the laboratory of one participant*). The method is standardized in the meantime as DIN V 38407-11. Bestimmung von Pflanzenschutzmittelwirkstoffen in Trinkwasser durch AMD. Teil IV: Grundlagen eines Ringversuchs Es wird eine Methode zur Bestimmung von funfzehn Pflanzenschutzmittelwirkstoffen in Grund-, Roh- und Trinkwasser beschrieben. Die Wirkstoffe werden durch Festphasenextraktion aus der Wasserprobe extrahiert und angereichert. Die erste Auftrennung des Extraktes erfolgt im sogenannten Screening-Gradienten an kieselgelbeschichteten Dunnschicht-Chromatographie-Platten durch AMD (Automated Multiple Development). Die Detektion der Wirkstoffe wird mit UV-Multidetektion durchgefuhrt. Positive Ergebnisse dieser ersten Bestimmung werden durch erneute Chromatographie abgesichert. Der Extrakt wird diesmal in einem AMD-Gradienten vollig anderer Selektivitat als beim Screening getrennt. Durch dieses Vorgehen ist es moglich, nahezu alle nichtfluchtigen, aus Wasser extrahierbaren marktgangigen Pflanzenschutzmittelwirkstoffe chromatographisch zu unterscheiden. Zur weiteren Absicherung positiver Resultate werden die UV-Spektren aufgenommen. In den meisten Fallen konnen die UV-Spektren auch an der Bestimmungsgrenze der Wirkstoffe bei 0.05 μg/L registriert werden. Alle gezeigten Chromatogramme und Spektren fielen, im Rahmen des Ringversuchs DIN 38407 Teil 11, im Labor eines der Teilnehmer an*). Die Methode ist mittlerweile als DIN V 38407–11 genormt.

The more sensitive an analysis method the more care must be given to sample preparation. Solid phase extraction (SPE) onto RP 18 phases has established itself as a general purification and enrichment technique for trace components in aqueous samples. Various factors may thereby influence the recovery rate. For example, the ratio amount of sorbent and sample volume should be taken into account to avoid breakthrough of the investigated substance. Extracting phenylurea herbicides from drinking water, the ratio should be 1 g RP 18 sorbent per 1 L water sample. Concerning the flow rate for the fungicides procymidone, vinclozolin, and iprodione, the recovery rate decreases using higher flow rates than 3 or 6 mL/min. In the case of phenylurea herbicides, a flow rate of 10 or 14 mL/min showed best results. Even the coefficient of variation is below ± 5% at these flow rates. Furthermore, the company dependence should be considered because the recovery rate might differ by up to 40% using the nominally identical material. The same applies to the batch dependence of quality from the same company. The concentration of the eluate must also be monitored and carried out with great care to avoid decreasing the recovery rate. Considering these factors of influence, SPE is a very useful tool in sample preparation, particularly if an automated system is used. Die Festphasenextraktion hat sich im Bereich der Wasseranalytik als leistungsstarkes Verfahren etabliert, vor allem, wenn Automaten zum Einsatz kommen. Einflusfaktoren, die die Effizienz dieses Clean-up an C18-Material bestimmen, wurden untersucht. Dazu gehoren Unterschiede im Sorptionsverhalten des RP-C18-Materials verschiedener Hersteller und Chargen, die Geschwindigkeit der Probenaufgabe und das Durchbruchsverhalten von Pestiziden sowie der daran anschliesende Einengprozes. Zudem wurde die Eignung der verwendeten Losemittel gepruft. Bei der Extraktion von Phenylharnstoff-Herbiziden aus Trinkwasser sollte ein Verhaltnis von 1 g RP-C18-Sorbens pro Liter Wasser eingehalten werden. Von Hersteller zu Hersteller differierte die Wiederfindung an nominell identischem RP-C18-Material um bis zu 40%. Ahnliche Abweichungen wurden fur verschiedene Sorbens-Chargen desselben Herstellers gefunden. Die Wiederfindungsrate bei der Extraktion der Fungizide Procymidon, Vinclozolin und Iprodion verschlechterte sich bei hoheren Flusraten als 3 bis 6 mL/min. Im Falle der Phenylharnstoff-Herbizide ergaben sich die besten Wiederfindungsraten fur Flusraten von 10 bis 14 mL/min. Bei diesen Flusraten lag der Variationskoeffizient der Wiederfindung bei unter ±5%.

The 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.

A newly developed thin-layer chromatographic method (TLC) for the quantitative determination of netilmicin in serum is described and compared with fluorescence polarization immunoassay (FPIA). The TLC procedure involves solid-phase extraction of the aminoglycoside from serum and chromatography on reversed-phase thin layer. Post-chromatographic derivatization is carried out using 2,2-diphenyl-1-oxa-3-oxonia-2-boratanaphthalene (DOOB) as fluorescence reagent. The calibration curve for netilmicin in serum is linear in the range 1.0–5.0 μg/ml and the detection limit is about 0.2 μg/ml (correlation coefficient r=0.9963, mean coefficient of variation VXO=±5.3 %). A total of 25 serum samples from patients treated with netilmicin was measured by TLC and the results were compared with those of FPIA (Abbott TDx). There is no statistically significant difference between the two procedures (y=−0.35+1.094·x, Passing/Bablok). Thus the TLC-procedure is an alternative for the determination of netilmicin in serum, that possesses the necessary sensitivity of other comparable methods.

The fundamentals of the AMD technique are discussed. Multiple and stepwise development combined with gradient elution is very suitable for the systematic detection and determination of crop-protection agents. The distance migrated is very reproducible (on the same HPTLC plate and on different ones), so that a narrow interval window is adequate. Screening and confirmation gradients, coupled with reflectance spectroscopy (multiwavelength scanning) and postchromatographic, microchemical derivatization make it possible to detect crop-protection agents in drinking, table and ground waters. Each step of the analysis leads to an increase in the probability of identification with a standard substance. Clean-up and enrichment by a factor of 5000 using solid phase extraction precedes chromatography, so that water samples containing only 20 ng impurity per 1 can be analysed for compliance with the German drinking water regulations. The analysis is computer-controlled. At least 100 substances can be checked for their presence on one HPTLC plate with only a few ml mobile phase and without environmental contamination.

A 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.

Bei der phytochemischen Untersuchung des atherischen Haselwurzoles wurde erstmals ein C15-Keton isoliert. Die Strukturaufklarung ergab, das es sich um das bicyclische Furopelargon A (1) handelte, dessen physikalisch-chemische und spektrale Daten zusammengestellt werden. Furopelargon A, a new Sesquiterpene Ketone from the Essential Oil of Asarum Europaeum L Furopelargon A (1) is a C15-ketone isolated from the volatile oil of Asarum europaeum L. It is the first time this substance has been found in the rhizome oils. Data characterizing its structure are reported.

Aus dem durch Wasserdampfdestillation bzw. Extraktion gewonnenen atherischen Krautol von Artemisia maritima L. ssp. maritima wurde ein C15-Alkohol isoliert, der bisher noch nicht genuin nachgewiesen werden konnte. Die Strukturaufklarung ergab, das es sich um das 2,6,10-Trimethyl-7,10-oxido-dodeca-3,11-dien-2ol-5on handelte, das im Anschlus an weitere Sesquiterpen-Derivate der genannten Species als Hydroxy-Davanon bezeichnet wird. New Substances from Essential Oils of Different Artemisia Species, IV: Hydroxydavanone, a Furanoid Sesquiterpene Alcohol Hydroxydavanone is one of the sesquiterpene alcohols which is isolated from the essential oil of Artemisia maritima L. ssp. maritima. It is for the first time, that this substance has been found in the volatile oil of this species. Elucidation of the structure showed that hydroxydavanone is 2,6,10-tri-methyl-7,10-oxidododeca-3,11-dien-2-ol-5-one. The trivial name was given in analogy to other sesquiterpene derivatives of that species.

Phytochemical investigations of the essential oil isolated from Asarum europaeum L. rhizomes led to the characterization of the tricyclic α–agarofurane. The structure was elucidated and the biochemical connections discussed to other compounds of the oil.

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.

β-d-Glucopyranoside von funf primaren Monound Sesquiterpenalkoholen (Citronellol, Nerol, Geraniol, cis-Myrtanol, Farnesol) wurden nach einem modifizierten Koenigs-Knorr-Verfahren hergestellt. Zum ersten Mal wird uber die Glucoside von l-cis-Myrtanol und Farnesol sowie uber ihre physikalisch-chemischen Daten, besonders die IR-, 1H-NMR, 13C-NMR und Massenspektren berichtet. Durch Anwendung nichtwasriger Bedingungen konnte die Ausbeute auf uber 80% erhoht werden. Ein Uberblick wird gegeben uber das naturliche Vorkommen der Monound Sesquiterpenalkoholglykoside in Pflanzen.

Aus dem atherischen Ol des Meeresbeifus, Artemisia maritima L. ssp. maritima L., wurde auf chromatographischem Wege ein heterocyclisches Sesquiterpenketon isoliert. Die Strukturaufklarung ergab, das es sich um Davanon(3) handelte, ein kopf-schwanz-verknupftes Tetrahydrofuran-Derivat, das bisher fur Ole dieser Species nicht beschrieben worden war. New Substances from the Essential Oils of Artemisia Species, II: Davanone, a Furanoid C15-Ketone The volatile oil of Artemisia maritima L. ssp. maritima L. has been separated chromatographically. One of the newly isolated compounds was the sesquiterpene ketone davanone, which till hitherto has not been described in essential oils of this species.

Aus dem durch Wasserdampfdestillation gewonnenen atherischen Krautol von Artemisia vulgaris L. var. vulgatissima wurde der tertiare Sesquiterpenalkohol α-Cadinol isoliert und mithilfe physikalisch-chemischer sowie spektroskopischer Methoden charakterisiert. New Substances from the Essential Oils of Artemisia-Species*), III: α-Cadinol α-Cadinol is a tertiary sesquiterpenic alcohol. For the first time it has now been found in the volatile oil of Artemisia vulgaris L. var. vulgatissima. Its identity was established by physicochemical and spectroscopic methods.

A 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

Four immunological assays (RIA, ELISA, EMIT, FPIA) for digoxin were characterized with respect to reproducibility, detection limit, selectivity, and accuracy, followed by the comparison with HPLC. Afterwards the serum samples of nearly 60 patients were analyzed by these five methods.

Aus den durch Wasserdampfdestillation gewonnenen atherischen Krautolen von Artemisia vulgaris L. sowie Artemisia dracunculus L. wurde erstmals der Sesquiterpenalkohol Spathulenol isoliert und charakterisiert. Substances First Isolated from the Essential Oils of two Artemisia-Species, 1. Spathulenol, an Azulenogenic C15-Alcohol Spathulenol is one of the sesquiterpenic alcohols which is isolated from the essential oils of Artemisia vulgaris L. and Artemisia dracunculus L. It is the first time this substance has been found in these volatile oils.

This 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%.

Die Methoden der direkten quantitativen Auswertung von Dunnschicht-Chromatogrammen besitzen gegenuber der Auswertung nach Elution der abgetrennten Substanzen u. a. den Vorteil einer groseren Nachweisempfindlichkeit bei der Messung. Die notwendigen Substanzmengen entsprechen den bei der qualitativen Chromatographie verwendeten (Nanogramm bis einige Mikrogramm). Es werden die verschiedenen Moglichkeiten der direkten Messung diskutiert, von denen a) der visuelle Fleckflachenvergleich mit seiner metrischen Variante nur semiquantitative Ergebnisse liefert. b) Bei der photometrischen Absorptionsmethode, die nicht nur auf den sichtbaren Spektralbereich beschrankt ist, sondern sowohl im UV-Bereich als auch im nahen Infrarot mit Erfolg eingesetzt werden kann, werden die apparativen und experimentellen Moglichkeiten der Reflexions- bzw. Transmissionsmessung diskutiert und die jeweiligen Vorteile herausgestellt. c) Zu den Methoden der Emissionsmessung im Hinblick auf quantitative Aussagen gehoren einerseits die direkten und indirekten Fluorescenzmessungen und die Auswertung der Phosphorescenz. Andererseits werden auch hier die Ergebnisse der direkten Radioaktivitatsbestimmung zusammengefast.

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.

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%.

Influence of chromatographic factors on remission measurements on thin-layer chromatograms . Part I. Choice of the method, properties of the stationary phase and application of substances Some introductory remarks are made concerning the choice of the determination method, and different methods are compared. Chromatographic factors affecting the spectrophotometric evaluation of thin-layer chromatograms are discussed. In connection with this, the layer thickness of the stationary phase, the grain-size distribution and the pore diameter of the adsorbent were investigated. In trace analyses using a stationary phase consisting of thin layers of finely granulated adsorbents having a small pore diameter, sensitivity can be increased by a factor of 10. It was found that there was no relationship between the direction in which the layer had been applied on the plates and that in which chromatography or measurement was carried out. The manner of applying the solutions to be examined is also discussed, and the advantages of applying the solution as streaks or round spots, respectively, are pointed out. For this purpose the polarity of the standard and test solutions should be considered so that no disturbances due to formation of zones differing in area will occur. To obtain calibration curves according to the Kubelka—Munk equation, the ratio of the measured area to the zone area should be kept constant.

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.

Im Verlaufe von. 5 Jahren wurden Hunderte von Haselwurzpflanzen (Asarum europaeum L.) zahlreicher Standorte Mitteleuropas mit speziellen Mikromethoden untersucht. Die arzneilich interessanten unterirdischen Pflanzenteile ergaben je nach Herkunft 1,8 bis 4,1% atherisches Ol. Die chemischen und die Vererbungs-Analysen zeigten, das es bei der bislang botanischsystematisch als einheitlich geltenden europaischen Haselwurz mindestens 4 verschiedene Chemische Rassen gibt. Neben Pflanzen, deren atherisches Ol uber 80% aus Asaron (= trans-Isoasaron) besteht, gibt es solche, deren Hauptbestandteil der trans-Isoeugenolmethylather ist und andere, die keine Methoxyphenylpropan-Derivate aber vorwiegend Sesquiterpenalkohole enthalten.

Digoxin ist eines der am h~iufigsten eingesetzten Cardenolide zur Therapie von Herzmuskelinsuffizienz, insbesondere wenn dieser eine Arteriosklerose, Hypertonie oder ein Klappenfehler zugrunde liegt. Allerdings ist die therapeutische Breite mit 0 ,5 -2 ng/ml Serum gering. Zur Kontrolle des CardenolidGehaltes im Blut werden fast immer immunologische Methoden (RIA, EMIT oder ELISA) eingesetzt. Das bislang einzige chromatographische Verfahren stammt aus der Gas-Chromatographie [1]. Es ist aufwendig und l~iBt die Wiederfindungsrate fiber einen radioaktiv markierten Standard bestim

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 10(4) whereas in the three liquid chromatographic methods it was 10(2). The reproducibility with a confidence limit of P = 95% had 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.