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compounds are shown in Figure 1 . In this study, a method of high-performance liquid chromatography with diode-array detection (HPLC–DAD) was established for simultaneous determination of multiple active compounds for the first time, which will contribute

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A high-performance liquid chromatography—diode-array detection method was developed and validated to determine simultaneously eleven major alkaloids in Corydalis decumbens (Thunb.) Pers. The alkaloids detected were corlumidine, protopine, coptisine, tetrahydrojatrorrhizine, palmatine, berberine, sanguinarine, papaverine hydrochloride, tetrahydropalmatine, bicuculline, and corydaline. Chromatographic separation was achieved using a C-18 column with a mobile phase composed of A (0.2% acetic acid solution, adjusted with triethylamine to pH 5.0) and B (acetonitrile), with stepwise gradient elution. Ultraviolet diode-array detection was used; chromatograms were examined at the wavelength of 280 nm. The regression equations showed a good linear relationship between the peak area of each marker and concentration (r = 0.9994–0.9999). The recovery values ranged between 93.66% and 100.54%. The method was fully validated with respect to detection and quantification limits, precision, reproducibility, and accuracy. The described high-performance liquid chromatography (HPLC) method was successfully used for the differentiation and quantification of the eleven major alkaloids in C. decumbens (Thunb.) Pers. and can be considered an effective procedure for the analyses of this important class of natural compounds.

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A novel liquid chromatographic technique, fully on-line TLC/HPTLC, is described. Although no forces other than capillary action are used, and evaporation of the mobile phase induces the linear mobile phase velocity, the method can be regarded as a type of forced-flow chromatography. The method integrates the idea of the continuous development, continuous-flow TLC, and diodearray detection. This paper describes the principle of the method, the design of the prototype apparatus, and the fundamental advantages of the novel technique.

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The objective of this study was the use of solid-phase extraction and planar chromatography with diode-array detection for analysis of dyes in beverages. The dyes were chromatographed in normalphase systems on silica, diol, cyanopropyl, and aminopropyl layers and in reversed-phase systems on cyanopropyl, diol, aminopropyl, and octadecyl silica wettable with water. Reversed-phase chromatography was performed with different mobile phase modifiers (tetrahydrofuran, dioxane, methanol, acetonitrile, and acetone) at different concentrations, containing different amines, cationic and anionic ion-pair reagents, buffers, and ammonia, again at different concentrations. On the basis of the results obtained it was possible to choose the best systems for analysis of tartrazine and sunset yellow in beverages. Identification of the analytes was confirmed by the comparison of UV spectra of drink components with those of standards, by use of the diode-array densitometer.

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Summary

Chestnut exhibits anti-inflammatory, styptic, anti-diarrhea, and analgestic effects as a traditional Chinese medicine. There is increasing evidence that shows that the consumption of chestnuts has become more important in human nutrition due to the health benefits provided by the antioxidants. The phenolic compounds are responsible for major bioactivities, such as anti-tumor and anti-oxidation. A high-performance liquid chromatography (HPLC) method with diode array detection (DAD) was established for the simultaneous determination of six phenolic compounds (gallic acid, GA; protocatechuic acid, PR; catechin, CA; epicatechin, EP; quercetin, QU; kaempferol, KA) in Chinese chestnut (Castanea mollissima blume) kernel. The sample followed by separation on Eclipse XDB-C18 column (150 × 4.6 mm, id., 5 μm) with gradient elution of methanol-1.0% acetate acid solution as a mobile phase, at a temperature of 30°C, under the ratio of 1.2 mL min−1, with 5 μL injection volume, and multi-wavelength synthesis was used with DAD. The correlation coefficients were larger than 0.999, the recoveries were 97.58% for GA, 100.41% for PA, 96.23% for CA, 101.38% for QU, 99.15% for EP, and 98.60% for KA, relative standard deviation (RSD) were 1.04% for GA, 1.21% for PA, 1.09% for CA, 1.19% for QU, 1.06% for EP, and 1.20% for KA. This method was applied for the determination of phenolics in chestnut kernel and was found to be fast, sensitive, and suitable.

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A high-performance liquid chromatographic method with diode-array detection (HPLC-DAD) has been developed and validated to establish the fingerprint of Turnera diffusa. Hydroalcoholic extracts were obtained from 19 raw herbal samples collected in different regions of México. Separation was performed on a 150 mm × 3.9 mm (4-μm particle) C18 column, using a gradient of methanol and 0.1% aqueous trifluoroacetic acid as the mobile phase. Chromatograms were recorded at 254 nm. To identify each peak, both retention time and peak spectrum were used. Intraday and interday relative standard deviations were <3% for retention time and <12% for relative areas. Extracts were stable in solution for up to 60 days. Results from a robustness study showed that the amount of ethanol in the mobile phase had a substantial effect on retention time. The relative areas of 12 peaks common to the chromatograms obtained from 19 authenticated T. diffusa samples were chosen to construct a principal-components analysis (PCA) model. The soft independent modeling of class analogy (SIMCA) method based on the PCA model was used to evaluate the quality of eleven commercial products.

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In-situ densitometry for qualitative or quantitative purposes is a key step in thin-layer chromatography. It offers a simple way of quantifying by measuring the optical density of the separated spots directly on the plate. A new TLC scanner has been developed which is able to measure TLC plates or HPTLC plates, at different wavelengths simultaneously, without destroying the plate surface. The system enables absorbance and fluorescence measurements in one run. Fluorescence measurements are possible without filters or other adjustments.The measurement of fluorescence from a TLC plate is a versatile means of making TLC analysis more sensitive. Fluorescence measurements with the new scanner are possible without filters or special lamps. Improvement of the signal-to-noise ratio is achieved by wavelength bundling. During plate scanning the scattered light and the fluorescence are both emitted from the surface of the TLC plate and this emitted light provides the desired spectral information from substances on the TLC plate. The measurement of fluorescence spectra and absorbance spectra directly from a TLC plate is based on differential measurement of light emerging from sample-free and sample-containing zones.The literature recommends dipping TLC plates in viscous liquids to enhance fluorescence. Measurement of the fluorescence and absorbance spectra of pyrene spots reveals the mechanism of enhancement of plate dipping in viscous liquids — blocked contact of the fluorescent molecules with the stationary phase or other sample molecules is responsible for the enhanced fluorescence at lower concentrations.In conclusion, dipping in TLC analysis is no miracle. It is based on similar mechanisms observable in liquids. The measured TLC spectra are also very similar to liquid spectra and this makes TLC spectroscopy an important tool in separation analysis.

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An effective, reliable, and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) with diode array detector (DAD) method was investigated for simultaneous determination of polydatin, isoquercitrin, resveratrol, and nicotiflorin in Tetrastigma hemsleyanum. The chromatographic separation of the four compounds was carried out on a Welchrom ODS column (4.6 mm × 250 mm, 5 μm) by gradient elution with phosphoric acid (H3PO4) aqueous solution (0.4%)–methanol as the mobile phase, at the temperature of 30 °C and a flow rate of 1.0 mL/min. The detection wavelength was set at 270 nm. Under optimum conditions, the baseline separation of these four compounds can be performed within 30 min. The developed method was validated in terms of detection limit, quantification limit, linearity, precision, and recovery tests. Eventually, the established HPLC–DAD method was successfully applied to the simultaneous determination of polydatin, isoquercitrin, resveratrol, and nicotiflorin in the extract of herb T. hemsleyanum.

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The newly developed experimental OPLC separation unit 100 (OSU 100) has been used for fully on-line multiple hyphenation using the one-channel flowing eluent wall (FEW) arrangement at the inlet. OSU 100-UV and OSU 100-DAD-ESI-MS with manual injector or autosampler were used to test the connected systems (OPLC-UV; OPLC-DAD-ESI-MS), using xanthine standards (caffeine, theophylline, theobromine) and green tea leaf extract as model compounds. The mobile phase chloroform-trifluoroacetic acid-acetonitrile-methanol, 76:4:6.67:13.33 ( v / v ) is suitable for rapid and selective separation, DAD, and ESI-MS of xanthines. The detection sensitivity of OPLC-UV or OPLC-DAD was increased by hyphenation with ESI-MS coupled in series. In contrast with DAD, the extracted ion chromatogram ( m / z = 181.1 Da) after background subtraction yields readily measurable peaks from the small amounts of theophylline and theobromine in tea leaf extract. Analysis of xanthines was achieved within 10 min with less than 2 mL mobile phase. Owing to the openable adsorbent layer in OPLC the bed conditions can, moreover, be checked off-line after single or multiple fully online separations.

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The purpose of this paper is to report a new procedure for analysis of analytes in complex mixtures by combination of different modes of multidimensional planar chromatography (MDPC) on monolayer or multiphase plates. The procedure described for separation of complex mixtures of compounds is inexpensive and can be applied to routine analysis of analytes in samples of natural origin, e.g., in water or plant extracts, after preliminary clean-up and concentration, e.g., by solid-phase extraction (SPE). Application of multidimensional planar chromatography (MDPC) and modern fiber optical TLC densitometric scanners with DAD is especially useful for correct identification of components of difficult, complicated mixtures, e.g., pesticides in plant extracts (clofentezine in Herba Thymi).

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