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  • Author or Editor: Mirko Prošek x
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Variation of antioxidant activity was measured for differently prepared extracts of pressed residues (marc) from red wine making and production of blackcurrant and redcurrant juice. On the basis of previous tests ethanol and water were chosen as suitable solvents for preparing phenolic extracts from selected samples. Antioxidant activity was also measured for commercially available standards of the anthocyanins present in grapes and red- and blackcurrants, for example malvidin 3-glucoside (Mv3G), cyanidin 3-glucoside (Cy3G), delphinidin 3-glucoside (Dp3G), peonidin 3-glucoside (Pe3G), and petunidin 3-glucoside (Pt3G). Each standard and a mixture of all standards (concentration 250 μg mL −1 ) were analyzed. Two different analytical methods were used, spectrophotometry and TLC. In both methods DPPH was used as a source of stable free radicals with which components with antioxidant activity react. The strongest antioxidant activity was found for Cy3G, then for Pe3G and Mv3G, and the lowest was for Pt3G and Dp3G (both methods). Relatively high antioxidant activity was determined for all samples. The most effective was blackcurrant extract prepared with 70% aqueous ethanol and the least effective were redcurrant extracts. Correlations between TLC and spectrophotometry were calculated for standards and for extracts. The results showed that the methods correlated well for pure standards ( r = 0.979) whereas for crude extracts the correlation was slightly lower (0.857) but still acceptable.Press residues from red wine making and from production of black-currant and redcurrant juice are rich sources of phenolic antioxidants and so could be used as natural antioxidants in the food industry.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Mitja Križman, Jernej Jakše, Mirko Prošek, Dea Baričevič, and Branka Javornik

Agarose gel electrophoresis is a basic separation tool used in molecular biology, mostly for qualitative DNA analysis. There are constraints limiting its use in quantitative analysis, namely low repeatability and a narrow linear range. However, by using an internal standard or internal normalization, repeatability and linear range could be significantly improved. In the work discussed in this paper it was shown that an approximately fivefold improvement in repeatability and an over threefold wider linear range could be achieved by applying internal normalization. Using the proposed approach, genetic markers, for example RAPD and PCR-RFLP, or even microsatellite markers, could be conveniently quantitatively assessed using agarose gel electrophoresis.

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Experiments and calculations show the importance of analytical management to reliable analytical results. A method with a measurement uncertainty of 10% (expressed as RSD ) is normally rejected whereas a sample uncertainty of 50% or more is normally tolerated. A special test sample was prepared. A solution of salicylic acid in methanol (2 mg mL −1 , 50 mL) was applied with a spraying device to a clean glass plate, dimensions 40 cm × 40 cm. After evaporation of the solvent 64 samples were taken from different positions on the test plate by use of swabs. The swabs were extracted on an ultrasonic bath and analyzed by HPLC and HPTLC. This experiment shows how random and thoughtful selection of swabbing positions affect the final results. It is possible to obtain reliable values only by careful selection of the number of sampling locations and their positions. The results obtained show that the median gives acceptable recovery whereas selection of maximum values (worst case) gives results which are too high, from 130% to 160%.

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A wide spread of measurements is typical in quantitative TLC. Improved reproducibility and speed can be achieved by automatic and controlled sample application, chromatographic development, and data acquisition and processing. Secondary chromatography is the main reason for poor precision in TLC. With RSD up to 10% this is by far the largest source of uncertainty. During the drying process mobile phase evaporates from the upper surface of the plate, and molecules of separated components inside the layer move up or down. Our experimental results show the strong dependence of the intensity of reflected diffuse light on the position of the spots inside the layer. Experimental results gave us an idea how to construct a device for drying and derivatization of TLC plates and a device which reduces uncontrolled propagation and non-homogeneous vertical in-depth distribution of spots during drying and derivatization was constructed. In addition the device designed is safer to use than a hair dryer. A laminar flow of air or inert gas constantly removes solvent vapor from the upper layer of the adsorbent and accelerates drying. Temperature is controlled and varies in a predetermined manner at predetermined intervals. Temperature gradient, which cannot be avoided in flow systems is controlled and is oriented in the direction of chromatographic development. The construction of the device results in identical drying conditions for substances with the same R F . Diffusion of the analyte is controlled and standardized and inhomogeneous in-depth distribution of compounds inside the adsorbent is minimized. Heating grade, heating intervals, pulses, switching, and other conditions are preset or programmable. The TLC dryer constructed reduces uncontrolled propagation and non-homogeneous vertical in-depth distribution of spots during drying and derivatization, which results in significantly improved reproducibility and precision. This is very important because in quantitative TLC most measurements are performed in reflectance mode.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Mirko Prosek, Luka Milivojevic, Mitja Krizman, and Maja Fir

A new on-line TLC-MS interface, with computer-controlled extraction of substances from selected spots on a TLC or HPTLC plate, has been constructed. The controlled collection of the sample and its programmed injection into the mass spectrometer is the advantage of this type of interface. The interface has been tested and validated with a standard solution of caffeine as test substance. The results were compared with those from a previously established and now routinely used off-line TLC-SPE-APCI-MS extraction procedure.

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TLC is often set aside as a result of unjustified simplification of analytical knowledge and, at the same time, over-emphasis of the use of metrological solutions. Some analysts assume that techniques with smaller measurement uncertainty automatically improve the reliability of their analytical results. This is not true. The reliability of some TLC techniques is demonstrated by analysis of an inclusion complex of coenzyme Q10 (CoQ10) with β -cyclodextrin, which was prepared because it is a water-soluble food additive. The products obtained were analyzed by one-dimensional, two-dimensional, and multi-dimensional TLC (according to Ebel ). These inexpensive, rapid, and very informative methods were used in-line during the preparation steps. Separation was performed in two steps, with dioxane-water, 50 + 50 ( v/v ), as the first mobile phase and chloroform-methanol, 55 + 45 ( v/v ), as the second. The development distance was 7.5 cm. The separated spots were visualized in two steps, first with 5% phosphomolybdic acid in EtOH and drying at 110°C, then with 50% H 2 SO 4 and heating at 120°C for 5 min. After the first treatment blue spots on yellow background were observed for CoQ10. After spraying with H 2 SO 4 dark blue spots of CoQ10 were observed on a light blue background. Images were acquired with a scanner and quantification was performed by use of TLC software. The synthetic yield was calculated from the concentrations determined. The results obtained were later confirmed by use of IR, NMR, and HPLC-MS.

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The ratio of lactulose/mannitol excretion in urine after their administration is of great importance for evaluation of malabsorption and intestinal permeability disruption in some diseases. An analytical method has been developed for determination of lactulose and mannitol in urine on the same amino HPTLC plate. The method enables densitometric quantification of lactulose by use of fluorescence mode, and mannitol by use of absorption mode after detection with AgNO 3 reagent.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Petra Jazbec, Andrej Šmidovnik, Mateja Puklavec, Mitja Križman, Jernej Šribar, Luka Milivojević, and Mirko Prošek

Coenzyme Q10, an essential factor of oxidative phosphorylation and an important antioxidant, is used as food additive in industrial production of poultry meat. In this study chickens were fed with water-soluble coenzyme Q10-β-cyclodextrin complex for 40 days and the distribution of CoQ10 and cholesterol in the chicken-breast cells was then measured. Cell fractions were analyzed by HPTLC and results were confirmed with a new HPLC-MS method. Both methods are suitable for quantitative evaluation, but HPTLC is particularly suitable for screening purposes. Validation revealed HPTLC is a reliable, sensitive, and flexible analytical tool in comparison with HPLC-MS, despite its inherently lower sensitivity and selectivity.

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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Mirko Prošek, Breda Simonovska, Alenka Golc-Wondra, Irena Vovk, Samo Andrenšek, Elizabeta Mičović, and Terezija Golob

HPTLC and HPLC-MS methods have been developed for quantitative determination of inulin in food products. Samples were applied to silica gel HPTLC plates and developed three times, twice with n -propanol-acetone-water, 45 + 30 + 25 ( v/v ), and the third time with n -propanol-acetone-water, 50 + 40 + 10 ( v/v ). Total development time was 150 min. Dried plates were dipped into DAP reagent for 10 s. After heating colored spots of the saccharides appeared. Quantitative evaluation of the colored spots was performed in transmission mode by means of a flatbed scanner and densitometer. The precision of measurements for the main inulin fractions was ±6.0%; the limit of quantitation ( LOQ ) varied from 0.1 to 1 µg per spot; and the ‘linear’ working range was between 0.5 and 4.0 µg per spot. The method was tested on real samples.

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