Overpressured Layer Research Articles

Advances and trends in analytical techniques in natural product research: Challenges and future perspective

Advances in analytical methods and bioassay development have helped to push forward the research in natural products. Their high chemical diversity and the effects of evolutionary pressure to create biologically active molecules could be attributed to success in drug discovery. Despite the availability of modern analytical instrumentation techniques, metabolic profiling covers the identification of a selected group of metabolites. The phytochemical analysis is commonly performed using standard techniques such as thin-layer chromatography, high-performance thin-layer chromatography, high-performance liquid chromatography, gas chromatography and more recently mass spectrometry and nuclear magnetic resonance spectrometry. Two-dimensional J-resolved Nuclear Magnetic Resonance (NMR) spectra and multivariate data analysis techniques were applied to avoid low resolution and overlapping signals hampering the identification of the individual components of botanicals. On-targeted metabolomic analysis via the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) was utilised to localise compounds belonging to various chemical classes (i.e. oxygenated fatty acids, flavonols, phenolic acids, and sinapoyl choline derivatives). Rotation planar chromatography over-pressured layer chromatography and electro planar chromatography are the other innovations. Parallel and serially coupled layers open up new avenues for the analysis of a large number of samples for high-throughput screening and very complex matrices in a natural product. Analytical strategies with applications to natural extracts and novel methods that have strong potential, regardless of how often they are used are discussed with respect to their potential applications challenges and future trends.

Forced flow, and physical field enhanced thin-layer chromatography

ABSTRACTPlanar chromatography was firstly introduced by Izmailow and Schreiber in 1938. Since then it has been used as a one of the basic techniques in liquid chromatography, but after introducing High Performance Liquid Chromatography in the 70’s of the past century by many investigators has been marginalized due to the worse separation capabilities and efficiency. The attempt of solving this problem was introducing forced flow planar techniques, and coupling it with other method of separation. In this article the ideas and history of Rotational Planar chromatography, Overpressured Layer Chromatography Planar Electrochromatography, and Thin Layer chromatography in Magnetic field have been presented. The short discussion on strong and weak sides of every presented technique and their further development possibilities was also included.

Layer chromatography-bioassays directed screening and identification of antibacterial compounds from Scotch thistle

The antibacterial profiling of Onopordum acanthium L. leaf extract and subsequent targeted identification of active compounds is demonstrated. Thin-layer chromatography (TLC) and off-line overpressured layer chromatography (OPLC) coupled with direct bioautography were utilized for investigation of the extract against eight bacterial strains including two plant and three human pathogens and a soil, a marine and a probiotic human gut bacteria. Antibacterial fractions obtaining infusion-transfusion OPLC were transferred to HPLC–MS/MS analysis that resulted in the characterization of three active compounds and two of them were identified as, linoleic and linolenic acid. OPLC method was adopted to preparative-scale flash chromatography for the isolation of the third active compound, which was identified after a further semi-preparative HPLC purification as the germacranolide sesquiterpene lactone onopordopicrin. Pure onopordopicrin exhibited antibacterial activity that was specified as minimal inhibitory concentration in the liquid phase as well.

Separation and detection of bioactive essential oil components by overpressured layer chromatography coupled with bioactivity tests

Overpressured layer chromatography (OPLC), ensuring pumpforced constant mobile phase flow and the possibility of overrun, offers the expanded exploitation of fine-particle adsorbent layers for a longer development distance. Using an infusion—transfusion OPLC method with a 26-cm long development, the separation of clove, rosemary, eucalyptus, tea tree, spearmint, thyme, and cinnamon bark essential oil components was achieved with good resolutions. In the combination of OPLC and Aliivibrio fischeri assay, the main essential oil components eugenol, borneol, (−)-R-carvone, thymol, and trans-cinnamaldehyde exhibited antibacterial effect. The OPLC—2,2-diphenyl-1-picrylhydrazyl (DPPH*) test showed two antioxidant components: eugenol and thymol.

A validated reversed-phase over-pressured layer chromatography-ultraviolet method for the quantification and optimum recovery of gallic acid inAnnona muricataL.

A validated online over-pressured layer chromatography (OPLC) method was developed for the rapid, cost-effective, and efficient separation of gallic acid. A reversed-phase (RP) ultraviolet (UV)–online OPLC analytical method was developed and validated as per the International Conference on Harmonization (ICH) guidelines. Methanolic extract of Annona muricata (fruit) was prepared by cold maceration. Separation of marker was achieved on RP-OPLC plates (5 × 20 cm) with isocratic solvent system of 0.1% acetic acid, water and methanol (30:70) at a flow rate of 0.15 mL min−1; detection was carried out at λmax 270 nm. The base peak of standard gallic acid was at 5.441 min with good linearity (r2 > 0.999), precision, and accuracy. The limit of detection (LOD) (521.84 mg mL−1) and limit of quantification (LOQ) (1581.336 mg mL−1) values reflect that the method is sensitive with high peak purity; the recoveries of analyte were 99 to 103%. The achievement of the method was the early retention time of gallic acid whic...

Applicability of Preparative Overpressured Layer Chromatography and Direct Bioautography in Search of Antibacterial Chamomile Compounds

In situ sample preparation and preparative overpressured layer chromatography (OPLC) fractionation on a 0.5 mm thick adsorbent layer of chamomile flower methanol extract prepurified by conventional gravitation accelerated column chromatography were applied in searching for bioactive components. Sample cleanup in situ on the adsorbent layer subsequent to sample application was performed using mobile phase flow in the opposite direction (the input and output of the eluent was exchanged). The antibacterial effect of the fractions obtained from the stepwise gradient OPLC separation with the flow in the normal direction was evaluated by direct bioautography against two Gram-negative bacteria: the luminescence gene tagged plant pathogenic Pseudomonas syringae pv. maculicola, and the naturally luminescent marine bacterium Vibrio fischeri. The fractions having strong activity were analyzed by SPME-GC/MS and HPLC/MS/MS. Mainly essential oil components, coumarins, flavonoids, phenolic acids, and fatty acids were tentatively identified in the fractions.

Simultaneous isolation of seven compounds fromGlehnia littoralisroots by off-line overpressured layer chromatography guided by a TLC antioxidant autographic assay

A combinative method using overpressured layer chromatography (OPLC) and TLC bioautography against O2(•-) was developed to separate antioxidants from Glehnia littoralis roots. Through target-directed isolation by the TLC bioautographic method, seven compounds including five antioxidants were rapidly isolated by OPLC and identified as 1-linoloyl-3-palmitoylglycerol, facarindiol, panaxynol, isoimperatorin, β-sitosterol, scopoletin, and umbelliferone from G. littoralis roots. This OPLC method is a very suitable separation technique for light-sensitive polyacetylenes (panaxynol and facarindiol) with higher isolated yields compared to conventional open column chromatography. This is the first report on the separation of polyacetylenes by OPLC.

Purification of vetiver alcohols and esters for quantitative high-performance thin-layer chromatography determination in Haitian vetiver essential oils and vetiver acetates

A simple, fast, and efficient High-Performance Thin-Layer Chromatography (HPTLC) method was developed for the simultaneous quantitative determination of alcohols and acetates in Haitian vetiver essential oils (Chryzopogon zizanioides) and its acetylated form. Analytes were separated by using a mixture of n-hexane–chloroform–ethyl acetate (8:6:0.5, v/v/v) as mobile phase under 47% humidity. Quantification was achieved by densitometric evaluation of the analytes in absorbance mode under visible light (λ=530nm) after staining with a vanillin–sulfuric acid reagent. Reference mixtures of alcohols and acetates were obtained by fractionation of Haitian vetiver oil or vetiver acetates, followed by purification of the fractions of interest by means of Over-Pressured Layer Chromatography (OPLC). The chemical composition of each reference fraction was determined by using GC–MS and GC×GC–MS, and their overall purity was determined by GC/FID and HPTLC. The TLC method provided compact spots for alcohols (Rf2=0.18±0.01 and Rf1=0.28±0.01) and acetates (Rf3=0.65±0.01). Calibration plots showed good linear correlation with r2=0.9995±0.0001 and r2=0.9995±0.0001 for alcohols and r2=0.9996±0.0001 for acetates in a 40–200ngspot−1 concentration range with respect to peak areas. The method was validated for precision and accuracy. Limit of detection (LOD) and quantification (LOQ) were determined. Method specificity was confirmed using retention factor (Rf) and GC–MS control of the standards reference mixtures.

OPLC comparison of methods for aqueous extraction ofSennae foliumandTiliae flosplant samples

A new, simplified open-vessel microwave extraction (OVME) method has been used to prepare aqueous extracts of Sennae folium and Tiliae flos . For comparison, extracts were also prepared by classic pharmacopeial methods — decoctum, infusion, and ultrasound extraction. The extracts prepared by the different extraction methods were compared by over-pressured layer chromatography (OPLC), followed by video and UV-densitometric evaluation. We demonstrated the applicability of rapid OPLC separation combined with UV densitometry for comparison of extracts prepared by different methods. OPLC-UV is suitable for rapid, non-destructive investigation of multicomponent preparations of biological origin.

Qualitative screening for basic drugs in autopsy liver samples by dual-plate overpressured layer chromatography

An overpressured layer chromatography (OPLC) method was evaluated for broad-scale screening of basic drugs in 5 g autopsy liver samples using two parallel OPLC systems. Sample preparation included enzymatic digestion with trypsin and liquid–liquid extraction with butyl chloride. Chromatographic separation was performed as dual-plate analysis, with mobile phases composed of trichloroethylene–methylethylketone– n-butanol–acetic acid–water (17:8:25:6:4, v/v) (OPLC1), and butyl acetate–ethanol (96.1%)–tripropylamine–water (85:9.25:5:0.75, v/v). Identification was based on automated comparison of corrected R f values (hR fc) and in situ UV spectra with library values by dedicated software. The identification limit was determined for 25 basic drugs in liver ranging from 0.5 to 10 mg/kg. The OPLC method proved to be well suited for routine screening analysis of basic drugs in post-mortem samples of varying quality, combining the benefit from moderately high separation power with the ease of disposable plates.

Separation and quantitative determination of aldoses and alditols by over-pressured layer chromatography (OPLC)

A new OPLC method has been established for separation and quantitative determination of three alditols (d -xylitol, l -arabitol, and d -glucitol) and four aldoses (d -xylose, l -arabinose, d -glucose, and l -rhamnose). The aldoses are present in hemicellulose hydrolyzates used as substrates in the production of d -xylitol from d -xylose by yeast, and all seven sugars could be found in the final fermented broth. The separation was performed in approximately sixteen minutes, on aluminum foil-backed silica gel OPLC-HPTLC plates with overrunning elution. Acetonitrile-acetic acid-water, 63 + 33 + 5 ( v/v ), was used as mobile phase. The upper limits of linearity were in the range 140–600 ng and detection limits were 15–50 ng per spot. The method has been used successfully to screen fermentation samples for aldoses and alditols.

Single‐ and Multi‐channel OPLC Separation on Non‐segmented Sorbent Bed Using Flowing Eluent Wall for Operating Segmentation

A new overpressured layer chromatography (OPLC) separation procedure has been developed for single‐ and multi‐channel separation using a non‐segmented sorbent bed and flowing eluent wall (FEW) for operating segmentation. The FEW detaches the sorbent bed into active and non‐active parts regarding separation during the process. Only mobile phase is introduced into the non‐active part, while, for the active part, eluent and also the sample can be admitted, thus the non‐homogeneous part of the sorbent bed is excluded from the separation process. The FEW helps the elimination of the edge effect of OPLC in case of single sample injection and abolition of the sample mixing effect of neighboring lanes in the case of a multi‐channel separation process. In the case of dirty samples, the one channel FEW‐OPLC system is well suited for quick isolation in different preparative ranges using preparative chromatoplates. The multi‐channel solution will be a tool for high throughput analysis using efficient fine, superfine, or monolithic layers. The four‐channel version can be applied for high throughput multi (parallel) analysis, as well as micro‐ and semi‐preparative parallel isolation using efficient analytical or preparative layers. The FEW provides the possibility for real multi‐channel liquid chromatographic separation on a non‐segmented sorbent bed.

Screening for basic drugs in 2-mL urine samples by dual-plate overpressured layer chromatography and comparison with gas chromatography-mass spectrometry.

A dual-plate overpressured layer chromatography (OPLC) method was evaluated for broad-scale screening of basic drugs in 2-mL autopsy urine samples. Extraction was carried out by mixed-mode solid-phase extraction, and identification was based on automated comparison of corrected Rf values (hRfc) and in situ UV spectra with library values by dedicated software. The day-to-day precision of hRfc values was good in both OPLC1 and OPLC2 systems with median relative standard deviations of 2.4% and 3.4%, respectively. Both Rf and hRfc values were independent of the amount of analyte (0.5-10 microg) applied to the plate. Detection limits were determined for 47 drug substances in 2-mL urine samples, and they varied between 0.05 and 3.5 mg/L with a median of 1.0 mg/L. The performance of OPLC was evaluated by analyzing 30 autopsy urine samples by both OPLC and gas chromatography-mass spectrometry (GC-MS). The majority of findings by OPLC were in agreement with GC-MS. Some substances with low concentrations were not detected by OPLC, whereas GC-MS failed to detect a few polar substances. The OPLC method thus provides an alternative for current planar and column liquid chromatographic drug screening methods with the possibility of lowering detection limits by using a larger sample size.

Planar chromatographic method development using the PRISMA optimization system and flow charts.

This study presents a modern planar chromatographic method-development procedure, based on the "PRISMA" optimization system, in which the optimum separation is achieved systematically and the structures and properties of the substances to be separated are not known. The procedure consists of three stages. In the first of these the basic conditions the stationary phase, vapor phase, and individual solvents are selected with a TLC procedure (generally in nonsaturated chromatographic chambers). In the second stage, the optimum combination of the selected solvents is determined with the PRISMA model. The third part of the procedure includes the selection of the development mode (circular, linear, or anticircular); the selection of an appropriate forced-flow chromatographic technique (over-pressured layer chromatography or rotation planar chromatography) with high-performance thin-layer chromatographic plates; the transfer of the optimized mobile phase to the various analytical, planar, or column preparative liquid chromatographic techniques; and the selection of the operating conditions. For practical reasons, the optimization process is presented with the help of flow charts.

Cleaning validation procedure eased by using overpressured layer chromatography

In the manufacturing plants of many pharmaceutical companies the reaction apparatus is suitable to produce different active pharmaceutical ingredients. After completing the production of a compound the equipment should be cleaned in order to avoid the cross contamination in the next lot of the other products. In the authors’ laboratory several chromatographic methods were introduced to measure the amount of the residual substances remaining on the surface of the apparatus after the cleaning procedure. A sensitive and fairly rapid overpressured layer chromatographic (OPLC) procedure — suitable to separate and control five steroid hormone compounds (allylestrenol, estradiol, ethynodiol diacetate, levonorgestel, norethisterone) produced in the same equipment at different times — was developed and validated.

Measurement of formaldehyde as dimedone adduct and potential formaldehyde precursors in hard tissues of human teeth by overpressured layer chromatography.

Simple, automatic overpressured layer chromatographic methods for the parallel determination of endogenous formaldehyde in the form of dimedone adduct and potential formaldehyde generators (betaines) in hard tissues of human teeth are described together with an efficient densitometric evaluation. These simple procedures involve the special preparation of teeth and extraction of formaldehyde, of different binding force in teeth, with methanol containing dimedone, and the isolation of betaines with an aqueous solution of methanol.

Distribution of cationic ion-pairing reagents on thin-layers after continuous overpressured layer chromatography

Ion-pair HPLC separations on layers were modelled by using continuous overpressured layer chromatography (OPLC). As a first step, the distribution of several cationic ion-pairing reagents on the layer was studied. The mobile phase containing different quaternary ammonium salts, such as tetramethylammonium (TMA), tetrabutylammonium (TBA), cetyltrimethylammonium (CTMA) and trioctylmethylammonium (TOMA) halides was continuously pumped through the stationary phase at a pressure of 40 bar. The volume of the mobile phase varied between 4.5 to 67.5 cm 3 which is equivalent to a 1 to 15 bed volume of the given silica-gel layer. The concentration of the reagents was measured after chromatography in ten samples of the stationary phase taken along the direction of the development. Silica F-gel and physically and chemically bonded reversed-phase layers (C 18) were used. In some cases the effluents also were analyzed. With 0.05 M CTMA in 50% methanol–water mixture, the layers acted as `spread out columns' and they could be conditioned after pumping through 22.5 cm 3 mobile phase. This means that there was no concentration gradient on the layer and the remaining reagent quantities on the layer did not change with increasing volumes of the mobile phase. In some cases, the behaviour of TMA was different from that of CTMA.

Statistical Method for Quantifying Mobile Phase Selectivity in One- and Two-Dimensional Overpressured Layer Chromatography

A method for selecting mobile phases for either one-dimensional (1-D) or two-dimensional (2-D) planar chromatography is described and is applied to the separation of steroids by overpressured layer chromatography [Formula: see text] a form of forced-flow thin-layer chromatography [Formula: see text] using both normal- and reversed-phase chromatography. Two metrics are used for evaluating the separation quality of simulated chromatograms for each of 100 (or more) subsets of a set of 30 steroids in each of 15 1-D, and 105 2-D systems. The subsets vary in size between five and 25 steroids. Butyl acetate/toluene on silica gel and aqueous 2,2,2-trifluoroethanol are, on average, the highest and second highest ranked 1-D systems, respectively, for separating all subset sizes. These two systems are the constituent members of the system that is, on average, the highest ranked 2-D system for all subset sizes. The probability of the above systems being highest ranked decreases with decreasing subset size. There is only a 17% probability of butyl acetate/toluene on silica being the best system for separating a subset of five steroids, while there is a 3% probability of this being the worst system for this subset size. The spot capacity of each system can be estimated by considering 100 subsets of each size and noting the largest subset size that yields an acceptable value of one of the metrics used for measuring separation quality. The mobile phase selectivity may be quantified using the actual values of either of the two separation metrics, or by a nonparametric approach. The latter is used in such a way that a difference of unity in the ranking (for a given subset size) of two systems corresponds to a 95% probability that the higher ranked system will yield the better separation.

Comparison of thin-layer chromatography and overpressured layer chromatographic techniques for the separation of ascorbigen and 1′-methylascorbigen

Simple and efficient methods are described for the separation of biologically active ascorbigen and 1′-methylascorbigen by normal-phase TLC, conventional as well as new personal overpressured layer chromatographic (OPLC) systems using the same eluent. Results obtained with these techniques were compared on the basis of the separation parameters with special emphasis on the presentation of the advantages of the automated personal OPLC system. For the characterization of the personal OPLC system dependence of average plate height and resolution on the external pressure, front velocity and development distance was examined on TLC and HPTLC sorbent layers using ascorbigen and 1′-methylascorbigen as model compounds.

Quantitative estimation of betaines in commercial seaweed extracts using overpressured layer chromatography

An off-line, overpressured layer chromatographic procedure has been developed for the evaluation of betaines in commercial seaweed extracts using Dragendorff's reagent for the detection of the compounds and densitometry for their quantitative estimation. Using continuous development and a low sample volume, the amounts of γ-aminobutyric acid betaine and δ-aminovaleric acid betaine can be estimated, but a larger sample volume is necessary for the estimation of glycinebetaine and minor betaines. The critical steps of this analytical method are the visualization and the quantitative evaluation of the spots produced. Temperature plays a major role in the resolution, sensitivity and precision after derivatization with Dragendorff's reagent; this has led to the adoption of standardized conditions.