Overpressured Layer Chromatography Research Articles

Reverse-Polarization High-Performance Layer Electrochromatography-A New Approach to Anion Separation.

High-performance layer electrochromatography (HPLEC) combines the advantages of overpressured-layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC) while overcoming some of their limitations. HPLEC equipment can work in various HPLEC, OPLC, and PPEC modes. The equipment enables HPLEC analysis also with an electroosmotic effect directed against the hydrodynamic flow of the mobile phase. The change in the electric field direction in the separation system does not result in a change in either the direction of the mobile phase flow or the direction of solute migration. The hydrodynamic flow generated by the pump dominates the electroosmotic effect and enables separation against the direction of the latter. Reversed-polarization HPLEC may be advantageous for the analysis of anionic compounds, as it facilitates faster and more selective separation than OPLC performed in similar conditions. This separation mode provides a new possibility to develop and optimize separation methods by performing separation against the electroosmotic effect and without need of any modification of the adsorbent surface. A drawback of this separation mode is the increase in the backpressure at the mobile phase inlet and the limitation of the mobile phase flow rate. Currently, contrary to the single-channel mode, multi-channel reverse-polarity HPLEC still requires some technical and methodological improvements.

Comparison of Overpressured-Layer Chromatography and High-Performance/High-Pressure Layer Electrochromatography Using the New Prototype Equipment in Various Operational Modes.

In our previous paper we have presented the new prototype equipment and introduced a new analytical technique—high-performance/high-pressure layer electrochromatography (HPLEC), a combination of overpressured-layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC). In this paper, the work of the equipment in various operational modes is investigated. Some difficulties and challenges related to various aspects of separation are discussed. The OPLC and HPLEC techniques are compared in terms of selectivity and performance. The results show that our equipment can be successfully used for singe- and multichannel OPLC and HPLEC separations in various sample application and detection modes. It includes the high-throughput, multichannel, and fully automated online separation of multiple samples simultaneously. The equipment allows for the independent optimization of various operational parameters. HPLEC combines the advantages of column/capillary and planar separation techniques while overcoming their limitations. It also combines the advantages and overcomes the drawbacks of OPLC and PPEC. It provides hydrodynamic flow of the mobile phase, irrespective of the voltage used and/or the mobile phase composition. Thus, any optimization of the composition and the voltage can be used independently. Both can be used to obtain the required selectivity of separation. The voltage can be used to facilitate the mobile phase flow and accelerate the analysis.

High Performance (High Pressure) Layer Electrochromatography Separation Technique: Equipment and Preliminary Results.

We present a new prototype device and propose a new analytical technique: high performance (high pressure) layer electrochromatography (HPLEC). The equipment provides a combination of overpressured layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC), yet it still enables researchers to perform each of these analyses separately. In comparison to PPEC, HPLEC provides hydrodynamic flow of the mobile phase, irrespective of the voltage used and the mobile phase composition. The advantages of HPLEC over OPLC include the possibility of the use of the electrophoretic effect to influence the selectivity of separation and the use of the electroosmotic effect to facilitate the mobile phase flow in order to decrease backpressure and increase the flow velocity. Many operational parameters can be freely adjusted and optimized independently. The equipment is fully automated and can work in various separation/operational modes, including combinations of online/offline sample application and detection. We present preliminary results of simultaneous, fully online, multichannel HPLEC separation of analgesic drugs (including acetaminophen, ibuprofen, and tramadol) as an example of increasing analysis throughput.

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.

Comparison between thin-layer chromatography and overpressured layer chromatography fingerprints of commercial essential oils and accelerated solvent extraction plant extracts

Comparison between thin-layer chromatography and overpressured layer chromatography fingerprints of commercial essential oils and accelerated solvent extraction plant extracts

Preliminary results of layer chromatography combined with bilateral band compression to search active minor components

Bilateral band compression (BBC) is a process where a solvent flow perpendicular to the direction of development squeezes bands into a smaller area. BBC of the 10 mm wide lanes of high-performance thin-layer chromatography (HPTLC) separation resulted in more than 6 times increase in peak height and peak area of Canadian goldenrod labdane diterpenes solidagenone and presolidagenone. Simultaneously, the signal-to-noise ratio increased only up to 2.2–2.4 times due to the higher noise level. The BBC increased the relative standard deviations (RSDs) of peak height and area reached three to four times of the original as well. It is known that overpressured layer chromatography (OPLC) separates more efficiently than conventional TLC/HPTLC techniques. In order to increase the bio-detection sensitivity, we combined bioautography, BBC, and fully offline infusion-OPLC using band-shaped sample application/separation. Thyme essential oil has been applied as a multi-component object to show the potential of this process. It is demonstrated that biologically active minor components, which may be missed in case of a conventional process, can also be detected by means of this combined method.

Why do not polyphenols of red wine protect against the harmful effects of alcohol in alcoholism?

The effect of polyphenolic bioactive substances, especially resveratrol (12.03 mg l−1), of an often consumed Hungarian red wine was investigated in a short term rat experiment. Male young Wistar albino rats were treated with high volumes of red wine (matching one bottle of wine/day for a 85 kg man) (N=5) and another alcoholic drink of the same alcohol concentration (N=5), corresponding to the circumstances of alcoholism, and 5 rats were in the control group. A total of 7 routine laboratory parameters were measured from the sera by kits. The changes of redox homeostasis (H-donor activity, induced chemiluminescence, diene-conjugates, GSHPx) were studied in blood plasma and/or in liver homogenates by spectrophotometric and luminometric methods. Transmethylation property of the liver was measured by overpressured layer chromatography (OPLC) technique. It was proven with in vitro OPLC analytical study that resveratrol reacted with methyl groups, and resveratrol was demonstrated to influence transmethylation processes as well as redox homeostasis. Red wine compounds do not protect from the harmful effects of alcohol, and even by high doses of resveratrol, the liver further deteriorates and the negative effect of alcohol increases. It has been confirmed that high doses of resveratrol do not provide protection against liver damage in those suffering from alcoholism.

Comparison of high-performance thin-layer with overpressured layer chromatography combined with direct bioautography and direct analysis in real time mass spectrometry for tansy root

Bioactive polyacetylenes in tansy (Tanacetum vulgare L.) root extract were separated by overpressured layer chromatography (OPLC) with better resolutions than achievable by capillary flow high-performance thin-layer chromatography (HPTLC). OPLC enabled a longer development distance in a shorter time (16 cm, 7 min) in comparison to conventional HPTLC (7 cm, 16 min). OPLC separations on HPTLC plates with spherical particles resulted in higher resolutions than on such with irregular particles. A slight distortion of zones sometimes occurred that could be eliminated by sonication of the mobile phase through removal of the dissolved air. However, zone distortion did not diminish the meaning of the qualitative outcome. The combination of OPLC with direct bioautography and direct analysis in real time-high resolution mass spectrometry (DART-HRMS) led to the determination of the individual polyacetylenes responsible for the diverse effects. In each recorded mass spectrum, the basepeak was the respective protonated molecule. The assignments confirmed the results of a previous study, however, compounds TR5a (tetradeca-2,4,6-triine-8-en-12-one) and TR5b (trans-2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4,5]dec-3-ene) that were not separable by HPTLC were partially separated by OPLC. Thus, it was proven that TR5b was not an oxidation product of TR5a and thus not formed as artefact during the HPTLC-DART-HRMS analysis, but originally present in the sample.

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...

Forced-flow planar chromatography in the rear view mirror.

Mobile phase progress in planar stationary phase can be evoked by either external or internal forces. An internal force is capillarity, while gravity, electric field, a pump and centrifugal forces belong to external forces. Overpressured layer chromatography gives a widely used special chapter of forced-flow planar chromatography, a special bridge between high-performance liquid column chromatography and thin-layer chromatography (TLC). A simple and special rule characterizes the progress of mobile phase. Optimal efficiency is composed by the doubled effect of flow resulting from the pump-forced mobile phase (convex profile of laminar flow) and capillary forces on the dry stationary phase (concave laminar flow). This review describes the most important aspects of forced-flow TLC, including how the set-ups are developed and also the progress of detection methods used.

Equipment and preliminary results for orthogonal pressurized planar electrochromatography

We report combination of overpressured layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC) techniques into a single technique in which both OPLC and PPEC processes proceed simultaneously and orthogonally. The separation process with this new technique is performed in adsorbent layer of a chromatographic plate, which is equipped with special sealing margin on its whole periphery and closed under pressure in special chamber. We have named this separation technique as orthogonal pressurized planar electrochromatography (OPPEC). Examples of analytical and micropreparative (continuous) OPPEC separations are demonstrated.

Characterization of Volatile Constituents from Origanum onites and Their Antifungal and Antibacterial Activity

Essential oils obtained by hydrodistillation (HD) and microwave-assisted HD (MWHD) of Origanum onites aerial parts were analyzed by GC and GCIMS. Thirty-one constituents representing 98.6% of the water-distilled oil and 52 constituents representing 99.6% of the microwave-distilled oil were identified. Carvacrol (76.8% HD and 79.2% MWHD) and thymol (4.7% HD and 4.4% MWHD) were characterized as major constituents in both essential oils. Separation of carvacrol and thymol was achieved by overpressured layer chromatography. HPTLC and TLC separations were also compared. Essential oils were evaluated for antifungal activity against the strawberry anthracnose-causing fungal plant pathogens Colletotrichum acutatum, C. fragariae, and C. gloeosporioides using a direct overlay bioautography assay. Furthermore, main oil components carvacrol and thymol were then evaluated for antifungal activity; only carvacrol demonstrated nonselective antifungal activity against the three Colletotrichum species. Thymol and carvacrol were subsequently evaluated in a 96-well microdilution broth assay against Phomopsis obscurans, Fusarium oxysporum, three Colletotrichum species, and Botrytis cinerea. No activity was observed against any of the three Colletotrichum species at or below 30 pM. However, thymol demonstrated antifungal activity and produced 31.7% growth inhibition of P. obscurans at 120 h and 0.3 pM, whereas carvacrol appeared inactive. Thymol and carvacrol at 30 pM showed 51.5 and 36.9% growth inhibition of B. cinerea at 72 h. The mechanism of antibacterial activity was studied in a bioautography-based BioArena system. Thymol and carvacrol showed similar inhibition/killing effect against Bacillus subtilis soil bacteria; the action could be enhanced by the formaldehyde generator and transporter copper (II) ions and could be decreased in the presence of L-arginine, a formaldehyde capturer. Results indicated that Origanum essential oils and its major components thymol and carvacrol appear to generate antimicrobial activity through a mechanism of action where formaldehyde and its reaction products are produced.

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.

Transmethylation and the redox homeostasis

Transmethylation modifies configuration and proceeds via formaldehyde. It has a significant role for example in epigenetic regulation. The whole methyl-pool can be evaluated by the measurement of bound formaldehyde. The bound formaldehyde was measured in wheat, bean, beetroot, cabbage, broiler- and rabbit liver. The relationship between transmethylation and redox homeosthasis was studied in the liver of domestic animals, and in the rat model of fatty liver. The diet of rats was enriched with cholesterol, sunflower oil and cholic acid. The bound formaldehyde was determined by overpressured layer chromatography. The hydrogen-donating ability was measured with 1.1-diphenyl-2-picrylhydrazylt free radical using spectrophotometric measurement. Beans had the most bound formaldehyde. The liver of broilers possessed significantly elevated hydrogen-donating ability and transmethylation ability. Rats with severe fatty liver had significantly less bound formaldehyde and the hydrogen-donating ability tendentiously decreased. These results draw attention to the diet, especially in obesity and obesity-related diseases.

OPLC separation and identification of some amino acids from different proteins

The basic of all proteins is constituted by approximately 20 common amino acids that differ only in the structure of their side chain. The purpose of our work was to use the overpressured layer chromatography (OPLC) technique for the separation and identification of the essential and non-essential amino acids from some most common proteins, like hen egg yolk protein, bovine serum albumin (BSA), and Type I Collagen from bovine achilles tendon. In this order, the target proteins were acid-hydrolyzed and derivatized. For the separation of essential amino acids from egg yolk, two successive OPLC developments (normal and overrunning) were performed, followed by direct derivatization with 4-hydroxybenzaldehyde and ninhydrin on the chromatoplate for identification. In the hydrolyzed egg yolk, nine essential amino acids were identified. Between lysine and histidine, a poor separation was obtained. The amino acids from BSA and Type I Collagen were separated and identified as phenylthiohydantoin (PTH) derivatives b...

Evaluation of hungarian wines for resveratrol by overpressured layer chromatography

A method, including solid phase extraction sample preparation, overpressured layer chromatographic separation, and subsequent densitometric evaluation, was developed for measurement of total resveratrol (cis- and trans-isomers) content of wine. The amount of resveratrol was determined in wine samples from different winemaking regions of Hungary. The total resveratrol was high in Hungarian red wines (3.6–11 mg L−1), and much lower in white ones (0.04–1.5 mg L−1).