A new process, bilateral band compression has been used to increase the sensitivity of detection of minor components of a mixture. After band-shaped sample application, fully off-line OPLC separation, and drying, the separated bands were compressed bilaterally, in parallel, perpendicular to the direction of development, by use of a strong eluent and capillary driven chromatography. To introduce the eluent on to the layer for band compression a simple manual tool equipped with parallel foam strips was constructed. A mixture of xanthine standards (caffeine 0.998 mg mL
, theophylline 1 μg mL
, and theobromine 1 μg mL
) and black tea leaf extract were used to test the efficiency of detection after bilateral band compression. After band compression 20 ng theophylline and theobromine could be detected by densitometry in the xanthine standard mixture at a loading of 20 μg/10 mm whereas they were not visible in the original, uncompressed chromatogram.
A new concept, the flowing eluent walls (FEW) process, for segmentation of a non-segmented adsorbent bed, has been used for single- and multi-channel on-line overpressured-layer chromatography (OPLC). The FEW process leads to active and non-active regions on the adsorbent layer during the separation process. Mobile phase only is introduced to the non-active part of the layer whereas mobile phase and sample can be admitted to the active part, thus the non-homogeneous part of the adsorbent bed can be excluded from the separation process. The FEW configurations at the inlet side (FEW-I) eliminate the edge effect of OPLC in single-sample injections and the effect of sample-mixing between neighboring lanes in multi-channel separations. The FEW arrangements at the inlet and outlet sides (FEW-I/O) enable separate detection and collection of the components of the separation, and FEW lines solve the problems of joint detection of sample components and contaminants originating from the sealing material. One-channel OPLC with the FEW configuration is suitable for rapid isolation in different preparative ranges. Analytical and preparative TLC plates (20 cm × 20 cm) can be loaded with 5–25 mg and 25–125 mg, respectively. The multi-channel solution will be a tool for high-throughput analysis using efficient fine, superfine, or monolithic layers. The four and eight-channel versions can be used for high-throughput parallel analysis and for micro-preparative parallel isolation. The FEW concept provides the possibility of real multi-channel liquid chromatographic separation on a non-segmented layer and column shaped adsorbent bed.
The flowing-eluent-wall (FEW) procedure is suitable for single and multi-channel on-line overpressured-layer chromatographic (OPLC) separation by operational segmentation of a non-segmented adsorbent bed. One pump only is used to deliver mobile phase and the samples can be injected in parallel on to the non-segmented layer in specific areas determined by the positions of inlet troughs. To prevent mixing of samples from adjacent lanes, mobile phase and samples are introduced to the layer at the sample-introduction positions and at the sides, so the bed is divided into active parts, where separation is performed, and non-active parts, where there is no separation. The FEW arrangement can be used at the inlet (FEW-I) or at both inlet and outlet (FEW-I/O) enabling separate detection and collection of the separation and FEW lines. The OPLC 50 system with the four-channel FEW-I/O configuration is suitable for fully off-line parallel separations and for four-channel fully on-line separations. One or two-channel on-line separations can be achieved by inserting the appropriate cassette. The newly developed experimental OPLC 100 separation unit has the FEW-I/O arrangement and applies 10 MPa external pressure to the surface of 5 cm × 20 cm layers. By use of the appropriate cassette fully off-line and one or two-channel fully on-line separation can be performed. Because of the 10-MPa external pressure the system enables more efficient fully off-line separation than systems of previous generations. Fully on-line separation can also be performed with aliquot detection, a new type of on-line detection of partially mixed samples in which the regions where mixing of parallel samples occurs is excluded. This enables more efficient separation than the original FEW-I/O arrangement.
In the twenty-two years since a similar review in the first issue of this journal there has been much progress in forced-flow planar liquid chromatography (FFPLC). Innovative developments have resulted in extremely diverse new technical solutions. This review briefly summarizes techniques which are already in use as a result of this progress (overpressured-layer chromatography, OPLC, and rotation planar chromatography, RPC) and the future potential of other FFPLC techniques, for example electrochromatographic techniques and shear-driven chromatography (SDC). It seems that efforts in FFPLC provide real possibilities of efficient analytical and preparative separations of different complex mixtures exploiting, with increasing success, the objective advantages of planar layer chromatography. This review summarizes, first, the unique opportunities resulting from modern biological detection on the adsorbent layer (BioArena) and some recent results obtained. These results include — among others — the indirect detection of small key molecules (e.g. formaldehyde, HCHO, and ozone, O3) in chromatographic spots. Practical applications are mainly in-vivo investigations — already without chromatographic separations — with special emphasis on disease resistance and cell proliferation. It seems, however, that in the future BioArena-guided in-vivo investigation will enable characterization of biologically active substances.
Authors:Imre Klebovich, György Morovján, István Hazai, and Emil Mincsovics
Glyceryl trinitrate (nitroglycerin), useful in the prevention and treatment of acute anginal attacks characteristic of ischemic heart disease, has been used as a model compound to evaluate the applicability of overpressured layer chromatography (OPLC) coupled with several radioactivity detection methods in metabolism research. Radioactivity detection methods studied included digital autoradiography (DAR), flow-cell solid scintillation radioactivity detection (RD), and the phosphor-imaging technique.After treatment of rats with
C-labeled glyceryl trinitrate, glyceryl trinitrate and its metabolites 1,2- and 1,3-glyceryl dinitrate and 1- and 2-glyceryl mononitrate were isolated from plasma by liquid-liquid extraction. OPLC separation of metabolites and glyceryl trinitrate was performed on fine-particle silica gel-coated TLC plates with acetonitrile-dibutyl ether mixtures as mobile phases and stepwise gradient elution.Detection of radiocarbon-labeled parent drug and its metabolites was performed off-line by digital autoradiography and the phosphor-imaging technique. On-line detection was performed by flow-cell solid scintillation radioactivity detection.OPLC coupled with RD combines the selectivity of radiochemical detection with the separation efficiency of overpressured liquid chromatography (OPLC); this is of great importance during metabolite screening and isolation. After sample extraction and application to the plate the selectivity of the system could be optimized. The optimization could be monitored by DAR, which could be chosen as detection method for the off-line analysis of multiple samples simultaneously separated on a single plate. Isolation of separated sample constituents could be performed after applying the sample as a single band to the plate and elution of metabolites of interest followed by their detection by use of on-line RD. Strongly retained compounds could be detected by DAR and removed mechanically, with the adsorbent, and extracted with a strong solvent.The recently developed on-line OPLC-RD method and the previously reported OPLC-DAR and HPLC-RD techniques are rapid, economic, and effective separation systems for application in metabolism research.
Authors:Emil Mincsovics, Éva Sárdi, István Velich, György Kátay, and Ernő Tyihák
A new OPLC procedure, infusion-transfusion OPLC, has been developed and compared with conventional transfusion OPLC. Spot and/or band deformation caused by the total wetness front (which results from pore filling) was reduced, as was the bubble effect in on-line detection. Both techniques were used for rapid micro-preparative OPLC isolation on analytical adsorbent layers. In-situ clean-up and separation were used to isolate trigonelline from Leuzea extract. Modeling of loading capacity for isolation of ascorbigen was accomplished by fully off-line OPLC. Under optimized conditions ascorbigen of high purity was isolated from cabbage extract by transfusion and infusion-transfusion OPLC.
Authors:Ernő Tyihák, Emil Mincsovics, György Kátay, Zsuzsa Király-Véghely, Ágnes Móricz, and Péter Ott
The BioArena system, which integrates the modern technique and biological results of bioautography with layer liquid chromatography is especially suitable for investigating biochemical interactions. Formaldehyde (HCHO) and its reaction products play a crucial role in the antibiotic activity of
-resveratrol and other molecules — when HCHO-capturing molecules are used in culture media the antimicrobial activity of antibiotic-like compounds decreases substantially. HCHO and hydrogen peroxide are present as normal endogenous compounds in cells, so there is a possibility of interaction in which singlet oxygen (
) and excited HCHO can be formed. The
can oxidize water molecules and so H
can be formed, from which, by disproportion, among other reactions, ozone (O
) also can be formed in the chromatographic spots. Elimination of HCHO and/or O
from the spots results in a decrease in the antiproliferative effect.
Authors:Ádám Dávid, Emil Mincsovics, István Antal, Éva Furdyga, Zsolt Zsigmond, and Imre Klebovich
We report a new combination of overpressured-layer chromatography (OPLC) with near-infrared (NIR) spectroscopy for pharmaceutical analysis. Different pharmaceutical preparations containing caffeine, paracetamol, and acetylsalicylic acid as model compounds were separated by OPLC. The band density in the solid phase after OPLC was suitable for study of the separated components directly on the layer by NIR spectroscopy.We have demonstrated the applicability of rapid OPLC separation combined with UV densitometry and NIR spectroscopy for both qualitative and quantitative analysis. This OPLC-UV-NIR technique is thus suitable for rapid, nondestructive investigation of multicomponent pharmaceutical preparations and enables a different type of pharmaceutical analysis, e.g. starting-material tests, in-process control, end-product control, stability testing, etc. Another benefit of this newly developed combination of rapid off-line techniques is the possibility of simultaneous collection of qualitative and quantitative chromatographic and spectral information.
Authors:Ádám Dávid, Emil Mincsovics, Katalin Pápai, Krisztina Ludányi, István Antal, and Imre Klebovich
A new, simplified open-vessel microwave extraction (OVME) method has been used to prepare aqueous extracts of
. 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.