MINCSOVICS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 82, No. 3,1999 587 CHROMATOGRAPHIC STUDIES Personal Overpressured-Layer Chromatography (OPLC) Basic System 50, Flexible Tool in Analytical and Semipreparative Work EMIL MINCSOVICS, MARTA GARAMI, LASZL6 KECSKES, and BARNABAS TAPA OPLC-NIT Engineering Ltd., Andor u. 60, H-1119 Budapest, Hungary ZOLTAN VEGH Chemical Works of Gedeon Richter Ltd., PO Box 27, H-1475 Budapest 10, Hungary GYORGY KATAY and ERNO TYIHAK Plant Protection Institute, Hungarian Academy of Sciences, Herman O. ut 15, H-1525 Budapest, Hungary A new automated overpressured-layer chroma- tographic (OPLC) system called the Personal OPLC Basic System 50 is suitable for analytical and semi- preparative separations. The automatic microproc- essor-controlled system ensures rapid and repro- ducible off-line isocratic and stepwise gradient separations. High external pressure (5 MPa) makes the sorbent layer more homogeneous, yielding more efficient off-line separation compared with those by early Chrompres chambers. A theoretical plate height of 10-30 fim can be achieved on an analytical high-performance thin-layer chroma- tographic (HPTLC) layer made of irregular silica gel with an average particle size of 5 jim if an optimal linear velocity (20-40 mm/min) and a nonviscous solvent system are used. On an analytical layer of 3 |im spherical silica gel, a theoretical plate height of 6-15 |im can be reached. Rapid analytical separa- tions of resveratrol (1555 s) and xanthine by one- (498 s) and two-directional (274 s) off-line develop- ments were accomplished. On-line separation and detection combined with off-line sample applica- tion and fully on-line processing (including on-line sample application, separation, and detection) were fulfilled with a TLC plate for xanthine separa- tion. Semipreparative isolation of xanthines was achieved through a fully on-line OPLC operating mode and scaled-up chromatography. O verpressured-layer chromatography (OPLC) refers to techniques involving a pressurized ultramicro (UM) chamber and a pump system that delivers eluent into the chamber, which contains analytical or preparative chroma- tographic plates under pressure. The ancestor of this technique is the UM chamber (1), from which we have developed the experimental pressurized UM chamber. Here, an external pres- sure is applied to the surface of the sorbent layer by means of a Received August 3, 1998. Accepted by JS December 23, 1998. cushion system, forcing the eluent to flow (by overpressure) through the sorbent layer (2-4). The first commercially available OPLC instrument (Chrom- pres 10) is a completely off-line system. All the principal chro- matographic steps, such as sample application, separation, quantitative evaluation, and preparative isolation, are per- formed independendy. The advantages of OPLC are revealed by this system (5-10). The second-generation instrument (Chrompres 25) is suit- able for both off-line and on-line separations. With a selective eluent to increase the resolution of compounds running with low R f values, continuous development can be used. Separated components are evaluated quantitatively by densitometry on the plate. If the eluent outlet of the chamber is connected to a flow-cell detector, eluting solutes can be detected on-line, and fractions can be collected. The entire chromatographic process can be performed by a fully on-line mode connecting a loop injector to the eluent inlet and a UV detector to the eluent outlet, as in column liquid chromatography (LC; 11-14). Different combinations of off-line and on-line steps can be used within this system (15), and it can be applied to various analytical and preparative applications (16-25). For example, separations of ascorbigens, amino acids, biogenic amines, and steroids demon- strate the efficacy of the personal OPLC system (25-28). Further- more, the combination of OPLC and digital autoradiography is a powerful method in metabolite research (29). Experimental Materials Mobile phases were made from chromatographic-grade chemicals (LiChrosolv, Merck Darmstadt, Germany). Stationary phases of analytical TLC, HPTLC, and 0.5 mm thick preparative sorbent layers (20 x 20 cm) with irregular sil- ica gel 60 and Raman plates (10 x 10 cm) made of 3 |im, spherical, superfine silica gel 60 (Merck) were used. The layers were sealed on 4 sides by a robot (OPLC-NIT Ltd., Budapest, Hungary) to ensure perfect closing of the planar sorbent bed during separation. PTH-Valine (phenylthiohydantoin derivatives of Valine; Pierce Chemical, Rockford, IL) dissolved in eluent (dichlo- Downloaded from https://academic.oup.com/jaoac/article/82/3/587/5683803 by guest on 01 March 2023