Emil Mincsovics

New planar liquid chromatographic technique: overpressured thin-layer chromatography

Abstract A novel planar liquid chromatographic technique using a pressurized ultramicro chamber (PUM chamber) has been developed. The sorbent layer is completely covered by a membrane under external pressure, so that the vapour phase above the layer is virtually eliminated. Solvent is admitted under pressure by means of a pump system. The main advantage of this technique, termed overpressured thin-layer chromatography (TLC), is the substantially shorter time required for separation than in classical column chromatography and TLC, with velocities of the mobile phase about as stable as in high-performance liquid chromatography, and aggressive reagents being applicable as in classical TLC. The method appears to be suitable for the accurate modelling of column chromatographic methods.

Liquid chromatographic determination of aflatoxins

Abstract The difurancoumarin derivatives known as aflatoxins are highly toxic fungi metabolites belonging to the vast class of mycotoxins, which can contaminate foods and feeds when storage conditions favor fungal growth. Because of potential health hazards for humans, levels of aflatoxins are monitored throughout the world. During the past two decades several chromatographic and other methods were developed for identification and determination of aflatoxins in agricultural and food products. This paper is a review of the overpressured-layer chromatographic (OPLC) and high performance liquid chromatographic methods most often used for the analysis of aflatoxins. However, emphasis is placed on summarizing the OPLC methods developed for determination of aflatoxins in maize, wheat, fish meat, peanut samples, rice and sunflower seeds spiked with aflatoxins B 1 , B 2 , G 1 and G 2 in concentration of 2–10 μg/cm 3 , which were developed in our laboratory. The results of the proposed validation procedure, whose development was based on the guideline of the International Conference on Harmonization (ICH) for pharmaceutical products (1994, Brussels), for the determination of the above-mentioned aflatoxins in wheat samples are also presented.

Resolution and retention behaviour of some dyes in overpressured thin-layer chromatography

Abstract The performance of conventional and overpressured thin-layer chromatographic techniques was compared in different chamber systems (normal, ultramicro and pressurized chamber), on commercial normal and high-performance thin-layer chromatography (HPTLC) silica gel plates. Data obtained with three dyes demonstrated that separation in the pressurized chamber is similar to those observed in normal and ultramicro chambers, but the diameters of the spots are significantly smaller in separations carried out in pressurized chambers, principally owing to decreased diffusion. It was also observed that the velocity of the moving phase is very stable in the pressurized chamber, and has a linear relationship to solvent flow-rate. Owing to the substantially shorter separation time and stable flow-rates in the pressurized ultramicro chamber, the resolution values obtained on HPTLC plates are also better in the case of longer solvent migration distances.

Optimization of operating parameters in overpressured thin-layer chromatography

Abstract By adjusting the solvent by means of a pump system in overpressured thin-layer chromatography using a pressurized ultramicro (PUM) chamber it is possible to separate substances with optional development distances. In the PUM chamber the external pressure on the flexible cover membrane must always be higher than the input pressure of the solvent. The input pressure of the solvent increases linearly with increasing solvent migration distance. An increase in the solvent flow velocity always results in higher input pressures, which must be taken into account by choosing an appropriate external pressure on the membrane. The number of theoretical plates and the separation numbers obtained with a PUM chamber of the linear type on fine-particle sorbent layers are also better with longer solvent migration distances than in a normal TLC chamber. The advantages and the necessity for development with a longer migration distance are demonstrated with the example of the separation of amino acids on a fine-particle silica gel chromatoplate.

Circular-Development with Overpressured Thin-Layer Chromatography

Abstract A novel type modification of circular thin-layer chromatography has been developed, in which the layer is tightly covered by a membrane, eliminating the vapour phase over the sorbent layer. The developing solvent is pumped through the apparatus.

Overpressured multi-layer chromatography

Abstract A new version of overpressured layer chromatography using two, three or more chromatoplates during one separation was developed. The admission of the eluent to the multi-layer system as a critical step is performed by making a perforation in the chromatoplates at the eluent inlet of a suitable size and shape. The technique, called overpressured multi-layer chromatography, is the most up-to-date version of layer liquid chromatography and is very attractive because a large number of samples (50–100 or more) can be separated during one development. It can be used effectively, e.g., in plant breeding, clinical laboratories and industrial control laboratories and for the sequence analysis of proteins and nucleic acids.

Overpressured-Layer Chromatography

Abstract In the 1960s, the development of an ultramicro (UM) layer liquid chromatographic (LLC) separation chamber brought about a special combination of the two basic liquid chromatographic (LC) techniques, column LC and LLC. In this simple chamber the adsorbent layer of the chromatoplate was covered by a glass plate and/or plastic film during the separation process such that the end of the cover plate was not immersed in the mobile phase (eluent). Elimination of the vapor phase above the adsorbent layer in LLC was first achieved by use of this UM chamber. Further possibilities of this type of closed-layer chamber—for example, the use of a pumping system to increase and optimize the flow velocity through an optional development distance and the use of a special chromatographic plate—were subsequently realized by the development of an experimental pressurized UM (PUM) chamber. In this unique solution an external pressure is applied to the surface of the adsorbent layer by means of a cushion, forcing the mobile phase to flow (by overpressure) through the adsorbent layer as in HPLC. The PUM chamber is the basic instrument in so-called overpressured-layer chromatography (OPLC). The first commercially available OPLC instrument (Chrompres 10) was a completely off-line system. The second generation instrument (Chrompres 25) was suitable for both off-line and on-line separations. However, these conventional OPLC instruments and methodological solutions were suitable in general for of the advantages of OPLC versions over TLC and HPTLC in analytical and preparative separations. A new automated microprocessor-controlled separation system, new technology in the field of LLC, ensures rapid, efficient, and reproducible off-line and on-line isocratic and stepwise gradient separations. These technological solutions have opened new horizons in the field of LLC and exploit more attractively the advantages of layer liquid systems. This chapter summarizes the latest results obtained with conventional and modern OPLC versions. (The positive results of the BioArena system are demonstrated in Chapter 7 in this book.)

Overpressured layer chromatography: From the pressurized ultramicro chamber to BioArena system

The pressurized ultramicro (UM) chamber as a closed adsorbent layer chamber enables the use of a special chromatoplate and a pump to increase and optimize the mobile phase flow velocity through an optional development distance in an adsorbent layer. This chamber is the basic instrument of overpressured-layer chromatography (OPLC), which is a separation technique that combines the advantages of conventional TLC/HPTLC with those of HPLC. The versions of OPLC instrument, the character and achievement of off-line and on-line OPLC systems in analytical and preparative use are described. The development of BioArena as a complex bioautographic system means an exploitation of the unique advantages of planar-layer system for detection, isolation and identification of new antimicrobials, antineoplastics, biopesticides and other biologically active substances as well as for studying fundamental biochemical reactions and mechanisms.

Micro-preparative OPLC — rapid isolation by transfusion and infusion-transfusion processes

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.

Forced-flow planar liquid chromatographic techniques (after twenty-two years)

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.