Ágnes M. Móricz

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.

Separation and detection of aflatoxins using overpressured-layer chromatography and bioautography

It has been established, by use of bioautographic detection with the phytopathogenic bacteria Pseudomonas savastanoi pv. phaseolicola, that the common aflatoxins have antimicrobial activity after OPLC separation. Our preliminary results suggest this antimicrobial activity originates from the activity of formaldehyde formed by the bacterial cells and/or from the methoxy groups of aflatoxin molecules.

Tracking and identification of antibacterial components in the essential oil of Tanacetum vulgare L. by the combination of high-performance thin-layer chromatography with direct bioautography and mass spectrometry

Two tansy (Tanacetum vulgare L.) essential oils were obtained by steam distillation of the capitula with subsequent liquid-liquid extraction (oil 1) or with use of an auxiliary phase for the trapping of the steam components (oil 2). These oils were investigated against Bacillus subtilis F1276, B. subtilis spizizenii (DSM 618), Xanthomonas euvesicatoria, Pseudomonas syringae pv. maculicola, Ralstonia solanacearum strain GMI1000 and Aliivibrio fischeri, using the coupling of high-performance thin-layer chromatography to direct bioautography (HPTLC-DB). Using this method with the potato and tomato pathogen R. solanacearum is shown for the first time. Due to the advanced extraction process, oil 2 was richer in components and provided more inhibition zones. The main bioactive components were identified by scanning HPTLC-Direct Analysis in Real Time mass spectrometry (HPTLC-DART-MS) and solid-phase microextraction gas chromatography electron impact MS (SPME-GC-EI-MS) as cis- and trans-chrysanthenol as well as trans-chrysanthenyl acetate. cis-Chrysanthenol exhibited antibacterial effects against all tested bacteria, whereas trans-chrysanthenol inhibited B. subtilis, R. solanacearum and A. fischeri. trans-Chrysanthenyl acetate was an inhibitor for X. euvesicatoria, R. solanacearum and A. fischeri. Although HPTLC-DART-MS resulted in a comparable fragmentation, the ionization characteristics and the recorded mass spectra clearly showed that DART is a softer ionization technique than EI. It is also more affected by ambient conditions and thus prone to additional oxidation products.

Separation and identification of antibacterial chamomile components using OPLC, bioautography and GC-MS

Components of 50% aqueous ethanol chamomile (Matricaria recutica L.) flower extract, previously found antibacterial in a TLC-bioautographic study, were separated and isolated by the use of on-line overpressured layer chromatography (OPLC). This system consisted of an OPLC 50 BS system, an on-line coupled flow-through UV detector, and a manual fraction collector. The collected fractions were investigated by GC-MS analysis and by TLC re-chromatography with subsequent visualization, performed after use of the vanillin-sulphuric acid reagent, or under UV illumination, or applying bioautographic detection. The main compounds of the collected 11 fractions were identified by GC-MS. The results showed that the antibacterial effect of 50% aqueous ethanol extract of chamomile is ascribable to cis-, trans-spiroethers, and the coumarins like herniarin and umbelliferone.

Usefulness of Transgenic Luminescent Bacteria in Direct Bioautographic Investigation of Chamomile Extracts

Direct bioautography performed with luminescence gene-tagged bacteria enables almost real-time detection of antimicrobial compounds in plant extracts. This method for the detection of chamomile (Matricaria recutita) components with antibacterial effect against Bacillus subtilis soil bacteria was more sensitive than commonly used bioautographic visualization by staining with a tetrazolium salt. Some compounds had a strong inhibiting effect only via the bioluminescence measurement. Extraction of antibacterial components of chamomile flowers was most effective with 50% ethanol; slightly lower efficiency was achieved with acetone and methanol, and hexane was least effective. The results were confirmed by using luminescent Pseudomonas syringae pv. maculicola plant pathogen bacteria.

Applicability of the BioArena System to Investigation of the Mechanisms of Biological Effects

Bioautography can be extended to a complex system called BioArena by linking different steps to it, for example dissolving a variety of compounds in the cell suspension to affect biological activity, measuring putative mediators of antibiosis, for example formaldehyde (HCHO) and hydrogen peroxide (H2O2) in the inoculated layer, and performing densitometric and ex and in situ spectroscopic examination to follow the changes in the inhibition zones and active compounds (e.g. antibiotics and toxins). Possibilities of the basic elements of BioArena system are illustrated in this paper by results with aflatoxin B1 (AFB1). Target bacterial cells in the logarithmic growth phase were found to be the most sensitive for direct bioautography. Densitometric signals of bioautograms (negative densitometry) of 0.125–1 μg AFB1 spots showed logarithmic correlation with the amount of AFB1. The HCHO capturer L-arginine decreased whereas the HCHO generator-mobilizer Cu(II) ions increased the antibacterial-toxic effect of AFB1. The latter effect was also confirmed by negative densitometry. Besides higher levels of HCHO, a decrease of H2O2 in the toxin spot was found. HCHO could also originate, among other sources, from demethylation of AFB1, which is apparent from the Fourier transform Raman spectra obtained in situ from the AFB1-containing spots. These results support the suggested role of HCHO and its reaction products with H2O2 (e.g. singlet oxygen (1O2), ozone (O3)) in the antibacterial-toxic effect of AFB1.

BioArena: An unlimited possibility of biochemical interactions in the adsorbent layer after chromatographic separation

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 trans-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 (1O2) and excited HCHO can be formed. The 1O2 can oxidize water molecules and so H2O3 can be formed, from which, by disproportion, among other reactions, ozone (O3) also can be formed in the chromatographic spots. Elimination of HCHO and/or O3 from the spots results in a decrease in the antiproliferative effect.

The potential of BioArena in the study of the formaldehydome

New results with BioArena as a complex separation and detection (evaluation) system support earlier observations that formaldehyde (HCHO) and its reaction products play a special and crucial role in the effects of antibiotic in general. It has been established that antibiotic-like compounds (e.g. trans-resveratrol, Cu(II) ions) have a duplicate inhibiting effect on pathogen cells as a result of the action of HCHO. HCHO as a key molecule of the formaldehydome participates in series of interactions which can be screened by means of different spectroscopic and chromatographic techniques; accumulation of HCHO and its reaction products in TLC spots is limited, however, so indirect detection is advantageous. In BioArena the planar stationary phase bed after TLC/HPTLC and, mainly, OPLC separation can be used for manifestation of deprivation of HCHO from antibiotic spots, for observation of the duplicate effect of substances with a direct effect, and for demonstration of cell proliferation promotion and retardation.

Antibiosis, Antibiotics, and the Formaldehyde Cycle: The Unique Importance of Planar Chromatographic Techniques to Progress in these Fields

The BioArena system, which integrates the up-to-date methodology and biological results of bioautography with OPLC as an efficient planar separation technique (compact spots, etc.), is especially suitable for investigating biochemical interactions in an adsorbent bed after chromatographic separation. The first results from BioArena show that formaldehyde (HCHO), which can originate from pathogen cells in some situations, can play a special role in the antibiotic activity of trans-resveratrol. When L-arginine and glutathione were used as endogenous HCHO-capturing molecules in the culture medium the antimicrobial activity of trans-resveratrol on the adsorbent layer decreased substantially. It has been observed that trans-resveratrol generates a time-dependent, and therefore concentration-dependent, duplicate inhibiting effect on the pathogen, and that the BioArena system was suitable for illustration of this new phenomenon. It is probable that this effect occurs as a result of HCHO, with special emphasis on the possibility of interaction between the HCHO and H2O2 of endogenous origin on the adsorbent layer incubated with pathogen cells. It seems that the BioArena system will be an important, indispensable complement to the basic separation technique OPLC.