Erno Tyihák

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.

Dose-dependent effect of resveratrol on proliferation and apoptosis in endothelial and tumor cell cultures

Experimental data suggest that Resveratrol, a compound found in grapes and other fruits may influence cell proliferation and apoptosis. The aim of our experiments was to study the effect of Resveratrol on tumor cell cultures and an endothelial cell culture in order to examine the effect of various doses of this compound on active cell death and cell proliferation. Human tumor (HT-29, SW-620, HT-1080) and endothelial (HUV-EC-C) cells were treated with various doses of (0.1 to 100.0 µg/ml) Resveratrol in vitro. Cell number, apoptotic and mitotic index was measured 24, 48 and 72 h after treatment. Low doses (0.1-1.0 µg/ml) of Resveratrol enhance cell proliferation, higher doses (10.0-100.0 µg/ml) induce apoptosis and decrease mitotic activity, which is reflected in changes of cell number. Resveratrol influences dose dependently the proliferative and apoptotic activity of human tumor and endothelial cells. The possible role of formaldehyde in the mechanism of action of Resveratrol is discussed.

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.

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.

Gas-liquid chromatographic analysis of dimedone derivatives of formaldehyde and other aliphatic aldehydes on capillary columns

Abstract The quantitative determination of small amounts of aliphatic aldehydes (formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde) in biological samples is difficult by the classical methods. The dimedone derivatives of these aldehydes were prepared and proved to be stable under the conditions of gas chromatography. The thermal properties of these derivatives were studied by thermal gravimetry, differential scanning calorimetry and thermal emission analysis, and the gas chromatographic peaks were identified by mass spectrometry. An analytical method was developed for the separation and determination of the dimedone adducts of aliphatic aldehydes by gas chromatography using capillary columns.

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.

Role of arginine and its methylated derivatives in cancer biology and treatment

Both L-arginine supplementation and deprivation influence cell proliferation. The effect of high doses on tumours is determined by the optical configuration: L-arginine is stimulatory, D-arginine inhibitory. Arginine-rich hexapeptides inhibited tumour growth. Deprivation of L-arginine from cell cultures enhanced apoptosis. The pro-apoptotic action of NO synthase inhibitors, like NG-monomethyl-L-arginine, is manifested through inhibition of the arginase pathway. NG-hydroxymethyl-L-arginines caused apoptosis in cell cultures and inhibited the growth of various transplantable mouse tumours. These diverse biological activities become manifest through formaldehyde (HCHO) because guanidine group of L-arginine in free and bound form can react rapidly with endogenous HCHO, forming NG-hydroxymethylated derivatives. L-arginine is a HCHO capturer, carrier and donor molecule in biological systems. The role of formaldehyde generated during metabolism of NG-methylated and hydroxymethylated arginines in cell proliferation and death can be shown. The supposedly anti-apoptotic homozygous Arg 72-p53 genotype may increase susceptibility of some cancers. The diverse biological effects of L-arginine and its methylated derivatives call for further careful studies on their possible application in chemoprevention and cancer therapy.

Determination of endogenous formaldehyde level in human blood and urine by dimedone-14C radiometric method

Radiometric method was developed for the analysis of endogenous formaldehyde in human blood and urine using dimedone-14C reagent. The method based on that dimedone /5,5-dimethyl-cyclohexane-1, 3-dione/ can very easily condense with formaldehyde and other aldehydes. With the help of this radiometric method a sensitive technique and a more accurate evaluation have been made possible. Applying this method it could be established that the formaldehyde level in the blood varies between 0.4–0.6 μg ml−1 and in urine 2.5–4.0 μg ml−1. The error of determination and the chemical bonds of formaldehyde in the biological fluids are discussed.