Antal Szabó

Densitometric determination of some bioactive guanidinium compounds without post-derivatization

In this paper we present thin layer chromatographic methods suitable for determination of bioactive guanidinium compounds, saturated antibiotics with UV absorbance only in the extremely short wavelength region, below 200 nm. For elimination of disturbing impurities, arising mostly from binding agents in the precoated silica gel plates, an efficient prewashing method with methanol-formic acid, 50:50 (v/v) was used. In this way a smooth and straight baseline was obtained in the region of measurement even if polar mobile phases had been used. Of the compounds investigated, special attention was devoted to primycin, a macrolide antibiotic agent. Owing to the complexity of this antibiotic good separation has been a real challenge. An efficient separation system, suitable for both the quality control of the end product and for monitoring the efficiency of isolation from the fermentation liquor, has been established. Further compounds containing guanidinium groups, for example streptomycin, dihydrostreptomycin, arginine, and protected arginine have also been investigated. The peaks obtained by direct evaluation were checked by a modified version of the well known Sakaguchi reaction, a selective reaction for the identification of compounds containing guanidinium groups.

Simultaneous monitoring of target compounds and carbohydrate patterns during pharmaceutical fermentations

A simple thin-layer chromatographic method suitable for monitoring microbiological fermentation processes is reported. In addition to compounds related to the target compound the method enables acquisition of data on the formation and degradation of a series of carbohydrates from starch to monosaccharides. The target compounds and the carbohydrates can both be analyzed by use of a single method using one layer only. Both sets of data are useful for optimization of different biosyntheses or bioconversions. The method involves one, effective, thin-layer chromatographic separation, subsequent charring with sulfuric acid solution as detection reagent, heating, and evaluation by scanning the colored spots.

TLC/HPTLC and HPLC methods for monitoring microbial transformation processes

TLC methods have been developed for monitoring microbial enantioselective reduction and regioselective oxidation. The work discussed in this paper focused on preparation of aromatic chiral alcohols by bioreduction of their respective acetone derivatives by biocatalysis using yeast strains in medium containing added resin. A simple method and suitable device were developed for sampling from such heterogeneous fermentation media. Methods and chromatograms are presented for quantitation by TLC and for enantiomer ratio determination by HPLC. With regard to oxidation procedures, a TLC method was established for monitoring compactin hydroxylation during fermentation processes–as a representative example. Improvement of the method and use of HPTLC plates resulted in high resolution and separation capacity equal to that of the HPLC method. By means of the method by-products usually formed as minor components in the biotransformation could also be detected. Repeated clogging of the HPLC column as a result of impurities usually present in considerable amounts in fermentation samples is another reason for using TLC for analysis of microbial media.

Thin-layer chromatographic method for separation of wheatgrass pigments on sucrose impregnated silica gel plates

One of the most important aspects of the selected analytical method is avoiding the degradation of the target compounds during the analysis of the samples. Chromatographic techniques, particularly thin-layer chromatography (TLC), are widespread in this field. However, difficulties arise in applying chromatographic methods, such as separation efficiency, and stability of target compounds. The application of silica gel as stationary phase might be an appropriate choice [2], but at the same time, the degradation of the pigment compounds is fast, so the analysis has to be done relatively quickly [3]. In order to avoid degradation, a column filled with sucrose was used as adsorbent for isolation of chlorophylls [4, 5] and home-made thin-layer plates for analysis [6–8]. However, the resolution was rather limited. The aim of this investigation was to find a rapid and simple analytical method for the separation of the pigments originated from commercial wheat grass extracts without any degradation product generated during the analysis of the samples. The sucrose immersed silica gel system fulfilled all of these requirements. 2 Experimental

TLC method for monitoring the formation and degradation of bacterial exo-polysaccharides

A simple thin-layer chromatography (TLC) method for continuous determination of metabolism of carbohydrate and production of polysaccharides in the course of bacterial fermentations is presented in this paper. Samples were taken from the supernatants of decanted fermentation broth and from the hydrolysates of both the supernatants and the fermentation broth. Hydrolysis of the exo-polysaccharides (EPSs) to the constituent monosaccharides was carried out by 10% v/v hydrochloric acid at 100°C. These samples were applied directly onto a precoated nano silica plate besides a series of mono-, oligo-, and polysaccharide reference solutions. The plates were developed in a twin-trough chamber previously saturated with a solution of chloroform-toluene-35% formic acid-methanol (10:2:2:7 v/v) for 1 h. The plates were placed in the empty trough of the chamber in order to be saturated and then transferred to the trough containing the solvent. After reaching a migration distance of 160 mm, the plates were dried out and placed in a chamber saturated with I2 vapor in order to detect starch-type polysaccharides. After drying, the plates were dipped into a dish containing a solution of 10% v/v concentrated sulfuric acid in n-propanol-toluene (1:1 v/v). The excess reagent was eliminated with filter papers. Visualization was achieved in an electric oven at 115°C for 10 min. Evaluation was accomplished by comparison of the RF values, and the intensities of the spots were evaluated instrumentally using a CAMAG TLC Scanner II. The presented method is suitable for monitoring the bacterial EPS production and determining the monosaccharide constituents.

TLC method for quantitation of methylenedioxyphenylacetone and its derivative formed in a resin-added bioreduction process

A novel class of orally active 2,3-benzodiazepines has recently been discovered and patented by Hungarian researchers [1]. Biological activity studies led to the discovery of (8R)-7-acetyl5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo[4,5h]-[2,3]benzodiazepine (Talampanel; structure I in Figure 1) a new drug, under investigation, with antiepileptic, neuroprotectant and skeletal muscle relaxant effects, as described in a review by F. Andrasi [2].