The role of planar chromatography in medicinal plant research.

This paper summarizes the role of planar chromatography (PC) in medicinal and aromatic plant (MAP) research and development, and demonstrates the importance of the technique, after extraction, in the analysis of MAP (identification and quantitative determination of the separated compound/s), in the purification and isolation process, and in different types of screening procedure. Special attention is paid to analytical, micropreparative and preparative forced-flow techniques, for example overpressured-layer chromatography (OPLC) and rotation planar chromatography (RPC). The special features of analytical, micropreparative, and preparative layer chromatography (PLC), OPLC, and RPC are compared in tables. Purification and isolation procedures using forced-flow techniques are shown in flowcharts. Some applications, relating to different classes of substance, are presented to demonstrate the versatility of various planar chromatographic techniques.

[Transfer possibilities of the mobile phases between different liquid chromatographic techniques for the analysis and isolation of compounds of biological matrices]. / A mozgó fázisok különbözó folyadék-kromatográfiás technikák közötti átviteli lehetóségei biológiai mátrixok komponenseinek analízise és izolálása céljából.

After the survey and characterisation of the solid/liquid chromatographic methods, the author summarized the features of overpressure layer chromatography; the disturbing zone and the multi-front effect as well as the elimination of their influence. In light of these effects, the strategy of the mobile phase transfer possibilities is demonstrated between the various analytical and preparative liquid chromatographic methods, with the OPLC playing a central role. The main point of this strategy is that the examination of biological matrices is always begun with unsaturated TLC chamber, in which the compounds to be separated are placed between the Rf values of 0.3 and 0.8. The optimized TLC mobile phase is transferred without changes to the OPLC technique where a prerun is applied. For separation of nonpolar compounds, the prerun can be performed with hexane; for separation of polar substances the prerun can be performed with any component of the mobile phase in which the components are unable to migrate. The selection of this solvent might be considered during optimization of the mobile phase. Using HPTLC chromatoplate and analytical OPLC technique, highly effective separation can be achieved. The scaling-up for the various preparative chromatographic systems can be performed on basis of the applied chromatographic circumstances. The dry-filled preparative (FC, LPLC, MPLC) columns can be equilibrated with the solvent used for the prerun in analytical OPLC, while in case of filling with slurry technique, the slurry has to be prepared using the same solvent as was used for the prerun of OPLC. The air bubbles can be eliminated in both cases by pumping over the appropriate quantity of the solvent used for prerun, afterwards the preparative separation can be started with the optimized unsaturated TLC mobile phase. The author deals separately with the mobile phase transfer possibilities between the different analytical and preparative planar (OPLC and RPC with various chamber types) chromatographic techniques, where the saturation of the vapour phase is the determinant. The strategy is demonstrated through various plant extracts. The mobile phase transfer possibilities between the different solid-liquid chromatographic methods are summarized in comprehensive figures. Figure 13 demonstrates the direct transfer possibilities, different lines show those applicable with various methods: dotted line stands for off-line and thin line for on-line methods, while the ones marked with thick line ensure the transfer of the optimized mobile phase without change between different solid-liquid planar and column chromatographic techniques in case of both--off-line and on-line--procedures.

[Strategies for identifying compounds of biological matrices, such as extracts of medicinal plants, using liquid chromatography and UV/VIS spectroscopy without isolation]. / Növényi kivonatok ismert komponenseinek, izolálás nélküli folyadékkromatográfiás és UV/VIS spektroszkópiás azonosítási stratégiája.

For a satisfactory identification of known compounds of biological matrices, such as extracts of medicinal plants--using liquid chromatographic methods (HPLC, TLC) as well as on-line and in-situ off-line UV/VIS spectroscopy--not only the chromatographic, but also the spectroscopic data have to be identical. Using reversed phase (RP) high performance column liquid chromatography (HPLC) the k' values of the reference compound and the substance have to be correlated to each other. The probability of chromatographic identity is high, if the difference between the k' values are less than 0.5%. Between 0.5-2% the identification is on the medium level. If the difference is higher than 2%, the identification level is low, the identification assurance is inadequate. The normal phase (NP) thin-layer chromatographic (TLC) identification probability [IP(Chr)] is defined as the area of the triangle formed by three mobile phases characterised with different total solvent strength (ST) and total selectivity value (SV), where the hRf values are practically identical with the standard (reference) substances. The higher the value of chromatographic identification probability, the best is the probability that two compounds are chromatographically identical. Using HPLC instruments with dioden array detection (DAD) system, with modern software, the identity or the difference of the UV/VIS spectra can be determined. For the in-situ off-line spectroscopic identification probability [IP(Sp)] two criteria have to be fulfilled. First of all, every minima and maxima of the UV and/or VIS spectra have to be practically identical, secondly the ratio of the locale absorbance minima and maxima has to be identical. Therefore all values of locale minima and maxima [gamma min, gamma max], as well as the relative absorbance ratios have to be depicted, respectively. The illustrated correlations between the reference and the substance in the sample to be identified have to give linears in the applied coordinate systems. The regression coefficients (r2) show the goodness of the spectroscopic identification probability [IP(Sp)]. The chromatographic and spectroscopic identification probability data can be classified in 3 levels. If at least one of the criteria is in the low probability level, the compounds to be identified is not in agreement with the reference substances. If all criteria are placed in the medium or high identification level, the two compounds have to be identical with a sufficient probability.

[Combinations of liquid chromatography methods for the breeding of high alkaloid-containing poppies]. / Folyadékkromatográfiás módszerek kombinált alkalmazása a magas alkaloid tartalmú mákfajták nemesítésében.

An analytical strategy for the determination of alkaloid content of poppy capsules was developed for the efficient plant breeding. The first step of the strategy is a screening by circular multilayer (ML) overpressured layer chromatography (OPLC) with large number (28 samples/chromatoplate) of samples under UV (254 and 366 nm) light. The next step is a TLC separation with densitometric evaluation for quantitative determination of the selected samples (ca. 50% of all samples). These results are confirmed by ionpair reversed-phase (RP) high performance liquid chromatography (HPLC) for the most interesting (20% of all samples) poppy capsules. If the difference of the two applied quantitative methods is higher than 10% the investigations have to be repeated by new mobile phase systems with different solvent strength and selectivity factor values to eliminate the disturbing effect of the minor compounds which appear during the plant breeding. On the basis of the analytical results two new populations were formed, one of them ("MAMOR") has high morphine (approximately 20 mg/g) content, the other one ("TEMAX") is characterized by ability of tebain (approximately 16 mg/g) accumulation. The two new poppy sorts are verification for the effectiveness of the combination of different separation techniques (OPLC, TLC, HPLC) and UV detection modes (off-line and on-line) for medicinal plant breeding.

Glucosylceramides of oat root plasma membranes--physicochemical behaviour in natural and in model systems.

Glucosylceramides from oat root plasma membranes have been characterized using HPLC-particle beam-mass spectrometry, differential scanning calorimetry, low angle X-ray diffraction and surface balance technique. 24:1-OH was dominating fatty acid (90%) together with 24:0-OH and 22:1-OH. The sphingosine base was sphingadienine isomers and the monosaccharide alpha and beta glucose. Differential scanning calorimetry of an aqueous dispersion of glucosylceramide revealed during heating an endothermic gel-liquid crystalline transition with double peaks at 47 degrees and 51 degrees C, the lowest known for naturally occurring glucosylceramides. A cooling scan after the endothermic gel-liquid transition showed one exotherm at 15 degrees C and if this was followed by another heating scan a large exotherm appeared with a peak at 18 degrees C. During the second heating the matrix was hydrated and the exotherm at 18 degrees C reflects then the transition between the supercooled metastable gel phase and the corresponding hydrated form. The calorimetric data indicate a lamellar phase which during the cooling scan appeared as an supercooled liquid crystalline phase. Low angle X-ray diffraction confirmed these calorimetric data. The surface pressure-area-curves of pure oat glucosylceramides were more expanded than those of bovine origin. Mixtures of oat glucosylceramides and phosphatidylcholine species similar to those present in oat root plasma membranes showed molecular miscibility but no interaction.

[Gas chromatographic analysis of cannabinoids on tandem columns]. / Kannabinoidok gázkromatográfiás vizsgálata tandem oszlopokon.

A simple, isotherm packed column gas chromatographic method was developed for the quantitative determination of neutral cannabinoids using 4-androstene-3,17-dion as internal standard. In order to achieve the best resolution and to avoid the evaluation of the disturbing hydrocarbon peaks a method was developed using "tandem" column made of 3% OV-1 (90%) and 3% OV-17 (10%) stationary phases. The psychotropic cannabinoids delta 1-tetrahydrocannabinol (delta 1-THC) and delta 1(6)-tetrahydrocannabionol (delta 1(6)-THC), as well as, their main metabolites cannabidiol (CBD) and cannabichromene (CBC) were baseline separated except the cannabigerol (CBG) and cannabinol (CBN) pair, however for these compounds the separation was also satisfactory for the quantitative determinations. The Kováts retention indices were calculated for the most important cannabinoids and established the detection limits, respectively (20-50 ng range). The reproducibility was found excellent cv% = 1.06 for delta 1-THC and the analysis time was 55 minutes. The practical usefulness of the method was demonstrated by the comparative analyses on hashish- and fibre type hemps.

Planar chromatography: current status and future perspectives in pharmaceutical analysis--I. Applicability, quantitation and validation.

The most important features of capillary action planar chromatographic methods are summarized. Certain properties such as ease in operation, sensitivity to experimental conditions, stationary and mobile phase selection, phase system optimization, separation efficiency and detection possibilities are discussed only briefly. Other aspects such as the applicability of planar chromatographic methods in pharmaceutical analysis, quantitation and validation are considered in more detail. The advantages and limitations of different evaluation techniques (visual comparison, spot elution techniques and in situ densitometry) are also discussed. Validation steps specific to planar chromatographic methods are emphasized.

Planar chromatography: current status and future perspectives in pharmaceutical analysis (short review)--II. Special techniques and future perspectives in planar chromatography.

The state of the art of various special analytical planar chromatographic methods is summarized especially for forced-flow planar chromatography (FFPC), overpressured-layer chromatography (OPLC) and rotation planar chromatography (RPC) as well as for the automated multiple development (AMD) technique. The connection between analytical planar and column liquid chromatographic methods and the identification of separated compounds with chromatographic and spectroscopic data are summarized. Some aspects of future perspectives, such as parallel connected multi-layer FFPC and long distance OPLC (LD-OPLC) are given. A combination of OPLC with the AMD method is predicted as the method of the future. Strategies using FFPC techniques are suggested in the form of a flow chart.

HPLC Analysis of the Main Furanocoumarins from Peucedanum palustre.

The main furanocoumarins from the roots of PEUCEDANUM PALUSTRE were separated with a RP-HPLC method. The mobile phase for the separation of the eight main compounds (oxypeucedanin hydrate, bergapten, oxypeucedanin, isobyakangelicin angelate, ostruthol, imperatorin, isoimperatorin, and columbianadin) was optimized with the "PRISMA" model, which is a multi-solvent optimization system. The chromatographic method can be applied for quantitative determination, as was demonstrated for different samples of P. PALUSTRE. The purity and identity of the peaks were controlled by diode-array detection and comparison with standards.