Attila Felinger

Numerical determination of the competitive isotherm of enantiomers

A numerical method was developed and used to determine adsorption isotherms in chromatography. The numerical parameters of an isotherm model were derived from the recorded band profiles of the racemic mixture of the 1-phenyl-1-propanol enantiomers, by means of a nonlinear least-squares method. We used the equilibrium-dispersive model of chromatography with several isotherm models. The numerical constants of the isotherm models were tuned so that the calculated and the measured band profiles match as much as possible. We show that this numerical inverse method can be applied even without the knowledge of the individual band profile of the pure enantiomers. The isotherms determined from the--usually unresolved--overloaded band profiles matched extremely well the isotherms determined by frontal analysis. Several isotherm models were used and tested--such as Langmuir, biLangmuir, Tóth, Langmuir-Freundlich. The best-fit isotherm was selected by means of statistical evaluation of the results.

Comparing the optimum performance of the different modes of preparative liquid chromatography.

A comparative study of the optimization of the different modes of the preparative separation of binary mixtures by liquid chromatography is presented. Band profiles were calculated by means of the equilibrium-dispersive model of chromatography in the cases of isocratic elution, gradient elution, and displacement chromatography. The objective function to be maximized was the product of the production rate and the recovery yield. The production rate was calculated using the same definition of the cycle time in all cases. This common definition accounts for column regeneration after each run in each mode of the separation. The calculations reveal that the number of experimental parameters to be adjusted to achieve optimum separations is relatively small. The major parameters are the loading factor and the number of theoretical plates, besides the displacer concentration in displacement chromatography, or the gradient steepness in gradient elution. The relative advantages of the different modes of preparative chromatography are discussed.

Determination of polyphenolic compounds by liquid chromatography-mass spectrometry in Thymus species

Polyphenolic compounds represent a wide group of phytochemicals, including well-known subgroups of phenolic acids, flavonoids, natural dyes, lignans etc., which are produced by plants. These natural bioactive compounds possess a variety of beneficial effects including antioxidant and anticarcinogenic activities, protection against coronary diseases as well as antimicrobial properties. Thymus species have already been reported as sources of different phenolic acids and flavonoids. Moreover, the composition and content of flavonoids in Thymus species play important role as taxonomic markers providing distinction of species. High-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) and on-line mass spectrometry (ESI-MS) method was used for analysis. The method was evaluated for a number of validation characteristics (repeatability and intermediate precision, LOD, LOQ, calibration range, and recovery). The polyphenolic pattern of five native Hungarian Thymus species (T. glabrescens Willd., T. pannonicus All., T. praecox Opiz, T. pulegioides L., and T. serpyllum L.) was characterized. The dominant compound was rosmarinic acid, which ranged between 83.49 μg g(-1) and 1.436 mg g(-1). Other phenolic acids (ferulic acid, caffeic acid and its other derivatives, chlorogenic acid and p-coumaric acids) were present in every examined Thymus species, as well as flavanones: naringenin, eriodictyol and dihydroquercetin; flavones: apigenin and apigenin-7-glucoside, flavonols: quercetin and rutin. The polyphenolic pattern was found to be a useful additional chemotaxonomic tool for classification purposes and determination of the locality of origin.

Determination of the single component and competitive adsorption isotherms of the 1-indanol enantiomers by the inverse method

The inverse method of isotherm determination consists in calculating the numerical values of the coefficients of an isotherm model that give a set of chromatographic profiles in best possible agreement with the set of experimental profiles available. This method was applied to determine the adsorption isotherms of the 1-indanol enantiomers on a cellulose tribenzoate chiral stationary phase. Both single-component and competitive isotherms were determined by using no more than one or two overloaded band profiles. The isotherms determined from the overloaded band profiles agreed extremely well with the isotherms determined by frontal analysis. Several isotherm models were used and tested. The best-fit isotherm was selected by means of statistical evaluation of the results. The results show that the adsorption is best characterized with a model describing heterogeneous adsorption with bimodal adsorption energy distribution.

Optimization of the experimental conditions and the column design parameters in overloaded elution chromatography

Abstract A theoretical study of the optimization of the separation of a binary mixture in overloaded elution chromatography was performed. The elution band profiles were calculated using the semi-ideal model of chromatography and assuming competitive Langmuir isotherms. In a first step, the experimental (operating) conditions (i.e., the reduced velocity of the mobile phase and the loading factor) were optimized using a simplex algorithm. In a second step, the column design parameters (i.e., the column length and the average particle diameter) and the operating conditions were optimized for the maximum production rate of either the more or the less retained component. The optimum value of the capacity factor of the first component, was also determined. Binary mixtures having relative retentions between 1.1 and 1.8 and relative concentrations of 1:3 and 3:1 were studied. The maximum production rates were obtained for very low values of the capacity factor of the first component, of the order of 0.3-0.5, depending slightly on the relative retention.

Optimizing preparative separations at high recovery yield

A new objective function is introduced for the optimization of the design and operation conditions in preparative chromatography. Instead of the production rate, we propose to maximize the product of the production rate and the recovery yield. This new objective function leads to optimum experimental conditions under which the production rate is almost as high as it would be if only the production rate were maximized, but the recovery yield is significantly improved. The advantages of the novel objective function are demonstrated by calculations based on both the ideal and the equilibrium-dispersive models of chromatography. The application of this novel objective function is beneficial in isocratic overloaded elution, in overloaded gradient elution and in displacement chromatography.

Determination of tropane alkaloids atropine and scopolamine by liquid chromatography-mass spectrometry in plant organs of Datura species

Hyoscyamine (atropine) and scopolamine are the predominant tropane alkaloids in the Datura genus, occurring in all plant organs. The assessment of the alkaloid content of various plant parts is essential from the viewpoint of medical use, but also as a potential risk of toxicity for humans and animals. Therefore, a reliable method for the determination of tropane alkaloid content is of high importance. The present work aimed at the elaboration of a rapid method for determination of the most abundant Datura alkaloids by LC-MS technique using a new generation of core-shell particle packed column. Tropane alkaloid content was investigated in various plant organs of four Datura taxa (D. innoxia, D. metel, D. stramonium, and D. stramonium var. tatula), grown under the same conditions, in two developmental stages. We have developed a rapid LC-MS method for the quantitative determination of atropine and scopolamine, which was successfully applied to quantify the alkaloids in different plant organs (leaves, flowers, stems, seeds) of thorn apples after a simple sample preparation step. Elaboration and validation of the method and analysis of plant extracts were done by UFLC-MS technique, employing an Ascentis Express C18 column. Detection was done in positive ionization mode (ESI+) and the method suitability was evaluated by several validation characteristics. Quantitation limits are 333 and 167 pgmL(-1) for scopolamine and atropine, respectively, and the method shows very good repeatability. The analysis of Datura extracts revealed significant differences depending on the species, the organ and the sampling period. Atropine was found to be dominant over scopolamine in three out of the four taxa investigated. D. innoxia showed the highest concentrations of scopolamine in all organs examined, whereas D. metel accumulated the lowest scopolamine levels. Hyoscyamine, measured as atropine, was the highest in D. stramonium var. tatula, and the lowest in D. innoxia. Samples collected in summer had higher scopolamine levels than autumn samples, concerning both stems and leaves.

Molecular dynamic theories in chromatography

The molecular dynamic model of chromatography is a microscopic model that consists of two fundamental processes: (i) the random migration of the molecules in the mobile phase, and (ii) the random adsorption-desorption of molecules on the stationary phase. The diffusion and drift of the molecules in the mobile phase is usually described with a simple one-dimensional random walk. The adsorption-desorption process is modeled most of the time by a Poisson process that assumes exponential sojourn times of the molecules in both the mobile and the stationary phases. The molecular dynamic model of chromatography can simply be used to characterize the chromatographic process on heterogeneous stationary phases. It has been applied to reversed phase, chiral, size-exclusion, and ion-exchange separations.

Solvent excess adsorption on the stationary phases for reversed-phase liquid chromatography with polar functional groups.

The measurement of acetonitrile and methanol adsorption was carried out on stationary phases with specific functionalities. The results were compared with the adsorption of those solvents on alkyl-modified adsorbents. This comparison allows us to describe the effect of polar groups on the adsorption of the organic modifiers. Our results clearly demonstrate how the functional groups modify the chromatographic properties of the homogeneous hydrophobic adsorbents.

Overloaded gradient elution chromatography on heterogeneous adsorbents in reversed-phase liquid chromatography.

Overloaded band profiles of phenol were measured on a C18-Kromasil column in gradient elution conditions. The mobile phase used was a mixture of methanol and water. The volume fraction of methanol was allowed to vary between 0 and 0.5. A general adsorption model, which expresses the amount of phenol adsorbed q* as a function of both its concentration C and the composition phi of the organic modifier (methanol) in the mobile phase, was empirically derived from previous independent adsorption experiments based on frontal analysis (FA) and frontal analysis by the characteristic point (FACP). Accordingly, the general model was an extension of the simplest heterogeneous model, the Bilangmuir model, to non-isocratic conditions. The low-energy sites followed the classical linear solvent strength model (LSSM), but not the high-energy sites whose saturation capacity linearly decreased with phi. The general model was validated by comparing the experimental and simulated band profiles in gradient elution conditions, in linear and non-linear conditions, as well. The band profiles were calculated by means of the equilibrium-dispersive model of chromatography with a finite difference algorithm. A very good agreement was observed using steps gradient (delta phi) from 0 to 50% methanol and gradient times t(g) of 20, 25, 30, 40, 60, 80 and 100 min. The agreement was still excellent for steps gradient from 5 to 45% (t(g) = 25 min), 5 to 35% (t(g) = 50 min), 5 to 25% (t(g) = 50 min) and 5 to 15% (t(g) = 50 min). Significative differences appeared between experience and simulation when the slope of the gradient (delta phi/t(g)) became too strong beyond 3.3% methanol per minute. This threshold value probably mirrored the kinetic of arrangement of the G18-bonded chains when the methanol content increased in the mobile phase. It suggested that the chromatographic system was not in a full thermodynamic equilibrium state when very steep mobile phase gradients were applied.