Josef F. K. Huber

Determination of Cu, Fe, Mn, and Zn in blood fractions by SEC-HPLC-ICP-AES coupling.

The binding of Cu, Fe, Mn, and Zn to proteins in blood and in blood fractions was investigated, since their interactions in free radical metabolism in humans is of great interest. An HPLC-ICP-AES technique was developed allowing adequate separation of metalloproteins and of inorganic and organic metal species. For the separation of metalloproteins in erythrocytes and blood plasma a Merck Superformance Fractogel EMD BioSEC 650 (S) column was used. Size exclusion chromatography (SEC)-HPLC was hyphenated to ICP-AES both on-line and off-line for the detection of trace elements in the fractions resulting from HPLC separations. HPLC parameters, pH, temperature, flow rate and salt concentration were optimized for the protein separation and the optimal conditions were applied for the hyphenation to the ICP-AES detector. The separation column was calibrated with five standard proteins. For the element determination by ICP-AES a line selection with respect to the sensitivity was performed. Three different methods were used for the determination of trace elements in blood: direct determinations, on-line and off-line SEC-HPLC-ICP-AES measurements. For the optimizing experiments blood samples of one female subject were used. The direct determination by ICP-AES of the elements was performed in blood and blood fractions of ten different subjects to obtain the average concentration ranges. From the results the identification of the protein Cu/Zn superoxide dismutase in erythrocytes was possible. The LOD were 0.03 microgram mL-1 for Cu, 0.026 microgram mL-1 for Fe, 0.8 ng mL-1 for Mn, and 0.09 microgram mL-1 for Zn in a synthetic blood matrix.

Effect of phase system selectivity in preparative column liquid chromatography

Abstract In the application of chromatography in preparative work, the sample load and the resolution are the most important characteristics. An equation describing both the sample load and the chromatographic resolution as a function of the other process variables is derived. The influence of the selectivity coefficient is discussed on the basis of this equation. The exploitation of the selectivity of the phase system in preparative work was investigated experimentally. The influence of the feed volume on the maximum and the width of the elution function as well as on the resolution of two components is described. For constant resolution, the maximum allowable sample load increases with increasing selectivity coefficient. It is demonstrated that the maximum sample load for simple mixtures can be very large if a highly selective column is chosen, and can be further increased by two-column operation.

Quantitation of the information content of multi-dimensional gas chromatography and low-resolution mass spectrometry in the identification of doping drugs☆

On the basis of information theory, a comparison of the performance of the identification of compounds by multi-dimensional gas chromatography and binary-encoded low-resolution mass spectrometry is made for doping drugs. This group of compounds was not selected at random but according to their pharmocological effects. For these compounds it was observed that the m/e values of the peaks in the mass spectra are highly correlated, while a number of chromatographic columns can be found on which the retention data are only slightly correlated. As a result, the information content of two-dimensional gas chromatographic retention data on such columns equals the information content of binary-encoded low-resolution mass spectra. It is shown that the accuracy and precision of retention data in gas--liquid chromatography, and therefore the information content, can be significantly improved if the data are corrected for adsorption effects of the solid support and extrapolated to a limiting value in the case of asymmetric peaks.

Characterization and selection of stationary phases for gas-liquid chromatography by pattern recognition methods

Abstract The classification of stationary phases in gas—liquid chromatography and the quantitation of their retention characteristics, which is generally described as ‘polarity’, were investigated by pattern recognition methods, especially by the hierarchical clustering and the minimum spanning tree techniques. It is demonstrated that the hierarchical clustering with a distant function and the minimum spanning tree method gives similar results with respect to the ranking and the differentiation of liquid stationary phases. New measures of the retention characteristics of liquid stationary phases were defined and tested. The potential of the various measures of solvent polarity is discussed. The mean retention index was found to be the best polarity characteristic. With a view to the rationalization of experimental work, an optimized procedure for the classification of liquid stationary phases and the calculation of their polarity with the minimum number of characteristic test solutes was elaborated. The set of characteristic solutes selected gives the best representation of the total number of solutes and covers all types of molecular interactions included in the term polarity.

Ultra-trace analysis of phenols in water using high-performance liquid chromatography with on-line reaction detection

Abstract An ultra-sensitive HPLC method for the determination of phenols in water based on flow-through reaction is described. The phenols are concentrated by a two-step liquid-liquid extraction with dichloromethane and after evaporation of the organic solvent the separation is carried out on an octadecyl-silica column by a gradient of water-acetonitrile. Detection is performed by an on-line twin detection system. First dinitrophenols are detected by UV-absorption measurement followed by oxidation of phenols with cerium(IV) in a tubular flow-through reactor and fluorescence measurement of cerium(III). Detection limits in the lower ppt range can be achieved. The influence of reaction time, temperature and reagent concentration on reaction yield was investigated. Identification and quantitation is improved by use of internal standards. The method was applied to samples of surface and drinking water.

Adjustment of the capacity factor by means of the specific surface area of the adsorbent for optimal separation in liquid-solid chromatography

Summary In the optimization of a chromatographic separation with respect to resolution, speed and dilution, the capacity factor is an important parameter. Its influence on the chromatographic characteristics is discussed theoretically. The capacity factor is determined by the distribution coefficient and the phase ratio. Only the phase ratio can be used, however, to adjust the value of the capacity factor without affecting the value of the selectivity coefficient at the same time. Micro-particulate silica and octyl silica with different specific surface areas were investigated experimentally as adsorbents for liquid-solid chromatography. It is shown that the selectivity of these materials is fairly constant for specific surface areas varying over two orders of magnitude. The effect of the adjustment of the specific surface area is demonstrated in the separation of dyestuffs and polynuclear aromatic hydrocarbons by high-pressure liquid chromatography. The application of column switching for optimal separation using two or three columns packed with the same adsorbent but with different specific surface areas is shown.

Enrichment of trace components from liquids by displacement column liquid chromatography

Abstract In trace analysis an enrichment has to be carried out in general. In order to achieve an enrichment the trace components have to be transferred to a volume which is orders of magnitude smaller than the original one. Such a process is displacement chromatography which has not yet been investigated systematically as enrichment procedure. In the present paper the fundamentals of enrichment by means of adsorption chromatography are discussed theoretically and investigated experimentally. The relationship between the enrichment factor and the change of the distribution coefficient is discussed for gradient elution and displacement chromatography. The limiting volume to which a trace component can be condensed is shown to be dependent on the mixing characteristics of the column. It is demonstrated that enrichment factors of the order of ten thousand can be obtained for polar as well as non-polar compounds using adsorbents with hydrophilic and hydrophobic surfaces, respectively.

Theorie des chromatographischen Prozesses

The results of the theory of chromatography are described using the mass balance model without entering into the details of mathematical treatment. The equation of the elution function describing the concentration in the column effluent as function of time is discussed with special emphasis on the theoretical plate height. The choice of the experimental parameters is discussed by means of the equation for the resolution of two components. Especially the optimation with respect to the time of separation and the dilution occurring at the separation is dealt with.ZusammenfassungAuf der Basis des Stoffbilanz-Modells werden die Ergebnisse der Theorie der Chromatographie dargestellt, ohne auf die Details der mathematischen Ableitung einzugehen. Die Gleichung für die Eluierungsfunktion, welche die Konzentration im Säuleneluat als Funktion der Zeit beschreibt, wird mit besonderer Berücksichtigung der theoretischen Trennstufenhöhe diskutiert. Mit Hilfe des Ausdrucks für die Auflösung zweier Komponenten wird die Wahl der Arbeitsbedingungen besprochen und insbesonders die Optimierung bezüglich Trennzeit und Verdünnung behandelt.

Adjustment of stationary phase selectivity in gas—liquid chromatography by using mixed liquid phases

Abstract Contrary to liquid—liquid chromatography, where binary, ternary and quarternary mixed solvents have been systematically investigated and extensively applied, very few systematic studies on mixed stationary liquid phases have been undertaken in gas—liquid chromatography (GLC). Except for mixtures of chemically analogous polymric constituents resulting from production processes, little use is made of mixed solvents in GLC. In this work, “binary” mixed solvents were studied as stationary liquids in GLC. Linear and non-linear relationships between the capacity factor and phase composition were found experimentally. In the non-linear case curves with minima were observed. It was confirmed experimentally that adsorption on the solid support is negligible. The shape of the relationship is discussed in terms of thermodynamic theory. The exploitation of the selectivity of mixed solvents in GLC separations is demonstrated. It is shown that columns with composite solvents as stationary phases and coupled columns with single phases aer equivalent if there is a linear relationship between the capacity factor and the statinary liquid phase composition. It is demonstrated that switching of columns can be successfully empployed in achieving or optimizing a separation of GLC.

Determination of inorganic and organic anions in one run by ion chromatography with column switching

Abstract A method for the simultaneous determination of low-molecular-mass organic and inorganic anions in aqueous solutions was developed using isocratic ion chromatography (IC) with suppressed conductimetric detection and column switching. Owing to the large differences in distribution coefficients between sulphate, nitrate and phosphate and the other species, these ions are separated in the first stage on a medium-capacity anion exchanger, whereas the other anions are led through a second column packed with a high-capacity anion-exchange resin via a column-switching valve. After optimization of the switching procedure a spiked drinking water sample was analysed. Fluoride, acetate, butyrate, formate, nitrate, nitrite and phosphate could be determined in addition to the main anions (chloride and sulphate). The time for a complete analysis is less than 20 min and the method can easily be automated. The precision and detection limit are as usual in IC with background suppression.