Katarzyna Krupczyńska

New generation of chromatographic packings and columns for determination of biologically active compounds

Analysis of biologically active substances is particularly important in the pharmaceutical and biomedical area. For separation of polar compounds or complex mixtures by normal (NP) or reversed phase liquid chromatography (RP-HPLC) and/or electromigration techniques, it is necessary to apply a new generation of packings and columns with strictly defined properties. It is connected to the definition of chromatographic behavior and determination of compounds that are described by its structure, as well as chemical and physical properties. One of the factors playing a predominant role in the separation process is the interaction between analyte and stationary phases. During recent years a large variety of stationary phases have become available and have been also applied in routine and research chromatographic separations. Bonded phases are in widespread use and popular because of the great number of available packing materials, allowing the solution of a scale of different separation problems. At the present time analytical methods cannot be restricted only to the so-called “black box.” To meet the requirements of modern analytical techniques, strong demands are put on further, deeper understanding of the essence of the separation process, among other things, on the basis of conclusions about interactions between solute and stationary phase surfaces. For surface characterization different physicochemical methods such as CP/MAS, NMR, FT IR, DCS, chromatography, etc., have been described. The resolution, as reflected by efficiency, selectivity, and retention patterns on these materials, has been demonstrated. The effect of structure of stationary phases on retention of a model series of test analytes has been proved and numerically expressed by means of the Quantitative Structure-Retention Relationship (QSSR). The main aim of this paper is to present possibilities of determining different biologically active compounds (e.g., vitamins, steroids, nucleosides, peptides) in complex chromatographic methods (sample preparation, final analysis, validation) using new generation of stationary phases, columns, and chips divides. Special attention is dedicated to the advantage of packing materials imitating natural membranes because of the possible examination of the interaction between drug membrane. They can permit to the design of new pharmaceuticals and observation of processes taking place on the border of phases without interfering in natural systems.

Combined effects of mobile phase composition and temperature on the retention of homologous and polar test compounds on polydentate C8 column.

Combined effects of temperature and mobile phase on the reversed phase chromatographic behavior of alkylbenzenes and simple substituted benzenes were investigated on a Blaze C8 polydentate silica-based column, showing improved resistance against hydrolytic breakdown at temperatures higher than 60 degrees C, in comparison to silica-based stationary phases with single attachment sites. For better insight into the retention mechanism on polydentate columns, we determined the enthalpy and entropy of the transfer of the test compounds from the mobile to the stationary phase. The enthalpic contribution dominated the retention at 80% or lower concentrations of methanol in the mobile phase. Entropic effects are more significant in 90% methanol and in acetonitrile-water mobile phases. Anomalies in the effects of mobile phase on the enthalpy of retention of benzene, methylbenzene and polar benzene derivatives were observed, in comparison to regular change in enthalpy and entropy of adsorption with changing concentration of organic solvent and the alkyl length for higher alkylbenzenes. The temperature and the mobile phase effects on the retention are practically independent of each other and--to first approximation--can be described by a simple model equation, which can be used for optimization of separation conditions.

Bronopol as an Ingredient of a New Test Mixture for Evaluation of HPLC Columns

Abstract Different tests, including analytes or mixtures characterized by specific properties, can be used for the evaluation of chromatographic columns. High performance liquid chromatographic (HPLC) columns with chemically modified surfaces, purchased from different producers, were tested in our studies. Characteristic chromatographic parameters, such as: number of plate theoretical (N T ), reduced plate height (h), the retention factor (k), resolution (R S ), and asymmetry factor (f AS ) were determined. Bronopol (2‐bromo‐2‐nitrpropane‐1,3‐diol) was suggested for column evaluation, especially as regards the homogeneity of adsorbent surface coverage by chemically bonded phase and spacing of residual silanols. This analyte was used separately, and in a mixture consisting of other analytes (uracil, acetophenone, benzene, toluene) with different physicochemical properties.

Effect of stationary phase structure on retention and selectivity tuning in the high-throughput separation of tocopherol isomers by HPLC.

Four stationary phases containing different groups such as: C18, C30, alkylamide, and cholesterolic, were presented for simultaneous HPLC analysis of structural isomers of tocopherol. Especially, the influence of stationary phase structure and properties on tuning of the highly selective HPLC separation of beta- and gamma-tocopherol pair demonstrating, respectively, para- and ortho- arrangement of methyl substituents on the 6-chromanol ring, has been elucidated. It was pointed out that selectivity of each stationary phase has been a result of modulation in the mass transfer and set of unspecific interactions in the tertiary system comprising analyte <==> stationary phase <==> mobile phase. Differences in observed retention and specific selectivity of tocopherols together with the stationary phase structure investigations indicated that a spatial organization changing of chemically bonded ligands as predominantly a solvation consequence. Additional molecular modeling studies preliminary explained some of these complicated supramolecular phenomena which caused that cholesterolic stationary phase offered beneficial performance in screening of tocopherols by HPLC and biomimetic studies of not completely recognized interactions of tocopherol isomers and biological membranes.