Some remarks on characterization and application of stationary phases for RP-HPLC determination of biologically important compounds

Abstract

Biologically active compounds such as vitamins, steroids, nucleosides, peptides and proteins play a very important role in coordinating living organism functions. Determination of those substances is indispensable in pathogenesis. Their complex structure and physico-chemical properties cause many analytical problems. Chromatography is the most common technique used in pharmaceutical and biomedical analysis. The interaction between analyte and stationary phase plays a major role in the separation process. The structure of the packing has a significant influence on the results of the separation process. Various types of spectroscopic techniques, such as nuclear magnetic resonance spectroscopy, infrared spectroscopy, fluorescence spectroscopy and photoacoustic spectroscopy can be useful tools for the characterization of packings. Surface area measurements, elemental analysis, thermal analysis and microcalorimetric measurements are also helpful in this field. Part of the paper contains a description of chromatographic tests used for the determination of column properties. The description of the possibilities of surface characterization is not complete, but is focused on the most popular techniques and practical chromatographic tests. All the presented methods made possible the design and quality control of a new generation stationary phases, which are the future of high-performance liquid chromatography.

Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography

“…Figure 6 depicts the most commonly used techniques for chromatographic column characterization. 416 2.4.1. Surface Topology Characterization.…”

“…Therefore, of all the available tests, the ones based on chromatography are the most important for day-to-day end-users who do not have access to the instruments, expertise, or time for in-depth analyses of the stationary phases to be used for a particular analysis. Figure depicts the most commonly used techniques for chromatographic column characterization …”

Physico-chemical methods for the chromatographic materials heterogeneity evaluation

“…In recent years interest has grown in a deeper understanding of surface phenomena beyond the usual description of surface area and adsorption isotherm. This has lead to an introduction of more sophisticated approaches, which allow for a study of thermodynamic and kinetic information [6].…”

“…polar groups in hydrophobic chains) and mixed phases containing various functional groups have become the newest solutions. Such stationary phases are derivatives of amino materials and are used for chromatographic separation, but also as specific support for bonding biological compounds like cholesterol and phospholipids [3][4][5][6]. Different kinds of silica gels are frequently used for the preparation of chemically bonded stationary phases.…”

“…Analyte varieties and retention mechanisms based on hydrophobic, hydrophilic, size exclusion and ion-pairing interactions resulted in many specific and selective chromatographic columns, but no ideal one [6]. The selection of a proper column for solving a separation problem is sometimes very difficult.…”