Generation Of Stationary Phases Research Articles

Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry

Supercritical fluid chromatography (SFC) is frequently used for the analysis and separation of non-polar metabolites, but remains relatively underutilised for the study of polar molecules, even those which pose difficulties with established reversed-phase (RP) or hydrophilic interaction liquid chromatographic (HILIC) methodologies. Here, we present a fast SFC-MS method for the analysis of medium and high-polarity (−7≤cLogP≤2) compounds, designed for implementation in a high-throughput metabonomics setting. Sixty polar analytes were first screened to identify those most suitable for inclusion in chromatographic test mixtures; then, a multi-dimensional method development study was conducted to determine the optimal choice of stationary phase, modifier additive and temperature for the separation of such analytes using SFC. The test mixtures were separated on a total of twelve different column chemistries at three different temperatures, using CO2-methanol-based mobile phases containing a variety of polar additives. Chromatographic performance was evaluated with a particular emphasis on peak capacity, overall resolution, peak distribution and repeatability. The results suggest that a new generation of stationary phases, specifically designed for improved robustness in mixed CO2-methanol mobile phases, can improve peak shape, peak capacity and resolution for all classes of polar analytes. A significant enhancement in chromatographic performance was observed for these urinary metabolites on the majority of the stationary phases when polar additives such as ammonium salts (formate, acetate and hydroxide) were included in the organic modifier, and the use of water or alkylamine additives was found to be beneficial for specific subsets of polar analytes. The utility of these findings was confirmed by the separation of a mixture of polar metabolites in human urine using an optimised 7min gradient SFC method, where the use of the recommended column and co-solvent combination resulted in a significant improvement in chromatographic performance.

Chromatographic evidence of silyl ether formation (SEF) in supercritical fluid chromatography.

In this article, we propose that silyl ether formation (SEF) is a major contribution to retention and selectivity variation over time for supercritical fluid chromatography (SFC). In the past, the variations were attributed to instrumentation, but high performance SFC systems have shed new light on the source of variation. As silyl ethers form on the particle surface, the hydrophilicity is decreased, significantly altering the retention and selectivity observed. SEF is expected to occur with any chromatographic particle containing silanols but is slowed on hybrid inorganic/organic particles. The SEF reaction is between alcohols on the particle surface and in the mobile phase solvent. We have found that storage conditions of a column are paramount, which can either prevent or accelerate the process. Because SEF exists as an equilibrium between the liquid phase and the particle surface, the process is also reversible. The silanols can be hydroxylated (regenerated) to their original state upon exposure to water. The next generation of stationary phases will either advantageously utilize SEF or effectively mitigate its effects. Mitigation of SEF would be a significant improvement in SFC that has the potential to vault their performance to levels of similar reproducibility and reliability observed for high performance liquid chromatography (HPLC). Further research in SEF may lead to a better understanding of the mechanism of interaction between the solutes and chromatographic surface.

Recent developments and applications of molecularly imprinted monolithic column for HPLC and CEC

As a new generation of stationary phases, monolithic supports have attracted significant interest in high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) because of their ease of preparation, high reproducibility, versatile surface chemistry and rapid mass transport. Molecularly imprinted polymers (MIPs) are synthetic materials with high specific recognition ability to template molecule. The combination of monolithic column and MIPs integrates the high efficiency of modern chromatography and the high selectivity provided by MIPs. This review focuses on the recent developments and applications of all kinds of monolithic matrix, i.e. organic polymer-based and silica-based MIP monolith in HPLC and CEC mode.

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.