Search

Highlights • An SFC method has been developed for the analysis of polar urinary metabolites. • 12 stationary phases, 9 modifier additives and 3 temperatures were evaluated. • DIOL and 2-PIC columns provide highest peak capacity and overall resolution. • Ammonium formate, ammonium hydroxide and water considerably improved separation. • Alkylamine additives should be strongly considered for polar SFC-UV analysis., 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 7 min gradient SFC method, where the use of the recommended column and co-solvent combination resulted in a significant improvement in chromatographic performance.

In order to prepare zwitterionic HILIC monolithic columns with high polarity, the highly hydrophilic monomer N,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)ammonium betaine (SPDA) and crosslinker N,N'-methylenebisacrylamide (MBA) were employed for developing a novel sulfoalkylbetaine type stationary phase. The polymerization parameters were systematically optimized in order to obtain a satisfactory performance for column permeability, mechanical stability, hydrophilicity, efficiency and selectivity. Compared to the previously reported poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine-co-ethylene dimethacrylate) (poly(SPE-co-EDMA)) monolith and the poly(SPDA-co-EDMA) monolith that we developed, a significantly enhanced hydrophilicity was obtained on the poly(SPDA-co-MBA) monolithic column, illustrated by the lowered critical composition of the mobile phase corresponding to the transition from the HILIC to the RP mode. Excellent permeability, reproducibility and stability were achieved on this optimized poly(SPDA-co-MBA) monolith. A column efficiency of 70,000plates/m was obtained for the analysis of bases at a linear velocity of 1.95mm/s. As expected, by studying the influence of mobile phase pH and salt concentration on their retention, a weak electrostatic repulsion interaction for negatively charged analytes was also observed at low organic solvent content on the poly(SPDA-co-MBA) monolithic column. The final optimized poly(SPDA-co-MBA) monolith exhibited good selectivity for a series of polar compounds, such as phenols, bases, benzoic acid derivatives, small peptides, urea and allantoin.

Hydrophilic interaction chromatography (HILIC) has experienced increasing attention in recent years. Much research has been carried out in the area of HILIC separation mechanisms, column techniques and applications. Because of their good permeability, low resistance to mass transfer and easy preparation within capillaries, hydrophilic monolithic columns represent a trend among novel HILIC column techniques. This review attempts to present an overview of the preparation and applications of HILIC monolithic columns carried out in the past decade. The separation mechanism of various hydrophilic monolithic stationary phases is also reviewed.

Solvent systems for use with LC–MS often result in a compromise between chromatographic performance and mass spectrometric detection, exemplified here by a LC–MS/MS method development for the analysis of ephedrines in doping control. Ephedrines, frequently found in therapeutic and nutritional preparations, are among the most commonly administered doping agents in competitive sport. Improved separation of these hydrophilic, basic compounds, some of which are diastereoisomers, is achieved in reversed-phase LC by the use of a high pH mobile phase in order to suppress analyte ionisation, and thus alter their polarity, resulting in reduced peak tailing and enhanced retention. However, when coupled to an ESI-MS detector, this eluent composition generated a non-linear and poorly reproducible signal. APCI yielded greater stability and reproducibility and is here presented as an ion source for the analysis of basic compounds under conditions that suppress their ionisation. Errors as large as 49.3% were observed with ESI, compared with 15.4% generated using APCI, for pseudoephedrine over the calibration range (25–400 μg/mL) in urine with a simple dilution and injection of samples. These data highlight the importance of suitable MS conditions for stable performance, necessary for accurate quantification, without undue compromise to the LC separation.

Capillary electrochromatography suffered in its development because of difficulty in producing stable columns with good permeability. Variability in frit characteristics gave rise to non-reproducible capillaries whose fabrication was extremely difficult and time consuming. Monolithic stationary phases gained popularity in the early 1990s due to the fact that they were easy to fabricate and required no retaining frits. They were also able to be manufactured in a wide variety of chemistries which made them very interesting to the analytical chemist who is constantly looking for materials with different selectivity to the popular silica-based stationary phases.

The use of wide-pore stationary phases in capillary electrochromatography has shown exceptional increases in separation efficiency in conjunction with high electroosmotic flow. These effects are due to the perfusive flow mechanism which is primarily controlled by the ionic strength of the mobile phase. Good correlation between calculated values of electrochemical double-layer thickness and efficiency data have also been obtained. Reduced plate height values of0.5 have been observed with pore sizes of 4000 A. In addition, electroosmotic flow mobility twice that of 3 microm Spherisorb ODS-1 has been obtained.

Over 95% of all papers published to the present time on capillary electrochromatography (CEC) have reported on the use of C 18 and to a much lesser extent, C 8 stationary phases. The relationship between the electroosmotic flow and pH is of considerable importance in CEC because it allows the analyst to develop methods under optimum conditions, especially with respect to analysis times. Although the electroosmotic flow dependence on pH for C 18 phases has been reported, there have been very few comparable studies carried out on other phases, and for this reason a systematic study of the performance of a wide range of stationary phases was initiated. The phases studied were Waters 3 μm Spherisorb ODS-1, propyl SCX, phenyl SCX, Symmetry SCX and a C 6 /SCX mixed-mode. Important new knowledge regarding the performance of CEC columns has been realised through this study, the first results of which are now reported.