RESUMEN
Ultra-high performance size-exclusion chromatography (UHP SEC) is a newly developed disruptive technology that allows the high-resolution separation of synthetic polymers in as little as 2 min. The capability of UHP SEC for the characterization of synthetic polymers in organic solvents has been demonstrated. Using the Waters ACQUITY UPLC® H-Class system and ethylene-bridged hybrid size-exclusion chromatography (SEC) columns packed with 1.7 to 2.5-µm particles with pore sizes ranging from 45 to 900 Å, size-based separations of polystyrene and poly(methyl methacrylate) standards in tetrahydrofuran and poly(ethylene oxide) standards in 20 mM ammonium acetate in methanol are achieved within 2-4 min. The speed of analysis is about ten times faster than conventional SEC separations, and greater resolution is achieved. Average molecular weights of selected commercial polymers have been determined using ultra-high performance and conventional SEC. Average M data of analyzed samples are in good agreement using the two approaches. An inherent limitation of SEC in UHP mode is the characterization of very high M polymers (above ca. 2 million Da) due to the deformation and/or mechanical shearing of large molecules at high flow rates.
RESUMEN
A comprehensive retention and selectivity characterization of several hydrophilic interaction chromatography (HILIC) stationary phases was performed with 28 test probes in order to study the influence of particle type, surface chemistry, and mobile-phase pH on chromatographic retention, selectivity, and MS response. Selectivity differences were compared for columns operated at both low and high pH, while ESI-MS was used to study the effects of mobile-phase pH on signal response. Additionally, acetone was explored as a potential alternative to ACN as the weak HILIC solvent. Moderate differences in selectivity were observed on the same column operated at different pH, mostly due to acidic compounds. In addition, the MS response increased when a high pH mobile phase was used, particularly for analytes that were ionized with negative ESI-MS. Even larger selectivity differences were observed for different stationary phases evaluated with the same mobile phase. Acetone was not a suitable replacement for ACN in routine HILIC separations due to differences in selectivity and MS response. Finally, the data from this study were used to establish guidelines for rapid HILIC method development of polar compounds, which is demonstrated with a mixture of histidine dipeptides and organophosphonate nerve agent metabolites.