RESUMEN
In spatial multi-dimensional liquid chromatography (LC) devices the flow of each dimension has to remain in the corresponding region, otherwise the separation efficiency is undermined. Adequate flow-confinement measures are necessary. Here, the use of permeability differences across different compartments of spatial two-dimensional (2D) and three-dimensional (3D) LC devices as a method to guide fluid flow and reduce analyte loss during the first, second- and third-dimension development was investigated with computational fluid dynamics (CFD) simulations. In case of 2DLC devices, it was shown that porous barriers with a permeability on the order of 10-12 m2 suffice to keep the total sample spillage from an open 1D channel under 1%. In case of 3DLC devices, it was shown that flow confinement could be achieved using an open 1D channel in combination with a highly-permeable monolith (permeability on the order of 10-12 m2) in the second-dimension (2D) and a less permeable packing with a permeability on the order of 10-15 m2 (e.g. 1⯵m particles) in the third-dimension (3D). Additionally, the impact of the 3D flow-distributor has been studied and a novel design, capable of limiting the spillage to the other dimensions to the absolute minimum, is proposed.
Asunto(s)
Cromatografía Liquida/métodos , Cromatografía Liquida/instrumentación , Diseño de Equipo , Hidrodinámica , Dispositivos Laboratorio en un Chip , Permeabilidad , PorosidadRESUMEN
Conventional one-dimensional column-based liquid chromatographic (LC) systems do not offer sufficient separation power for the analysis of complex mixtures. Column-based comprehensive two-dimensional liquid chromatography offers a higher separation power, yet suffers from instrumental complexity and long analysis times. Spatial two-dimensional liquid chromatography can be considered as an alternative to column-based approaches. The peak capacity of the system is ideally the product of the peak capacities of the two dimensions, yet the analysis time remains relatively short due to parallel second-dimension separations. Aspects affecting the separation efficiency of this type of systems include flow distribution to homogeneously distribute the mobile phase for the second-dimension (2D) separation, flow confinement during the first-dimension (1D) separation, and band-broadening effects during analyte transfer from the 1D separation channel to the 2D separation area. In this study, the synergy between computational fluid dynamics (CFD) simulations and rapid prototyping was exploited to address band broadening during the 2D development and analyte transfer from 1D to 2D. Microfluidic devices for spatial two-dimensional liquid chromatography were designed, simulated, 3D-printed and tested. The effects of presence and thickness of spacers in the 2D separation area were addressed and leaving these out proved to be the most efficient solution regarding band broadening reduction. The presence of a stationary-phase material in the 1D channel had a great effect on the analyte transfer from the 1D to the 2D and the resulting band broadening. Finally, pressure limit of the fabricated devices and printability are discussed.
Asunto(s)
Cromatografía Liquida/instrumentación , Dispositivos Laboratorio en un Chip , Simulación por ComputadorRESUMEN
This review focuses on the preparation of organic polymer-based monolithic stationary phases and their application in the separation of biomolecules, including antibodies, intact proteins and protein isoforms, oligonucleotides, and protein digests. Column and material properties, and the optimization of the macropore structure towards kinetic performance are also discussed. State-of-the-art liquid chromatography-mass spectrometry biomolecule separations are reviewed and practical aspects such as ion-pairing agent selection and carryover are presented. Finally, advances in comprehensive two-dimensional LC separations using monolithic columns, in particular ion-exchange×reversed-phase and reversed-phase×reversed-phase LC separations conducted at high and low pH, are shown.