RESUMEN
We report on a direct comparison of the separation performance in capillary nano-LC between commercial packed bed columns and the small-domain silica monoliths in applications. Octadecylsilylated monolithic silica capillary columns with a 50 and 100 µm inner diameter (i.d.) were prepared with a procedure providing domain sizes in the sub-2 µm range. The fabricated monolith columns could provide plate heights (H) of 4.0â4.2 µm for hexylbenzene (retention factor (k) = 3.6) at an optimal linear velocity range under an isocratic condition, while showing a column permeability (Kv0 = 1.6â1.8 × 10-14 m2) comparable to that of a column packed with 3â3.5 µm particles. When the peak capacity (np) for a cytochrome C digest was compared for variable gradient times (tG = 15, 30, 60, and 120 min) and constant gradient steepness (b'), the present monolith columns could show a 30â40% higher np-value than the packed capillary column with 2 µm particles (e.g. np = 180 versus np = 259 at tG = 30 min). The produced monolith columns showed a high chromatographic repeatability for both isocratic and gradient elution (e.g. relative standard deviation (n = 3, RSD (%)) = 0.5% for H, 2,6% for k, and 5.6% for Kv0 in the isocratic mode using the 100 µm i.d.-columns). The present results show that the domain sizes which can now be achieved for capillary silica monoliths are sufficiently small to result in separation efficiencies that can successfully compete with the commercial packed bed columns available for use in nano-LC applications.
Asunto(s)
Cromatografía Liquida/métodos , Nanotecnología/métodos , Tamaño de la Partícula , Dióxido de Silicio/química , Derivados del Benceno/química , Citocromos c/análisisRESUMEN
Monolithic silica materials (first unclad monolith rods, then monolithic capillary columns) were prepared using various amounts of polyethylene glycols (PEGs) with different molecular weight (MW). The monolith rods were used to examine the mesoporosity by argon physisorption technique, and the macroporosity by mercury intrusion porosimetry. Subsequently, silica-based monolithic capillary columns with an inner diameter of 100 µm were produced using the same preparation conditions as used for the rods. The results obtained with the monolith rods showed the following important findings: (1) it is feasible to fabricate monolithic silica rods possessing macropore size of 0.5-1.4 µm by tuning the amount of PEGs (independently of the MW), whereas the macropore volume and the mesoporosity remain similar. (2) the smallest macropore size (0.5 µm) rod prepared with PEG having a MW=20,000g/mol provided a narrower macropore size distribution than with PEG with MW=10,000g/mol. The monolithic capillary columns produced with the different PEG type showed similar retention factors for hexylbenzene (k=2.3-2.4) and similar t0-based column permeability (Kv0=2.3-2.4×10(-14)m(2)) in 20:80% (v/v) water:methanol, as expected from the results obtained with the monolith rods. The column prepared with PEG of MW=20,000g/mol gave a plate height of H=4.0 µm for hexylbenzene at an optimal linear velocity of u0=2.6mm/s in 20:80% (v/v) water containing 0.1% formic acid:acetonitrile. To the best of our knowledge, this is the lowest plate height ever recorded for a monolithic column. Comparing the kinetic performance at 30MPa shows that the best monolithic silica column obtained in the present study performs better than the second-generation monolithic silica columns developed up till now in the practically most relevant range of plate numbers (N≤40,000). In this range, the performance is now similar to that of 2.7 µm core-shell particle columns.