We report an experimental study of separation efficiency in microchip high-performance liquid chromatography (HPLC). For this study, prototype HPLC microchips were developed that are characterized by minimal dead volume, a separation channel with trapezoidal cross section, and on-chip UV detection. A custom-built stainless steel holder enabled microchip packing under pressures of up to 400 bar and ultrasonication. Bed densities were investigated with respect to the packing conditions and consistently related to pressure drop over the packed microchannels and separation efficiency under isocratic elution conditions. The derived plate height curves show a decrease of mobile phase mass transfer resistance with increasing bed density. High bed densities are critical to separation performance in noncylindrical packed beds, because only at low bed porosities does hydrodynamic dispersion in noncylindrical packings come close to that of cylindrical packings. At higher bed porosities, the presence of fluid channels of advanced flow velocity in the corners of noncylindrical packings affects hydrodynamic dispersion strongly. We demonstrate that the separation channels of HPLC microchips can be packed as densely as the cylindrical fused-silica capillaries used in nano-HPLC and that consequently microchip-HPLC separation efficiencies comparable to those of nano-HPLC can be achieved.

The average interparticle voidage or porosity (epsilon(inter)) in cylindrical capillaries is studied in dependence on the column diameter (d(c)) to particle diameter (d(p)) ratio for 5 < d(c)/d(p) < 50. Using optimized slurry and packing solvents, high pressure and ultrasonication, 5 mum-sized porous C18-silica particles were slurry-packed into fused-silica capillaries having ids from 30 to 250 mum. Packing densities are assessed by a polystyrene standard which is size-excluded from the intraparticle pore space of the packings. For d(c)/d(p) > 35 densely packed beds are realized (epsilon(inter) = 0.36-0.37), while for decreasing aspect ratios an exponential increase in epsilon(inter )is observed reaching epsilon(inter ) approximately 0.47 at d(c)/d(p) = 5. This behaviour is ascribed to a combination of the geometrical wall effect operating in the direct vicinity of the column wall, caused by the inability of the particles to form a dense packing against the hard surface of the column wall, and particle characteristics like the size distribution, shape and surface roughness. Results are compared with the literature data to address also the importance of absolute particle size in studying structure-transport relations in packed beds in dependence on the aspect ratio d(c)/d(p).

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Flow and transport in a particle-packed microchip separation channel were investigated with quantitative numerical analysis methods, comprising the generation of confined, polydisperse sphere packings by a modified Jodrey-Tory algorithm, 3D velocity field calculations by the lattice-Boltzmann method, and modeling of convective-diffusive mass transport with a random-walk particle-tracking approach. For the simulations, the exact conduit cross section, the particle-size distribution of the packing material, and the respective average interparticle porosity (packing density) of the HPLC-microchip packings was reconstructed. Large-scale simulation of flow and transport at Peclet numbers of up to Pe = 140 in the reconstructed microchip packings (containing more than 3 x 10(5) spheres) was facilitated by the efficient use of supercomputer power. Porosity distributions and fluid flow velocity profiles for the reconstructed microchip packings are presented and analyzed. Aberrations from regular geometrical conduit shape are shown to influence packing structure and, thus, porosity and velocity distributions. Simulated axial dispersion coefficients are discussed with respect to their dependence on flow velocity and bed porosity. It is shown by comparison to experimental separation efficiencies that the simulated data genuinely reflect the general dispersion behavior of the real-life HPLC-microchip packings. Differences between experiment and simulation are explained by differing morphologies of real and simulated packings (intraparticle porosity, packing structure in the corner regions).

This work demonstrates by direct visualization using confocal laser scanning microscopy that the application of electrical fields to a single-fixed, ionpermselective glass bead produces a remarkable complexity in both the coupled mass and charge transport through the bead and the coupled electrokinetics and hydrodynamics in the adjoining bulk electrolyte. The visualization approach enables the acquisition of a wealth of information, forming the basis for a detailed analysis of the underlying effects (e.g., ion-permselectivity, concentration polarization, nonequilibrium electroosmotic slip) and an understanding of electrohydrodynamic phenomena at charge-selective interfaces under more general conditions. The device used for fixing single beads in a microfluidic channel is flexible and allows to investigate the electrohydrodynamics in both transient and stationary regimes under the influence of bead shape, pore size and surface charge density, mobile phase composition, and applied volume forces. This insight is relevant for the design of microfluidic/nanofluidic interconnections and addresses the ionic conductance of discrete nanochannels, as well as nanoporous separation and preconcentration units contained as hybrid configurations, membranes, packed beds, or monoliths in lab-on-a-chip devices.

This work investigates the impact of conduit geometry on the chromatographic performance of typical particulate microchip packings. For this purpose, high-performance liquid chromatography (HPLC)/UV-microchips with separation channels of quadratic, trapezoidal, or Gaussian cross section were fabricated by direct laser ablation and lamination of multiple polyimide layers and then slurry-packed with either 3 or 5 microm spherical porous C8-silica particles under optimized packing conditions. Experimentally determined plate height curves for the empty microchannels are compared with dispersion coefficients from theoretical calculations. Packing densities and plate height curves for the various microchip packings are presented and conclusively explained. The 3 microm packings display a high packing density irrespective of their conduit geometries, and their performance reflects the dispersion behavior of the empty channels. Dispersion in 5 microm packings correlates with the achieved packing densities, which are limited by the number and accessibility of corners in a given conduit shape.

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