Microchip HPLC: Experimental investigation of separation efficiency, column characteristics, and coupling with mass spectrometry

This work deals with separation efficiency and column characteristics of particle-packed HPLC microchips. The impact of conduit geometry on the chromatographic performance of typical particulate microchip packings is investigated. For this purpose, HPLC/UV-microchips with separation channels of quad...

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Bibliographic Details
Main Author: Jung, Stephanie
Contributors: Tallarek, Ulrich (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2012
Chemie
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Summary:This work deals with separation efficiency and column characteristics of particle-packed HPLC microchips. The impact of conduit geometry on the chromatographic performance of typical particulate microchip packings is investigated. For this purpose, 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 µm 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. 3 µm packings display a high packing density irrespective of their conduit geometries and their performance reflects the dispersion behaviour of the empty channels. Dispersion in 5 µm packings correlates with the achieved packing densities, which are limited by the number and accessibility of corners in a given conduit shape. Interparticle void volumes and porosities of packed capillaries and of commercially available, analytical, reversed-phase HPLC columns have been determined using intraparticle Donnan exclusion of a small, unretained, co-ionic tracer (nitrate ions). The operational domain of this approach has been characterized for bare silica, reversed-phase, and strong cation-exchange materials with different particle sizes, intraparticle pore sizes, and construction (fully porous or core-shell) in dependence of the mobile phase ionic strength. At low buffer concentrations nitrate ions are completely electrostatically excluded from the intraparticle mesopore space, which is reflected by a plateau region in the elution curves. The elution volume in the plateau region equals the interparticle void volume. Clearly defined plateau regions were observed for all columns, even those densely packed with core-shell and sub-2 µm particles, enabling the accurate determination of interparticle porosities to three decimal places in a fast and convenient way. Interparticle porosities agree well with those analyzed by inverse size-exclusion chromatography (ISEC). Limitations to the use of Donnan exclusion (electrostatic exclusion) and ISEC (mechanical exclusion) arise as either type of exclusion becomes noticeable also in the cusp regions between particles, or as the intraparticle pores are so large that complete electrostatic and size exclusion are difficult to realize. Dynamic changes in mobile phase composition during high performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray modes. The impact of the eluent composition on spray stability and MS response by infusion and injection experiments with a small tetrapeptide in water-acetonitrile mixtures was investigated. The employed HPLC/ESI-MS configuration uses a microchip equipped with enrichment column, separation column, and make-up flow (MUF) channel. One nano pump is connected to the separation column, while a second one delivers solvent of exactly inverted composition to the MUF channel. Both solvent streams are united behind the separation column, before the ESI tip, such that the resulting electrosprayed solution always has identical composition during a gradient elution. Analyte peak parameters without and with MUF compensation are determined and discussed with respect to the electrospray mode and eluent composition. The post-column MUF significantly improves spray and signal stability over the entire solvent gradient, without compromising the performance of the HPLC separation column. It can also be conveniently implemented on microchip platforms.
DOI:https://doi.org/10.17192/z2012.0937