Instrumental and method development for multidimensional electrophoretic separations – coupling of capillary isoelectric focusing to mass spectrometry

Abstract

Multidimensional electromigrative separation techniques represent powerful tools meeting the complexity of analytical and bioanalytical challenges, especially regarding sample and matrix complexity as well as trace analysis. In this work a glass chip interface in a 2D capillary-chip system was further developed and optimized regarding stability and on-interface (intermediate) analyte detection sensitivity. To overcome limiting performance of previous approaches such as dead volume and capillary-chip connection, a new chip interface was addressed and compared to the previous designs. Selective Laser Etching (SLE) proved to be a promising alternative to established production processes giving rise to a nearly dead volume free capillary connection, enabling the use of screw-tight fittings at lower prototype costs. To ensure zero potential difference at channel cross-sections or ground potential (GND) at one channel of a 2D assays if mass spectrometry (MS) is used as the detection method, on-chip potential measurements were conducted via a passivated electrode inside the chip interface. Using a low current measuring prototype in combination with a SiN passivated Ti/Pt electrode inside the interface, disturbances of the electric field lines as well as electrolysis during potential measurements conducted along electrophoretic separations were successfully avoided; resulting in an excellent setup stability. On-chip fluorescence detection was implemented to properly monitor the analyte inside the interface and thus, to perform a proper sample transfer. Therefore, spectrally and spatially resolved fluorescence detection was implemented via a linear optical fiber array and a push broom imager. In this regard, desired sensitivity as well as spectral and spatial resolution was achieved for fluorescein isothiocyanate and labeled model proteins using flushing and capillary isoelectric focusing (CIEF) experiments. The optimized two-dimensional setup was used for the hyphenation of capillary isoelectric focusing to mass spectrometry using a 2D CIEF/CE-MS setup. For an optimized CIEF separation-based analyte positioning in the first dimension as well as separation efficiency of the protein and the ampholyte fraction within the second CE dimension, the combination of computer simulations and experimental data were used. Applying the on-chip fluorescence detection setup for intermediate analyte monitoring in the 2D setup, analyte carryover as well as peak broadening and peak splitting could be eliminated.