Temperature Gradient Elution and Superheated Eluents in Chip-HPLC.

Utilizing temperature as an active parameter for optimization in chip-based liquid chromatography is an important step toward high-speed and high-efficiency separations on the microscale. A device including a low thermal mass micro thermostat and a microfluidic glass chip as central elements were designed and evaluated for maximal heating performance of up to 4.7 °C s-1 at up to 200 °C. With this enabling technology, high-speed separations in temperature gradient mode were performed both in common reversed-phase eluents and environmental friendly ethanol-based alternatives.

On-chip integration of organic synthesis and HPLC/MS analysis for monitoring stereoselective transformations at the micro-scale.

We present a microfluidic system, seamlessly integrating microflow and microbatch synthesis with a HPLC/nano-ESI-MS functionality on a single glass chip. The microfluidic approach allows to efficiently steer and dispense sample streams down to the nanoliter-range for studying reactions in quasi real-time. In a proof-of-concept study, the system was applied to explore amino-catalyzed reactions, including asymmetric iminium-catalyzed Friedel-Crafts alkylations in microflow and micro confined reaction vessels.

Enantioselective reaction monitoring utilizing two-dimensional heart-cut liquid chromatography on an integrated microfluidic chip.

Chip-integrated, two-dimensional high performance liquid chromatography is introduced to monitor enantioselective continuous micro-flow synthesis. The herein described development of the first two-dimensional HPLC-chip was realized by the integration of two different columns packed with reversed-phase and chiral stationary phase material on a microfluidic glass chip, coupled to mass spectrometry. Directed steering of the micro-flows at the joining transfer cross enabled a heart-cut operation mode to transfer the chiral compound of interest from the first to the second chromatographic dimension. This allows for an interference-free determination of the enantiomeric excess by seamless hyphenation to electrospray mass spectrometry. The application for rapid reaction optimization at micro-flow conditions is exemplarily shown for the asymmetric organocatalytic continuous micro-flow synthesis of warfarin.

Evaluation of Pressure Stable Chip-to-Tube Fittings Enabling High-Speed Chip-HPLC with Mass Spectrometric Detection.

Appropriate chip-to-tube interfacing is an enabling technology for high-pressure and high-speed liquid chromatography on chip. For this purpose, various approaches, to connect pressure resistant glass chips with HPLC pumps working at pressures of up to 500 bar, were examined. Three side-port and one top-port connection approach were evaluated with regard to pressure stability and extra column band broadening. A clamp-based top-port approach enabled chip-HPLC-MS analysis of herbicides at the highest pressure and speed.

HPLC-MS with Glass Chips Featuring Monolithically Integrated Electrospray Emitters of Different Geometries.

We present and evaluate an approach for coupling liquid chromatography in glass chips with mass spectrometry via fully integrated electrospray emitters. We developed an instrumental platform which allows a robust and reproducible operation of high performance chip chromatography coupled to mass spectrometry. A comparison of differently shaped emitters, from flat over edged to pulled geometries, revealed that all types performed equally well for typical nano-HPLC flow rates. At very low flow rates below 50 nL·min(-1) very sharp, pulled nanospray emitters turned out to be mandatory for the generation of a stable electrospray.

Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.

We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and ß-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.

Chip-based high-performance liquid chromatography for high-speed enantioseparations.

In this work, the first high-performance chiral liquid chromatography in packed microfluidic chips is presented. The chromatographic separation was performed on a column integrated into the microfluidic glass chip and packed with the particulate chiral stationary phase. Cellulose tris(3,5-dimethylphenylcarbamate) coated on 5-µm fully porous silica was used as chiral stationary phase material. Several racemic analytes including pharmaceutical products were baseline separated into their corresponding enantiomers under reversed-phase, polar organic and normal-phase conditions, demonstrating the versatility of the glass chip in the field of chiral separations. Van Deemter plots revealed a reduced plate height of 2.2 and a trend to enhanced mass transfer processes for solutes under low retention conditions. The utilization of very short column lengths of down to 12 mm led to ultrafast separations of enantiomers within 5 s.