How electrospray potentials can disrupt droplet microfluidics and how to prevent this.

A pressure-resistant microfluidic glass chip that integrates a packed-bed HPLC column, a droplet generator and a monolithic electrospray emitter is presented. This approach enables a seamless coupling of chip-HPLC and droplet microfluidics with ESI-MS detection. For the electrical contacting of the emitter, an electrode was integrated into the channel, which reaches up to the emitter tip. The incidental finding that under certain circumstances, the electrospray potential can strongly disturb the droplet microfluidics by electrowetting, was investigated in detail. Strategies to avoid this are evaluated and include electrical shielding and/or chip layouts, where the droplet generator is positioned at a long distance from the emitter.

On-chip integration of normal phase high-performance liquid chromatography and droplet microfluidics introducing ethylene glycol as polar continuous phase for the compartmentalization of n-heptane eluents.

We introduce an integrated chip-approach for a postcolumn segmentation of normal phase liquid chromatography. This is achieved by the seamless integration of a chiral NP-chip-HPLC column, a flow-focusing droplet generator, and a segmented flow channel in a single microfluidic glass chip. This allows a continuous segmentation of the eluent into droplets which are picked up and transported via an immiscible continuous phase. The combination of NP-chip-HPLC and droplet microfluidics enables to fractionate and conserve chromatographic runs for further downstream processes at picoliter scale. An essential aspect is the proper choice of the continuous phase concerning polarity, wetting properties and viscosity. For this purpose, ethylene glycol is introduced which facilitates this first combination of normal phase chromatography and droplet microfluidics. By adjusting the flow rates and varying the generator geometry, the size and frequency of the droplets could be precisely controlled.

Seamless Combination of High-Pressure Chip-HPLC and Droplet Microfluidics on an Integrated Microfluidic Glass Chip.

We introduce an approach for the integration of high performance liquid chromatography and droplet microfluidics on a single high-pressure resistant microfluidic glass chip. By coupling these two functionalities, separated analyte bands eluting from the HPLC column are fractionated into numerous droplets in a continuous flowing oil phase. The compartmentalization of the HPLC-eluate in a segmented flow was performed with droplet sizes of approximately 1 nL and with droplet frequencies reaching up to 45 Hz. This approach prevents peak dispersion and facilitates post column processing of chromatographic fractions on chip. A reliable generation of droplets is also possible in reversed phase gradient elution mode as demonstrated by applying a solvent gradient from 20% to 100% acetonitrile. A chip design with an incorporated dosing unit enabled the directed postcolumn addition of reagents to individual droplet fractions. The capability of this dosing function was successfully evidenced by post column addition of a reagent which quenches the fluorescence signal of the analytes. The chip-integration of gradient HPLC, fractionation, detection and post column addition of reagents opens up new avenues to perform multistep chemical processes on a single lab-on-a-chip device.

Continuous on-chip fluorescence labelling, free-flow isoelectric focusing and marker-free isoelectric point determination of proteins and peptides.

We present a microfluidic platform that contains a micro flow reactor for on-chip biomolecule labelling that is directly followed by a separation bed for continuous free-flow electrophoresis and has an integrated hydrogel-based near-infrared fluorescent pH sensor layer. Using this assembly, labelling of protein and peptide mixtures, their separation via free-flow isoelectric focusing and the determination of the isoelectric point (pI) of the separated products via the integrated sensor layer could be carried out within typically around 5 minutes. Spatially-resolved immobilization of fluidic and sensing structures was carried out via multistep photolithography. The assembly was characterized and optimized with respect to their fluidic and pH sensing properties and applied in the IEF of model proteins, peptides and a tryptic digest from physalaemine. We have therefore realized continuous sample preparation and preparative separation, analyte detection, process observation and analyte assignment capability based on pI on a single platform the size of a microscope slide.