Stefan Ohla

Asymmetric organocatalysis and analysis on a single microfluidic nanospray chip.

The miniaturization of chemical processes onto so-called labon-a-chip devices has gained significant importance in different fields of chemistry. Besides the advantages of enhanced portability, reduced reagent consumption, and improved safety, a characteristic feature of miniaturized platforms is the possibility to achieve higher reaction and analysis rates. From their roots in analytical sciences, microfluidic systems have received much attention over the past decade. At present, microfluidics is becoming increasingly popular in inorganic and organic chemistry, as syntheses are performed on chip-based microreactors or capillary-based microflow reactors. While diverse reactions have been performed in microfluidic chip devices with impressive results, the analytical characterization is, however, usually carried out off-chip by conventional macroscopic instruments. However, this approach does not exploit the promise and the full potential of chip technology, namely, the integration of different functionalities such as chemical synthesis and analysis on one single device. Thus, it is desirable to develop, in analogy to microelectronics, integrated chemical circuits as new chemical tools, for example, for catalyst screening or for online monitoring of biological processes. In previous work we demonstrated a first approach for integrating chemical reactions and analysis on a single microchip for the screening of enantioselective biocatalysts. Nevertheless, this system was limited to aqueous media and native fluorescent molecules. Therefore we intended to develop an advanced chip system with a wider applicability in synthetic chemistry, including the utilization of non-aqueous reaction media and a more general detection system. In this context, the coupling to mass spectrometry appears to be very attractive, as it provides additional structural information for substance identification. To demonstrate our concept, we focused on organocatalysis, which has been one of the most innovative research fields in synthetic chemistry over the past years. Recently, Odedra and Seeberger described a first miniaturized approach, performing organocatalysis in a microfluidic flow reactor chip. The reaction products were, however, analyzed offline by traditional HPLC. Herein we present the, to our knowledge, first asymmetric organocatalytic reaction on a single chip with integrated analysis. As a model system we chose the enantioselective vinylogous Mannich reaction published in 2008, which is catalyzed by chiral phosphoric acid. The alcoholic solvent mixture and the nonfluorescent reaction products in this synthesis are quite challenging for chip integration. Thus, it was necessary to adapt the reaction media to the aqueous separation electrolyte on-chip to enable an undistorted electrophoretic analysis of the reaction products. Therefore, we developed a new microfluidic chip design containing different functionalities, including a reaction structure for the organic synthesis, a structure for aqueous dilution of the reaction mixture, a cross section for injection, and a separation channel for chip electrophoresis. Furthermore, the chip layout includes an integrated nanoelectrospray emitter with makeup-flow channels for dead-volume free coupling to mass spectrometry at the end of the separation channel. A schematic drawing of the chip with 50 mm wide channels and a photo is shown in Figure 1. Reaction and analysis processes on the single chip were carried out as follows: initially, the reaction structure was filled with the alcoholic solvent mixture for synthesis; then the remaining structure was replenished with the aqueous separation electrolyte. Afterwards, each reactant solution

Chip-based separation devices coupled to mass spectrometry

The hyphenation of miniaturized separation techniques like chip electrophoresis or chip chromatography to mass spectrometry (MS) is a highly active research area in modern separation science. Such methods are particularly attractive for comprehensive analysis of complex biological samples. They can handle extremely low sample amounts, with low solvent consumption. Furthermore they provide unsurpassed analysis speed together with the prospect of integrating several functional elements on a single multifunctional platform. In this article we review the latest developments in this emerging field of technology and summarize recent trends to face current and future challenges in chip-based biochemical analysis.

Monitoring On‐Chip Pictet–Spengler Reactions by Integrated Analytical Separation and Label‐Free Time‐Resolved Fluorescence

High-throughput screening for optimal reaction conditions and the search for efficient catalysts is of eminent importance in the development of chemical processes and for expanding the spectrum of synthetic methodologies in chemistry. In this context we report a novel approach for a microfluidic chemical laboratory integrating organic synthesis, separation and time-resolved fluorescence detection on a single microchip. The feasibility of our integrated laboratory is demonstrated by monitoring the formation of tetrahydroisoquinoline derivatives by Pictet-Spengler condensation. After on-chip reaction the products and residual starting material were separated enantioselectively on the same chip. On-chip deep UV laser-induced fluorescence detection with time-correlated single photon counting was applied for compound assignment. The system was utilized to screen reaction conditions and various substrates for Pictet-Spengler reactions on-chip. Finally, the microlab was successfully applied to investigate enantioselective reactions using BINOL-based phosphoric acids as organocatalysts.

Microfluidic chips for chirality exploration

Microfluidic chips applied to the investigation of chirality allow reaction, separation and analysis of minuscule amounts of enantiomeric molecules. Chiral chip technology is employed in fields as diverse as pharmaceutical high throughput screening and deep space exploration missions.

Improving sensitivity in microchip electrophoresis coupled to ESI‐MS/MS on the example of a cardiac drug mixture

A comprehensive study for a sensitivity optimization in MCE with mass spectrometric detection is presented. As a text mixture, we chose a mixture of the cardiac drugs propranolol, bisoprolol, lidocaine, procaine and studied the effect of different chip layouts and experimental parameters with the aim of achieving both high sensitivity in MS detection and adequate chip electrophoretic separation. An important aspect was a comparison of microfluidic layouts containing various sheath‐flow channels with that avoiding sheath‐flow junctions on‐chip. We utilized glass chips with monolithically integrated nanospray emitter tips coupled dead volume‐free to an IT mass spectrometer running in fragmentation mode (MSn). With this setup, detection limits down to 0.6 ng/mL for the model compound propranolol were achieved.

Label-free analysis in chip electrophoresis applying deep UV fluorescence lifetime detection

Herein we introduce deep UV fluorescence lifetime detection in microfluidics applied for label‐free detection and identification of various aromatic analytes in chip electrophoresis. For this purpose, a frequency quadrupled Nd:YAG (neodymium‐doped yttrium aluminum garnet) picosecond laser at 266 nm was incorporated into an inverse fluorescence microscope setup with time‐correlated single photon counting detection. This allowed recording of photon timing with sub‐nanosecond precision. Thereby fluorescence decay curves are gathered on‐the‐fly and average lifetimes can be determined for each substance in the electropherogram. The aromatic compounds serotonin, propranolol, 3‐phenoxy‐1,2‐propanediol and tryptophan were electrophoretically separated using a fused‐silica microchip. Average lifetimes were independently determined for each compound via bi‐exponential tail fitting. Time‐correlated single photon counting also allows the discrimination of background fluorescence in the time domain. This results in improved signal‐to‐noise‐ratios as demonstrated for the above model analytes. Microchip electrophoretic separations with fluorescence lifetime detection were also performed with a protein mixture containing lysozyme, trypsinogen and chymotrypsinogen emphasizing the potential for biopolymer analysis.

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.

Label-free fluorescence detection of aromatic compounds in chip electrophoresis applying two-photon excitation and time-correlated single-photon counting.

In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

Surface modification of PDMS microfluidic devices by controlled sulfuric acid treatment and the application in chip electrophoresis

Herein, we present a straightforward surface modification technique for PDMS‐based microfluidic devices. The method takes advantage of the high reactivity of concentrated sulfuric acid to enhance the surface properties of PDMS bulk material. This results in alteration of the surface morphology and chemical composition that is in‐depth characterized by ATR‐FTIR, EDX, SEM, and XPS. In comparison to untreated PDMS, modified substrates exhibit a significantly reduced diffusive uptake of small organic molecules while retaining its low electroosmotic properties. This was demonstrated by exposing the channels of a microfluidic device to concentrated rhodamine B solution followed by fluorescence microscopy. The surface modification procedure was used to improve chip‐based electrophoretic separations. Separation efficiencies of FITC‐labeled amines/amino acids obtained in treated and untreated PDMS‐devices as well as in glass chips were compared. We obtained higher efficiencies in H2SO4 treated PDMS chips compared to untreated ones but lower efficiencies than those obtained in commercial microfluidic glass devices.

Rapid prototyping of microfluidic chips for dead-volume-free MS coupling

A fast and straightforward method to prototype microfluidic chip systems for dead-volume-free hyphenation to electrospray-ionisation mass spectrometry is presented. The developed approach based on liquid-phase lithography provides an inexpensive and reliable access to microfluidic chips for MS coupling which can be manufactured in any laboratory with low technical demands. The rapid prototyping approach enables the seamless integration of capillaries serving as electrospray emitters with negligible dead volume. The high versatility of the presented prototyping method and the applicability of a variety of chip-based devices in different fields of lab-on-a-chip technology are established for analytical separations by means of chip-electrochromatography–MS and for continuous-flow synthesis using microreactor technology with MS detection.