(209b) In-Flow Reaction Monitoring by NMR on Nanoliter Samples in a µ-Fluidic Chip

The growing interest in chemistry on small sample volumes performed on chip sets the demand for analytical methods which can handle corresponding volumes. Nuclear Magnetic Resonance (NMR) is a powerful analytical tool not only for determining complex (bio)molecular structures, but also for monitoring molecular dynamics and reaction kinetics. Despite its versatility, NMR analyses currently require relatively large amounts of the relevant molecules because of its intrinsic low sensitivity. We report on the design and measurement results of a NMR microchip that can analyze nanoliter samples. Initial measurements have shown a very high sensitivity combined with an excellent resolution.

NMR is used to study the properties of molecules containing nuclear spins by applying a magnetic field and observing the precessing of excited spins after irradiation with an electromagnetic field. The chemical shift of resonance peaks in NMR spectra is correlated to the electron configuration around a specific group in a molecule. This means that peaks in a spectrum can be directly assigned to a chemical group. Furhtermore, fine structures like J-coupling and dipolar coupling give a measure of the magnetic interaction and distance between two nuclei. NMR is a non invasive technique and provides direct quantitative information about the molecule under study.

Currently, NMR on small sample volumes is performed by means of small solenoids wrapped around a capillary [1], or planar coils on glass chips containing microfluidic channels, [2] [3] [4]. Despite many efforts, these approaches have not yet penetrated mainstream NMR spectroscopy. The most important problem is the fact that the nearby copper windings of the rf-coil tend to induce static field distortions that limits the resolution and (indirectly) the signal to noise performance. We designed a novel route towards microchip integrated NMR analysis, [5]. The basic element in the design is an rf stripline which can be defined in a single layer lithographic process and which is fully scalable to smaller dimensions. This stripline functions as the rf 'coil' which excites the spins. Due to the non-complicated copper geometry, the static field distortion is very low, resulting in a high resolution detection system. A microfluidic channel is integrated, which makes this chip feasible for in-flow measurements, (see figure 1). A probe was designed which contains the fluidical and electrical connections. A microreactor chip was implemented in this probe, which was directly connected to the NMR-chip via a short capillary, (see figures 2-4). All our measurements were carried out in a 600 MHz superconducting wide-bore magnet.

Single shot experiments showed unsurpassed sensitivity and resolution. Initial measurements with a non-integrated system containing only 12 nl sample have been carried out succesfully, [5]. In our fully integrated microfluidic chip containing 600 nl of pure ethanol, a Signal to Noise Ratio (SNR) of 5500 and a linewidth of 0.0017 ppm (1 Hz) full-width-at-half-maximum (FWHM) were achieved. Typical J-multiplets were resolved down to the baseline. This high SNR is especially necessary for in-flow kinetics monitoring, where samples stay for a limited time in the detection area.

NMR has been coupled to separation methods such as liquid chromatography (LC) [6] and capillary electrophoresis (CE) [7], allowing hyphenated analyses. Furthermore, microcoil NMR was used as a method to follow reaction kinetics, [4] [8] [9]. However, due to its intrinsically low sensitivity, these in-flow monitoring experiments remain to be a challenge, especially for low sample volumes. With our chip we were able to perform reaction monitoring on 600 nl sample volumes with minimal averaging (4x). For the monitoring experiments, a Micronit microreactor chip was implemented in the probe. Real-time monitoring of the carbamate formation from toluene diisocyanate (TDI) (0.5M in toluene) and pure ethanol in the microreactor chip by NMR was demonstrated. By changing the relative flow rates, the product formation could be studied as a function of ethanol stoichiometry. Figure 5 shows spectra recorded at different flow-rates. The product peaks clearly rise as a function of the relative compound concentration. From these spectra the conversion as function of the relative concentration can be fastly determined. Other reactions such as Diels-Alder reactions, alcohol protections and oxidation reactions, which are performed in microreactor chips are currently under study using the NMR microfluidic device.

References

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