Continuous Flow Biocatalytic Reductive Amination by Co-Entrapping Dehydrogenases with Agarose Gel in a 3D-Printed Mould Reactor.

Herein, we show how the merge of biocatalysis with flow chemistry aided by 3D-printing technologies can facilitate organic synthesis. This concept was exemplified for the reductive amination of benzaldehyde catalysed by co-immobilised amine dehydrogenase and formate dehydrogenase in a continuous flow micro-reactor. For this purpose, we investigated enzyme co-immobilisation by covalent binding, or ion-affinity binding, or entrapment. Entrapment in an agarose hydrogel turned out to be the most promising solution for this biocatalytic reaction. Therefore, we developed a scalable and customisable approach whereby an agarose hydrogel containing the co-entrapped dehydrogenases was cast in a 3D-printed mould. The reactor was applied to the reductive amination of benzaldehyde in continuous flow over 120 h and afforded 47 % analytical yield and a space-time yield of 7.4 g L day-1 using 0.03 mol% biocatalysts loading. This work also exemplifies how rapid prototyping of enzymatic reactions in flow can be achieved through 3D-printing technology.

Introduction of Octadecyl-Bonded Porous Particles in 3D-Printed Transparent Housings with Multiple Outlets.

Microfluidic devices for comprehensive three-dimensional spatial liquid chromatography will ultimately require a body of stationary phase with multiple in- and outlets. In the present work, 3D printing with a transparent polymer resin was used to create a simplified device that can be seen as a unit cell for an eventual three-dimensional separation system. Complete packing of the device with 5-µm C18 particles was achieved, with reasonable permeability. The packing process could be elegantly monitored from the pressure profile, which implies that optical transparency may not be required for future devices. The effluent flow was different for each of the four outlets of the device, but all flows were highly repeatable, suggesting that correction for flow-rate variations is possible. The investigation into flow patterns through the device was supported by computational-fluid-dynamics simulations. A proof-of-principle separation of four standard peptides is described, with mass-spectrometric detection for each of the four channels separately. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04156-w.

Optimizing design and employing permeability differences to achieve flow confinement in devices for spatial multidimensional liquid chromatography.

In spatial multi-dimensional liquid chromatography (LC) devices the flow of each dimension has to remain in the corresponding region, otherwise the separation efficiency is undermined. Adequate flow-confinement measures are necessary. Here, the use of permeability differences across different compartments of spatial two-dimensional (2D) and three-dimensional (3D) LC devices as a method to guide fluid flow and reduce analyte loss during the first, second- and third-dimension development was investigated with computational fluid dynamics (CFD) simulations. In case of 2DLC devices, it was shown that porous barriers with a permeability on the order of 10-12 m2 suffice to keep the total sample spillage from an open 1D channel under 1%. In case of 3DLC devices, it was shown that flow confinement could be achieved using an open 1D channel in combination with a highly-permeable monolith (permeability on the order of 10-12 m2) in the second-dimension (2D) and a less permeable packing with a permeability on the order of 10-15 m2 (e.g. 1 µm particles) in the third-dimension (3D). Additionally, the impact of the 3D flow-distributor has been studied and a novel design, capable of limiting the spillage to the other dimensions to the absolute minimum, is proposed.

Experimental and numerical study of band-broadening effects associated with analyte transfer in microfluidic devices for spatial two-dimensional liquid chromatography created by additive manufacturing.

Conventional one-dimensional column-based liquid chromatographic (LC) systems do not offer sufficient separation power for the analysis of complex mixtures. Column-based comprehensive two-dimensional liquid chromatography offers a higher separation power, yet suffers from instrumental complexity and long analysis times. Spatial two-dimensional liquid chromatography can be considered as an alternative to column-based approaches. The peak capacity of the system is ideally the product of the peak capacities of the two dimensions, yet the analysis time remains relatively short due to parallel second-dimension separations. Aspects affecting the separation efficiency of this type of systems include flow distribution to homogeneously distribute the mobile phase for the second-dimension (2D) separation, flow confinement during the first-dimension (1D) separation, and band-broadening effects during analyte transfer from the 1D separation channel to the 2D separation area. In this study, the synergy between computational fluid dynamics (CFD) simulations and rapid prototyping was exploited to address band broadening during the 2D development and analyte transfer from 1D to 2D. Microfluidic devices for spatial two-dimensional liquid chromatography were designed, simulated, 3D-printed and tested. The effects of presence and thickness of spacers in the 2D separation area were addressed and leaving these out proved to be the most efficient solution regarding band broadening reduction. The presence of a stationary-phase material in the 1D channel had a great effect on the analyte transfer from the 1D to the 2D and the resulting band broadening. Finally, pressure limit of the fabricated devices and printability are discussed.

Two-dimensional insertable separation tool (TWIST) for flow confinement in spatial separations.

Spatial comprehensive two-dimensional liquid chromatography (xLC×xLC) may be an efficient approach to achieve high peak capacities in relatively short analysis times, thanks to parallel second-dimension separations [1,2]. A key issue to reach the potential of xLC×xLC is to achieve adequate flow control and confinement of the analytes to the desired regions, i.e. confinement in the first-dimension direction and subsequently homogeneous flow in the second dimension. To achieve these goals we propose the TWIST concept (TWo-dimensional Insertable Separation Tool), a modular device that includes an internal first-dimension (1D) part that is cylindrical and rotatable. This internal part features a series of through-holes, each of which is perpendicular to the direction of the 1D flow. The internal part is inserted in the cylindrical casing of the external part. The internal diameter of the casing is marginally larger than the external diameter of the internal part. The external part also comprises a flow distributor and second-dimension (2D) channels. During the 1D injection and development, the channel is placed in a position where the through-holes are facing the wall of the external part, such that the liquid remains confined within the 1D channel. Thereafter, to realize the transfer to the second dimension (2D injection), the 1D channel is rotated, so that the holes of the internal part are aligned with the holes on the external part, allowing a transversal flow of the 2D mobile phase from the distributor through the 1D channel and eventually into the 2D area.

Thermal decomposition of hydroxylamine: isoperibolic calorimetric measurements at different conditions.

Thermal decomposition of hydroxylamine, NH2OH, was responsible for two serious accidents. However, its reactive behavior and the synergy of factors affecting its decomposition are not being understood. In this work, the global enthalpy of hydroxylamine decomposition has been measured in the temperature range of 130-150 °C employing isoperibolic calorimetry. Measurements were performed in a metal reactor, employing 30-80 ml solutions containing 1.4-20 g of pure hydroxylamine (2.8-40 g of the supplied reagent). The measurements showed that increased concentration or temperature, results in higher global enthalpies of reaction per unit mass of reactant. At 150 °C, specific enthalpies as high as 8 kJ per gram of hydroxylamine were measured, although in general they were in the range of 3-5 kJ g(-1). The accurate measurement of the generated heat was proven to be a cumbersome task as (a) it is difficult to identify the end of decomposition, which after a fast initial stage, proceeds very slowly, especially at lower temperatures and (b) the environment of gases affects the reaction rate.