Non-aqueous micro free-flow electrophoresis for continuous

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Non-aqueous micro free-flow electrophoresis for continuous separation of reaction mixtures in organic media Benjamin M Rudisch, Simon A Pfeiffer, David Geissler, Elisabeth Speckmeier, Andrea A Robitzki, Kirsten Zeitler, and Detlev Belder Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on April 29, 2019

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Analytical Chemistry

Non-aqueous micro free-flow electrophoresis for continuous separation of reaction mixtures in organic media Benjamin M. Rudischa†, Simon A. Pfeiffera, David Geisslera, Elisabeth Speckmeierb, Andrea A. Robitzkic, Kirsten Zeitlerb and Detlev Beldera* a) Institute of Analytical Chemistry, Leipzig University, Johannisallee 29, 04103 Leipzig, Germany b) Institute of Organic Chemistry, Leipzig University, Johannisallee 29, 04103 Leipzig, Germany c) Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany ABSTRACT: The continuous separation mechanism of micro free-flow electrophoresis (µFFE) is a straightforward, suitable tool for micro-scale purification of reaction mixtures. However, aqueous separation buffers and organic reaction solvents limit the applicability of this promising combination. Herein, we have explored non-aqueous micro free-flow electrophoresis for this purpose and present its suitability for continuous work-up of organic reactions performed in acetonitrile. After successful non-aqueous FFEseparation of organic dyes, the approach was applied to continuously recover the photocatalyst [Ru(bpy)3]2+ from a homogenous, acetonitrile-based reaction mixture. This approach opens up possibilities for further downstream processing of purified products and is also attractive for recycling of precious catalyst species.

Free-flow electrophoresis (FFE) is a continuous, electrophoretic separation method. Due to an applied electric field, a perpendicular flowing sample stream can be fractioned continuously. With the advent of microfluidics and the ongoing development of miniaturized separation devices, micro-FFE (µFFE) was established.1 The advantages of the smaller dimensions like increased heat dissipation, laminar flow control, reduced sample and buffer amount and essentially the ease of combination with other microfluidic building blocks have led to a wide range of applications for µFFE such as continuous process monitoring,2–5 online coupling6– 9 and microscale purification.10–13 However, almost all developed µFFE methods rely on aqueous buffer solutions. While this is preferred for biological samples, it is a limitation concerning the separation of chemical synthesis products in reaction mixtures. Most chemical reactions require organic solvents or even run at the exclusion of water. In fact, the incompatibility of solvents used in organic and inorganic synthesis is described to be the main problem when coupling micro reactors with µFFE.11 It would thereby be highly preferable to have a continuous separation, purification and fractionation method for pure organic media. This could advance the area of automated synthesis platforms14 joining continuous flow synthesis with continuous product separation. The lack of experience with non-aqueous µFFE is quite remarkable, considering the broad use of non-aqueous electrolytes in capillary electrophoresis (CE). Because for both, µFFE and CE, the underlying separation mechanism is the same, CE can be a valuable tool for buffer optimization in µFFE. Unlike in µFFE, non-aqueous CE (NACE) has first been disclosed shortly after the first publication of high-resolution CE by Jorgenson and Lukacs in 1983.15,16 Since then, a vast number of publications reporting separations in nonaqueous electrolytes have been published and were summarized in a two-part review by Kenndler.17 The most frequently used organic solvents in non-aqueous CE are methanol, acetonitrile and mixtures

thereof. They offer good miscibility with most organic solvents, which makes them highly interesting for syntheses and corresponding work-up and purification applications. In microchip capillary electrophoresis DMSO was also reported as a solvent of background electrolyte.18 Further advantages of non-aqueous over aqueous systems can be enhanced analyte solubility, a wider selectivity window by alteration of pKa19 and effective mobility values as well as a reduced electric current. In spite of the beneficial features and the high need to develop nonaqueous applications, up-to-date there are only a few examples described in the literature on the topic. So far, only one truly nonaqueous electrolyte system based on methanol has been reported in a study on analyte stream stability of Rhodamine dyes in µFFE using various additives in mainly aqueous electrolyte systems.20 The use of organic solvents in µFFE has in part been addressed by others,11,21 but distinctive examples for practical applications are still missing. In that regard, an interesting area of application for µFFE is the integration as a downstream continuous flow synthesis element in line with the idea of an integrated chemical circuit.22 While there is significant progress in downsizing chemical reactions from flasks to microchannels,23–25 processing, work-up and purification of reaction components, which continuously stream out of a microreactor, is still challenging. This is of special importance for catalytic reactions, where the separation of the catalyst from the reaction mixture is mandatory for downstream processes. Catalyst recovery and reuse is highly beneficial,26 not only in terms of sustainability but to avoid potential toxic contamination of the products of interest. This might be straightforward for heterogeneous catalytic reactions,27–30 though, in cases where a homogenous catalysis is advantageous, e.g. for photocatalytic transformations requiring high transmittance of the mixture for the irradiation, this often remains an unmet challenge.31,32 In this regard, µFFE is an attractive tool for continuous-flow synthesis and micro-preparative separation, because there are only few truly continuous-flow purification techniques available11,33 and

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under ongoing improvement.34–39 Previously described applications of micro-preparative separations using µFFE were shown for protein analysis,40–42 aptamer isolation,43 enrichment of bacteria,44 organelle45 and cell studies46,47 as well as quantum dot purification.12 While all of these examples targeted biological samples or particles, applications in the context of organic reactions are scarce. While this combination bears great potential, especially the label-free detection of reaction components by sophisticated optical techniques48,49 and in fusedsilica devices50 is promising. Based on our recent activity in the microscale purification of organic synthesis products,6,10 our focus in the current contribution is on the development of a solely organic solvent-based electrolyte system for µFFE. By applying the method for the extraction of a catalyst from an organic reaction mixture, we aim to demonstrate the applicability of µFFE as a continuous separation method for micro synthesis applications in purely organic samples for continuous-flow purification. In the first part, we present a µFFE buffer system with pure organic solvent based on acetonitrile used for the separation of dyes. The buffer was tested on a microfluidic device fabricated using rapid prototyping liquid phase lithography (LPL) to examine its behavior and stability. Thereupon, the setup was used to successfully separate a transition metal photocatalyst directly from the reaction mixture of a typical photoreaction.51

EXPERIMENTAL SECTION Materials. For chip production, (3-Methacryloyloxypropyl) trichlorosilane, Polyethyleneglycoldimethacrylate (PEG-DA, MW~257 and MW~575) and Dimethoxy-2-phenylacetophenone (DMPA) were purchased from Sigma-Aldrich (St. Louis, USA). Samples were prepared using Rhodamine B, Sulforhodamine B and Rhodamine 123, also from Sigma-Aldrich as well as Alexa Fluor 647-Succinimidylester from Thermo Fisher Scientific (Waltham, USA). Acetonitrile (HPLC-grade) and sodium acetate from Merck (Darmstadt, Germany), acetic acid, sodium chloride from Carl Roth (Karlsruhe, Germany) and Triton X-100 (Sigma-Aldrich) were used for electrolyte preparation. The photocatalyst tris(2,2´-bipyridyl)dichlororuthenium(II)hexahydrate ([Ru(bpy)3]Cl2×6H2O) was purchased from ABCR chemicals (Strem) (Karlsruhe, Germany). Synthetic precursors were prepared according to known procedures 50 (see supporting information). µChip Manufacturing. Chips were produced using liquid phase lithography to create a structured polymer layer between two microscope glass slides (Carl Roth, Karlsruhe, Germany) like described elsewhere.52 Holes were sandblasted (Point II, Barth, Königsbach Stein, Germany) into a lid-slide, providing an interface for microfluidic connections. The glass surfaces of both, lid and bottom slide, were modified with (3-methacryloyloxypropyl) trichlorosilane to allow permanent bonding between glasses and polymer layer. To create the structure, a mixture of 99% (w/w) PEGDA (MW~257) and 1% (w/w) DMPA was filled between the two slides separated by spacers of adhesive copper foil tape (CFT-50, Conrad, Germany) resulting in a gap height of 55 µm. A negative photomask printed on foil (DTP-System-Studio, Leipzig, Germany) was aligned on top of the lid and the chip was exposed to UV-light for 1.3 s using a FE5 Flood Exposure (SüssMicroTec, Munich, Germany) equipped with an Hg lamp (13.6 mW/cm2 at 365 nm). The remaining liquid prepolymer was removed under reduced pressure. The structure was rinsed with ethanol before illuminating the chip again for 1.3 s as described above. To the four innermost holes on the lid-side silicone tube pieces (60°Shore, ESSKA, Germany) were attached with silicone rubber (E41, Wacker, Germany).

PEG-DA hydrogel walls were used to partition electrode channels from the separation bed. Therefore, the chip was filled with a solution of 50% (w/w) PEG-DA (MW~575) and 1% (w/w) DMPA in water and hydrogel walls were polymerized using an IX71 microscope with a 20x UPlan FLN objective (both from Olympus Europa, Hamburg, Germany) with a UV-LED (M365L2 with DC2100, Thorlabs, Newton, USA). The remaining prepolymer solution was removed under reduced pressure before the chip was rinsed with isopropanol. For storage, the chips were filled with and kept in water. Experimental setup. For operation, the chip was mounted on an IX71 microscope equipped with a 2x UPlanFLN objective and a FITC-Cy3-Cy5 filter set (Chroma, USA). As excitation source, a UULS100HG Hg lamp (Olympus) was used. Fluorescence light was collected by the same objective and transmitted onto a pco.1600 MOD CCD-camera (PCO AG, Kelheim, Germany) operated with µManager. To apply defined flow rates, the chip was connected to 1 and 2.5 mL glass syringes (ILS, Stuetzerbach, Germany) mounted in a neMESYS syringe pump device (cetoni, Korbusen, Germany) using 150 µm PEEK Tubing capillaries and N-12304 ferrules (Postnova, Landsberg, Germany) on the chip-side. The injection was performed using a 6-port valve (Knauer, Berlin, Germany) with a 75µL sample loop. For the electrode channels, 0.5 mm ID TFE Teflon tubes (Supelco, USA) were used. Electrical fields were applied via internal Pt electrodes sputtered onto the bottom slide before chip manufacturing (catalyst recovery) or internal electrodes consisting of a Cu braid polymerized into the electrode electrolyte channel (dye separations). As power source, a HVS4 D6000 highvoltage-sequencer (LabSmith, Livermore, USA) was used. UV-Vis spectra were recorded on a V-650 spectrophotometer (JASCO, Groß-Umstadt, Germany). Sample preparation. Samples were prepared using 10 mM stock solutions of Rhodamine B, Sulforhodamine B, Rhodamine 123 and Alexa Fluor 647-Succinimidylester in acetonitrile. The used dye mixtures were prepared in acetonitrile and afterwards diluted (1:10 v/v) with background electrolyte (BGE). As BGE, a solution of 1 M acetic acid and 10 mM sodium acetate in acetonitrile was used in all experiments. For the electrode channels (separated by hydrogel walls from the µFFE bed) an aqueous electrolyte solution of 1 M sodium chloride with 0.25 mM Triton X-100 was used. Separation of photocatalytic reaction mixture. In an oven-dried schlenk tube equipped with a magnetic stir bar 127 mg 2-oxo-1,2diphenylethyl acetate (0.50 mmol, 1.0 equiv), 7.5 mg tris(2,2´bipyridyl)-dichlororuthenium(II)hexahydrate (0.010 mmol, 2 mol%), 152 mg dimethyl-2,6-dimethyl-1,4-dihydropyridine-3,5dicarboxylate (0.60 mmol, 1.2 equiv) were dissolved in 3 ml acetonitrile. 60.7 mg triethylamine (0.60 mmol, 1.2 equiv) were added under nitrogen and the reaction mixture was degassed via the freeze-pump-thaw technique (3 cycles). Then the tube was irradiated with blue LEDs (8 OSLON LD H9GP deep blue (OSRAM, 455 nm) attached to a heat sink) for 1 h at room temperature to typically reach full conversion. This reaction mixture was then directly injected in the µFFE-device. Synthetic details, including the preparation of starting material and further information on the procedure is available in the supporting information. Safety concerns. Special care has to be taken, when working with high voltage sources. Furthermore, safety specifications and precautions have to be followed, while using organic solvents.

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RESULTS AND DISCUSSION Frequently used solvent systems for so-called non aqueous capillary electrophoresis are based on methanol, ethanol, acetonitrile, mixtures thereof, and often contain variable amounts of water.17,53,54 With our objective in mind of separating constituents of organic synthesis mixtures, we ruled out any water content and chose acetonitrile as the base solvent for initial studies. Acetonitrile is an attractive solvent for non-aqueous electrophoresis due to its physicochemical properties, e.g., the higher ratio of dielectric constant and viscosity compared to methanol or water. Furthermore, the lower surface tension and higher gas solubility of acetonitrile compared to water can promote the removal of gas bubbles caused by electrolysis, which is often an issue in free flow electrophoresis.55,56 However, in our setup, the resulting gas bubbles are excluded from the µFFE bed via hydrogel walls. In these electrode channels, we used an aqueous solution with high ionic strength to minimize the potential drop across the separation bed. Although, strictly speaking, pH values are only defined in aqueous solutions, similar concepts have been discussed for non-aqueous electrolyte systems.17,19,57 In the current work, we chose an acidic electrolyte to keep the reaction products of the photocatalytic deoxygenation uncharged. Therefore we supplemented the solvent with acetic acid and sodium acetate, which is widely employed as a background electrolyte (BGE) system in NACE separations.17 Our first investigation concerned the long-term stability of our developed µFFE devices against the non-aqueous electrolyte conditions. To evaluate the solvent resistance of the polyethylene glycol sandwich chips and the basic functionality of the BGE for

Figure 1. a) Layout of the µFFE device, flow direction from the sample inlet (yellow), BGE inlets (blue) and electrode channels (green) with hydrogel walls (red), outlets are numbered (1-4) where fractions were collected, b) Separation of Rhodamine B, Sulforhodamine B and Rhodamine 6G in an acetonitrile based electrolyte with 1M acetic acid and 10 mM sodium acetate at 2.1 kV (210 µA) and a linear flow velocity of 0.7 mm/s, below is shown a relative fluorescence intensity profile 2.5 mm behind the sample inlet, resolutions achieved are 3.0 and 1.1 for Rhodamine B/Sulforhodamine B and Rhodamine B/Rhodamine 6G, respectively.

non-aqueous separations, we investigated a model separation of fluorescent dyes. In figure 1a, the chip setup and the corresponding fluid streams are depicted schematically. The sample was introduced in the separation bed through two inlet holes prior to the meandering reaction channel. The BGE dissolved in acetonitrile was pumped in the channels flanking the sample stream. The electric field was established through the outer electrode channels, which were separated by hydrogel walls from the separation area and constantly flushed with an aqueous solution of 1 M sodium chloride with 0.25 mM Triton X100. After passing the µFFE bed, separated fractions can be collected at the outlets 1 to 4. For an initial proof of concept, a sample mixture consisting of Rhodamine B (RhB), Sulforhodamine B (SRhB) and Rhodamine 6G (Rh6G) was used. The separation of the mixture in a non-aqueous, acetonitrile based electrolyte is shown in figure 1b. The concentration of the dyes was 500 µM each and the applied voltage was 2.1 kV (210 µA). As shown, all three constituents could be separated, which proves the fundamental feasibility of the chosen BGE and solvent as a separation medium for non-aqueous µFFE. With regards to the compatibility of the microchip wall material (polyethylene glycol), optical inspection of the chip revealed minor damage of the channel walls after prolonged contact with acetonitrile. Especially the interface of glass and polyethylene glycol visually showed dissolution of the wall material after prolonged use. The limited stability of (in-part) polymeric devices towards organic media can be

Figure 2. Long-term stability experiments (40 minutes) of a µFFE separation of Sulforhodamine B (SRhB), Rhodamine 123 (Rh123) and Alexa Fluor 647 (AF647) in an acetonitrile based electrolyte with 1M acetic acid and 10 mM sodium acetate at 2.1 kV (210 µA), a) continuously acquired fluorescence intensity profiles 2.5 mm behind the sample inlet over 40 minutes, b) position of the peak maxima over time.

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Figure 3. Scheme of the doubly positive charged photocatalyst and the investigated photocatalytic deoxygenation reaction.

problematic for non-aqueous separations. Therefore, full-body glass or fused silica microfluidic substrates would be preferable to manufacture long term stable devices. To systematically study the long term stability of the separation and to assess the possibility to perform micro-preparative tasks, a separation of a second mixture of dyes (SRhB, Rhodamine 123 Rh123) and Alexa Fluor 647 succimidyl ester (AF647)) was investigated for 40 minutes. Therefore, the sample was injected into the device and by adjusting the separation voltage and flow velocity, the separated bands were positioned in front of the outlets of the device. Subsequently, fluorescence intensity profiles shortly before the outlets were collected over time. The results can be seen in figure 2. The voltage and current during the separation were monitored over 40 min until all of the 75 µL sample-loop passed the device, which also defined the measurement endpoint for our developed setup. As can be seen in figure 2a, a stable separation of the dyes over the measurement time could be achieved. To evaluate the stability, the band positions were monitored as well. Therefore, a cross section 2 mm in front of the outlet channels was selected, and the position of the band maxima was traced over time (figure 2b). The device showed excellent stability with less than 5% change in relative position, which corresponds to about 200 µm range of fluctuation. This is by far less than the 2 mm width each outlet channel can cover. Furthermore, we investigated diffusive band-broadening effects on SRhB and RhB by comparing peak widths closely behind sample inlet as well as in front of the device outlets within one run. We found a broadening effect of 65 % for SRhB and 95 % for RhB (more detailed information can be found in figure SI-5). However, since all our devices used fraction outlets much wider than the broadened bands, this effect had no impact on fraction purity. For our proof-of-concept study, the experiment duration was defined by the volume of the employed sample loop and syringes. The prototyped microfluidic device itself could be used up to two days after which the hydrogel walls started to decompose (see figure SI-4). Separation of a photocatalyst from a homogenous reaction mixture. After our successful proof-of-concept studies, the developed method was applied to the purification of a reaction mixture. Likewise, the continuous non-aqueous separation conditions were employed to extract a photocatalyst from a homogeneous reaction mixture. Many of the catalysts used for visible light photocatalysis are transition metal-based and permanently charged.58 Furthermore, when excited by visible light, their intrinsic fluorescence greatly facilitates and allows for easy monitoring of the separation. The separation and especially the recycling of transition-metal based catalysts is of great interest since it would not just contribute to reducing costs (especially for expensive iridium- and ruthenium-based catalysts), but also would allow for improved sustainability of the process and could further help to remove undesired metal contamination in the synthetic products.59 As the separation and recovery in heterogeneous catalysis

Figure 4. a) Deflection of the pure catalyst at 400 V (160 µA), b) Separation of the reaction mixture at 375 V (160 µA) with view on outlet 1, where the catalyst is retrieved. Each measurement in an acetonitrile based electrolyte with 1M acetic acid and 10 mM sodium acetate, c), d) Corresponding relative fluorescence intensity profiles at the chip position indicated as white, dashed lines in the images above (Figure c and d were recorded at different exposure times).

is rather straightforward, catalyst recycling is often troublesome or impossible in homogenous catalysis, which is why heterogeneous catalysis is often preferred in industry. For our proof-of-concept study, we chose the visible-light promoted deoxygenation of multifaceted benzoin derivatives using ([Ru(bpy)3]2+ as an effective photocatalyst, conducted in acetonitrile as solvent.51 In combination with triethylamine and Hantzsch ester as reductive quencher and H-atom donor α-carbonyl located C–O σ-bonds can reductively be cleaved to yield the corresponding deoxybenzoin as wells as the carboxylate. The specific reaction is shown in figure 3. As the catalyst species in this reaction is double positively charged, we envisioned its facile separation from the complex final reaction mixture and the products.

Figure 5. UV/Vis spectra of the fractions of the reaction mixture separation collected at the corresponding outlets 1-4. In fraction 1 the expected spectrum of catalyst is found, whereas the characteristic peak at 450 nm is missing in fraction 2-4. Correspondingly, the spectrum of the product mixture (exempt from the metal catalyst) is assigned to fraction 3.

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Analytical Chemistry To study the feasibility of the approach, we first injected the pure catalyst in the µFFE device. In figure 4a the corresponding deflection of the sample at 400V can be seen. The catalyst is easily deflected due to its charge. After this successful first step, we injected the solely organic acetonitrile-based reaction mixture (after 1 h reaction time) from the flask in the µFFE device and adjusted the flow rates and separation voltages to guide the fluorescent catalyst stream towards outlet 1 (figure 4b). To verify the success of the separation, fractions of the outlets 1-4 (see figure 1a) were collected in glass vials outside the chip and analyzed by absorbance spectroscopy and gas chromatography. For comparison, spectra of pure catalyst in the electrolyte, pure starting materials without catalyst as well as the reaction mixture before injected into the µFFE device were collected beforehand. The absorbance spectra of the fractions collected are shown in figure 5. Further additional spectra and gas chromatographic analysis of the corresponding fractions can be found in the supporting information (figures SI-1 and SI-2). As can be seen in figure 5, for the fraction collected at outlet 1, to which the catalyst stream was deflected, a spectrum with two distinct peaks at 287 nm and 451 nm is acquired. This corresponds nicely to the premeasured spectra of the pure catalyst as well as respective spectra described in literature.60 Using the absorption maximum at 451 nm and the volume of the fraction, a recovery rate of 83 % for the catalyst was calculated (see SI-3 for further information). On the other hand, in fractions 2-4 no absorption peaks are found at these wavelengths. The µFFE setup was adjusted in such a way, that the neutral reaction mixture was guided onto outlet 3. Hence, as expected, only for fraction 3 a significant peak at 246 nm is measured, whereas for the samples from outlet 2 and 4 only minor absorption is detected. Notably, the characteristic peak at 451 nm, measured for the catalyst species, is completely missing in the spectra of the other channel outlets (product mixture). These results verify that we could successfully separate the catalyst of a photoreaction from a homogenous reaction mixture and thus develop a non-aqueous µFFE method for microscale purification.

CONCLUSION We developed a microfluidic FFE approach for non-aqueous electrophoretic separations in a rapid-prototyped glass-polymer hybrid chip. The continuous and stable method was successfully applied for the recovery of a photo-catalyst from a homogenous reaction mixture. With our method, we were able to retrieve fractions of the reaction mixture and verify an effective separation of catalyst and reaction products. The presented method is a promising new method in the toolkit for micro-preparative lab-on-a-chip devices. The applicability of organic solvents enables µFFE for a wide range of reaction conditions. To improve the solvent stability full body glass chips should be preferable. By the use of fused silica material, a wide range of reaction products can be label-free tracked and separated. As a subsequent step of on-chip reactions, the continuous separation of catalyst and reaction products allows the streamlined addition of further functionalities and analytics downstream like chip chromatography or mass spectrometry.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. UV-Vis spectra and GC measurements, μFFE chips and separation, Synthesis protocol and NMR data

AUTHOR INFORMATION Corresponding Author *[email protected]

Present Address †Institut für Physikalische und Theoretische Chemie (IPTC), Universität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was funded by the Deutsche Forschungsgemeinschaft (DFG) FOR2177.

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