Integration of Microreactors with Spectroscopic Detection for Online

Review pubs.acs.org/IECR

Integration of Microreactors with Spectroscopic Detection for Online Reaction Monitoring and Catalyst Characterization Jun Yue,† Jaap C. Schouten,† and T. Alexander Nijhuis*,† †

Laboratory of Chemical Reactor Engineering, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. ABSTRACT: Microreactor technology has gained significant popularity in the chemical and process industry in the past decade. The development of microreactors either as innovative production units for chemical synthesis or as promising laboratory tools for reaction and kinetic studies relies highly on the capability of performing online analyses, which opens great opportunities for the integration of spectroscopic detection techniques. This paper gives an overview of the state-of-the-art in the combination of microreactors with spectroscopic analyses for online reaction monitoring and catalyst characterization. In this upcoming field, many studies have been carried out combining fluorescence, ultraviolet−visible, infrared, Raman, X-ray, and nuclear magnetic resonance spectroscopy. Current research progress is reviewed, with emphasis on the existing integration schemes and selected application examples that demonstrate the potential of online spectroscopic detection for rapid microreactor process analysis and optimization. An outlook on the future development in this area is also presented.

1. INTRODUCTION Microreactor technology as an efficient tool for chemical synthesis has attracted significant research attention over the past decade.1−4 There are two main driving forces for the application of microreactor technology in the chemical and process industry. The first driving force can be found in process intensification or advanced processing, which takes advantage of the excellent heat and mass transfer characteristics in microreactors.5 The enhanced rate of heat and mass transfer is capable of providing a wider operating window and a tighter process control. The former capability, for example, allows for safely operating a process in a regime which would be within the explosive regime in conventional reactors6−8 and the latter permits, for example, a higher selectivity of the target product for a reaction by preventing undesired consecutive or side reactions.9 The potential cost benefits with the move from labor-intensive batch processes to continuous (automated) flow processes also represent a big incentive for the application of microreactor technology, particularly in the fine chemical and pharmaceutical production.10 The second driving force lies in the fact that microreactor technology facilitates the switch from a large-scale production on centralized locations to a distributed production at multiple locations.11 This switch has the direct consequence of gaining more flexibility and safety. For instance, the transportation and storage of hazardous or unstable chemicals can be avoided by utilizing on-site microreactorbased production units. Furthermore, with this new production mode it is possible to construct a flexible multipurpose microplant which can produce chemicals on demand or, even more complex, produce different pharmaceutical products from a series of common starting materials. A main focal point of research into microreactor technology has been the development of microreactors for use in a variety of chemical reactions. The vast number of reactions performed in microreactors has been detailed in many reviews.2,4,12−20 Although the application potential of microreactors has been well demonstrated in terms of the merits addressed above, © 2012 American Chemical Society

there is a continuous requirement in the integration of analytical tools for online process monitoring.21 This opens a new way for a faster and more reliable process optimization in comparison with the traditional ‘black-box’ approach in which the microreactor performance is evaluated based on the off-line product analysis without the real-time acquisition of the reaction information inside the microreactor itself. For the development of small-scale autonomous microreactor-based production plants, online process monitoring is essential to verify if the desired product quality is being met, to verify if the feed is up to standards, and also to have information on the state of solid catalysts (if present) in the microreactor (e.g., if the catalyst is performing well or deactivated). This requirement is equally important when implementing microreactor technology on a pilot or industrial scale as this technology adopts a numbering-up methodology to increase the product capacity, that is, by using one or a number of microreactor stacks comprising a multitude of parallel microchannel units performing chemical tasks simultaneously. In the latter case, it is in general helpful to apply an online detection method as a diagnosis tool to monitor the working status of individual units, for example, to identify those units in which the reaction process does not go smoothly possibly arising from local catalyst deactivation, flow maldistribution, or even channel blockage. This would allow for a quick process diagnosis and a local replacement of the malfunctioned units can thereby solve the problem without a need to shut down the whole system, which translates into economic profits. The online integration of detection techniques is also essential when using microreactor technology as a powerful laboratory tool for reaction and kinetic studies. The advantages of microreactor technology in the precise control of the process Received: Revised: Accepted: Published: 14583

May 14, 2012 September 13, 2012 October 3, 2012 October 3, 2012 dx.doi.org/10.1021/ie301258j | Ind. Eng. Chem. Res. 2012, 51, 14583−14609

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Table 1. List of Review Articles Published on the Integration of Spectroscopic Analyses into μTAS spectroscopic methods

a

authors

year published

fluorescence

UV−vis

IR

Raman

NMR

othersa

Swinney and Bornhop32 Bruin33 Schwarz and Hauser30 de Beer et al.34 Mogensen et al.31 Uchiyama et al.35 van Bentum et al.36 Viskari and Landers37 Yi et al.38 Kuswandi et al.39 Götz and Karst40 Myers and Lee41 Hunt and Wilkinson42 Chen and Choo43 Schulze and Belder44 Huh et al.45 Harel46 Malic et al.47 Yin et al.48 Wu and Gu49 März et al.50 Gökay and Albert51 Jones and Larive52

2000 2000 2001 2002 2004 2004 2004 2006 2006 2007 2007 2008 2008 2008 2009 2009 2009 2010 2011 2011 2011 2012 2012

√ √ √ √ √ √ √ √ √ √ √ √ √ -

√ √ √ √ √ √ √ √ √ √ √ √ -

√ √ -

√ √ √ √ √ √ √ √ √ √ √ -

√ √ √ √ √ √ √ √

√ √ √ √ √ √ √ √ √ √ √ √ √ √ -

Chemiluminesence, refractive index detection, mass spectrometry, and other methods.

analyses into μTAS, as shown in Table 1. While it is admitted that the existing designs and integration schemes developed in the area of μTAS (as detailed in those reviews listed in Table 1) can be transferred to microreactor applications, further research challenges have to be addressed in the latter case during the integration of spectroscopic detection if one considers the fact that reaction processes carried out in microreactors are usually subject to elevated temperature or pressure, high throughput, and concentrated chemicals that are toxic or hazardous in nature.21 An increasing number of papers have appeared in recent years to highlight the obvious benefit of integrating spectroscopic measurements into continuous microreactor processes that such integration can tell a live story of what is occurring inside a microreactor, that is, not only the fluid compositions going in and out but also the state of the solid catalyst present in the microreactor.3,21−26,53−56 The purpose of this review is therefore to provide an overview of the spectroscopic detection techniques that are currently available or being developed in combination with microreactors for online reaction monitoring as well as catalyst characterization. The coupling of six representative spectroscopic analyses with microreactors are presented, which consist of fluorescence, UV−vis, IR, Raman, X-ray, and NMR spectroscopy. These spectroscopic methods are widely used for concentration measurements and species detection in conventional reactors, and now it becomes also important to explore their full potential in microreactor applications. An outlook is given about the future opportunities for the integration of spectroscopic analyses into microreactors. It may be noted that selected examples of microreactors integrated with spectroscopic analyses for online reaction monitoring have been briefly discussed in two excellent reviews by Löbbecke21 and Jensen’s group.56 The current review aims to provide a comprehensive overview of the-state-of-the-art in this

conditions, such as well-defined flow pattern, efficient mixing, uniform temperature distribution, and safe operation in a wide process window, make it an attractive means for the extraction of kinetic information. Apart from a fast data acquisition, online reaction monitoring can reveal the presence of short-lived species or unstable intermediates that might not be observed through off-line analysis.21 The capabilities of microfabricated reactors in investigating solid catalysts under realistic reaction conditions have been demonstrated recently thanks to the online integration of spectroscopic analyses.22−26 Such in situ investigation, in combination with the traditional ex-situ catalyst characterization under nonreaction conditions, can better elucidate the mechanistic aspects of catalytic processes, which thereby accelerates the process of catalyst development and also promotes the application of microreactor technology in solid-catalyzed reactions. In parallel to the burgeoning development of microreactor technology, there is a rapid growing area of micrototal analysis systems (μTAS) also called ‘lab on a chip’ which can be traced back to 1970s and has gained its momentum for significant growth since the early 1990s.27−29 Various detection techniques have been developed and integrated with miniaturized analytical devices to enable a direct online observation of physical or chemical events, mainly including optical, electrochemical, mass spectrometric methods, and some other unconventional methods.30,31 A large amount of these detection methods rely on spectroscopic analyses for measuring the analyte concentration, ranging from the widely used fluorescence spectroscopy to the increasingly applied ultraviolet−visible (UV−vis) absorbance, infrared (IR), Raman, and nuclear magnetic resonance (NMR) spectroscopy, where continuous research efforts are being taken to improve the system performance in terms of sensitivity, portability, and cost-effectiveness. This is supported by the appearance of many review articles from year 2000 onward detailing the integration of these spectroscopic 14584

dx.doi.org/10.1021/ie301258j | Ind. Eng. Chem. Res. 2012, 51, 14583−14609


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field and further addresses the recent advances in the coupling of spectroscopic analyses for in situ catalyst characterization in microreactors, where different aspects covering spectroscopic integration scheme and application examples are detailed. Although this review does not cover other spectroscopic methods (e.g., mass spectrometry) and process analytical techniques (e.g., chromatography), their capabilities of being integrated into microreactor processes for rapid online reaction monitoring or catalyst characterization are well recognized here and have been discussed already by many researchers.57−65 Several reviews addressing this integration are also available.21,56,66,67

a laser source is coupled to an inverted microscope within which a set of two filters and a beam splitter guide the incident excitation light into the microchannel through an objective. The fluorescence light from within the microchannel is then passed through the microscope and imaged onto a charge-coupled device (CCD) camera, while the reflected excitation light is blocked from reaching the camera by a filter. As the laser sheet illuminates the complete microchannel volume, the recorded fluorescence intensity is an average along the optical path. To obtain the species concentration field in three dimensions, the use of confocal fluorescence microscopy is required, which allows collecting the excited fluorescence light from various focus planes across the microchannel.72 The on-chip integration of optical components to excite and collect the fluorescence signal represents a further step toward the development of monolithically integrated, portable microchemical systems. This will eliminate the need of bulky optical equipment such as the lenses and microscopes used in traditional fluorescence measurements. A straightforward way of integration can be realized by embedding the optical fibers onto the microreactor chip with the fiber tip in direct contact with the fluidic microchannel.73,74 A more advanced design is to incorporate planar waveguides that intersect the microchannel giving the direct excitation of the analyte. On-chip planar waveguides made of various materials such as silica, PDMS, SU-8, soda lime glass, and acrylate-based polymer have been reported.75−81 Mazurczyk et al.79 fabricated the channel optical waveguides buried in soda lime glass-based microchips using an ion exchange method. The laser beam was injected into the waveguide of the chip via a coupled monomode optical fiber and then excited the fluorescence emission at the intersection between the waveguide and the microchannel filled with the fluorescent dye solution. Three different ways to collect the fluorescent beam were investigated: the first was to collect the beam by the facing on-chip waveguide; the others were to collect at the right angle with respect to the beam either by a microscope objective positioned above the chip or by a multimode fiber glued onto the chip surface. Fluorescence measurements with a model dye solution of rhodamine 6G revealed a limit of detection around 0.5 nM and 1 nM for the perpendicular configurations using microscope and optical fiber, respectively. However, the limit of detection was as high as 0.5 μM for the parallel configuration using the opposite on-chip waveguide, implying that in this configuration the excitation light component was much less eliminated from the detected beam than in the perpendicular configuration. Malic and Kirk80 designed an array of 10 multimode waveguides consisting of SU-8 core and two upper and lower PMMA cladding layers on a silicon substrate. The waveguides were perpendicular to the microchannel, and its optical alignment with the externally coupled optical fibers was implemented via etched V-grooves on the substrate (see Figure 1). Fluorescence emission measurements performed in a dark room for the aqueous solution of Alexa Fluor 633 dye showed a limit of detection less than 10 nM. A similar on-chip waveguide design was presented by Sheridan et al.,81 who developed a microchip consisting of two in-plane integrated waveguides and one microchannel that were fabricated in an acrylate-based polymer. The microchannel intersected with the two waveguides at 90°. A laser beam was coupled to the edge of one waveguide via a multimode fiber and light from the output of the other waveguide was collected via another multimode fiber to a spectrometer. The fluorescence spectrum for aqueous solutions of fluorescein flowing through the microchannel indicated a low

2. INTEGRATION OF MICROREACTORS WITH SPECTROSCOPIC DETECTION FOR ONLINE REACTION MONITORING Various means of integrating spectroscopic detection into continuous microreactor processes have been devised to allow an online reaction monitoring, which can be utilized for either optimizing the reaction parameters or extracting kinetic information. This section presents an overview of the existing schemes of integration between the spectroscopic detection methods described above and microreactors. Selected reaction examples are provided demonstrating the potential of such integration. In view of the fact that many spectroscopic integration schemes originally developed in the area of μTAS can be in principle applied to microreactor processes for monitoring the reaction progress, the pertinent achievements in this area are also included. 2.1. Reaction Monitoring with Fluorescence Spectroscopy. 2.1.1. Integration Scheme. Fluorescence spectroscopy is a widely used detection method in microchemical systems owing to its superior sensitivity, which allows the detection limit down to the nanomolar or picomolar range and allows single-molecule detection under some circumstances.28,39 It is based on the detection of light emission from excited singlet states of fluorescent substances (fluorophores). The emission spectra measured at a single constant excitation wavelength are indicative of the chemical structure of the fluorophore (e.g., the structure of the different vibrational levels), and the fluorescence intensity infers the concentration of the fluorophore.68 As most chemical applications do not involve the presence of fluorophores, the target analyte usually has to be labeled to introduce fluorescence. Fluorescence signal in a microreactor channel is usually detected using a microscope setup. The excitation sources to induce fluorescence include mainly laser, light emitting diodes (LEDs), and lamps. To ensure the optimal limits of detection, materials that show weak fluorescence background at the investigated wavelengths are preferred for the manufacture of the microreactor chip. Glass and quartz are good candidates but require the costly fabrication process. Low-cost plastic materials for chip fabrication such as poly(methyl methacrylate) (PMMA) and polydimethylsiloxane (PDMS) exhibit significant autofluorescence when excited by UV or even visible radiation.69 However, the fluorescence background interference from chips made of these materials can be mitigated by using improved optics or by labeling analytes with fluorophores excitable at longer wavelengths.70 Compared to fluorescence measurements in macroscopic flows, there is usually no room to span up a light sheet for microchannel flows. Matsumoto et al.71 presented an optical technique for measuring the height-averaged species concentration field in a microchannel based on the fluorescence intensity. In their design, 14585



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step, which allowed for the precise control of the laser launch angle into the waveguide to provide sampling via the evanescent field. The waveguide was placed orthogonally to microchannels. Fluorescence measurements were performed for a solution of AlexaFluor 647 flowing through the device, and the fluorescence signal was recorded using a microscope equipped with a CCD camera. Eleven microchannels could be measured simultaneously with a fairly uniform intensity for each (cf. Figure 2c). Besides solid-core optical waveguides, the integration of liquidcore optical waveguides (i.e., using liquid to deliver excitation light or collect fluorescence) onto microfluidic chips for fluorescence detection have been reported to yield promising results, for example, to achieve a sensitivity comparable to those commercially available confocal systems for laser-induced fluorescence measurements.83−85 2.1.2. Examples of Application. Fluorescence detection, particularly microlaser induced fluorescence (μ-LIF), is commonly used to directly visualize mixing patterns in microchannels, which contributes greatly to the fundamental understanding on mixing-sensitive reaction processes being carried out in microreactors. This is usually accomplished by the incorporation of a fluorescent dye into one inlet stream which is diluted or quenched by the other undyed stream moving through microchannels.72,86−90 The fluorescence intensity field of the dye excited by the light beam is measured to infer the concentration field, thus providing a qualitative description of the mixing quality. The mixing quality can also be characterized by using a chemical reaction in the presence of a dye having a quantum yield dependent on the local chemical environment (e.g., pH value).72,91 Hoffmann et al.72 performed the acid−base neutralization reaction in T-type micromixers. The acid solution as one inlet stream contained the dye disodium fluorescein that fluoresces above a threshold of pH = 4 when efficiently excited by a laser beam at a wavelength of 488 nm. The base solution as the other stream contained no fluorescein. A three-dimensional concentration field during the mixing/reaction along the microchannel could be constructed based on μ-LIF signals obtained using a confocal laser scanning microscope. Alternatively, the chemical reaction utilized to characterize mixing can involve the formation of a fluorescent product. Ismagilov et al.92 visualized the region of transverse diffusive mixing in a microchannel by observing the concentration of a strongly fluorescent complex formed near the interface between two laminarly flowing aqueous solutions of fluo-3 and CaCl2 using confocal fluorescent microscopy, as shown in Figure 3. The fluorescence experiments confirmed the theoretical predictions that the width of the reaction-diffusion zone at the interface adjacent to the wall of the microchannel scales as the one-third power of both the axial distance down the microchannel and the average velocity of the flow, as compared to the one-half power scaling that was measured in the middle of the microchannel. Another important application of online fluorescence detection is the measurement of chemical kinetics in microreactors.93−96 Nagai et al.93 performed the extraction of Ag (I) cations from the aqueous phase to the organic phase in a PDMS microreactor that contained a guide structure to form a stable liquid−liquid interface along the center of the microchannel. The organic phase was a 1,2-dichloroethane solution containing 1,4,7,10,13,16-hexathiacyclooctadecane (HTCO), while the aqueous solution contained silver sulfate, sodium sulfate, and a fluorescent anion, 2,4,5,7-tetrabromofluorescein disodium (eosin Y). When the two phases contacted under

Figure 1. Microfluidic chip with integrated optical waveguides for fluorescence spectroscopy. (a) Simplified device layout with two waveguides. (b) Image of fiber inserted in a V-groove and self-aligned to the waveguide. (c) Image of the whole device with laser turned on. Reproduced with permission from ref 80. Copyright Elsevier, 2007.

detection limit of 7 nM which is comparable to that of Malic and Kirk80 and significantly lower than that of Mazurczyk et al.79 in a similar configuration. They claimed that the improvement of the detection limit compared to that of Mazurczyk et al.79 might be due to the efficient light coupling and collection thanks to the device design which uses multimode waveguides to maximize input power and the matched dimensions between the optical fibers and the waveguides. All the above studies were on systems with a single-channel geometry. Okagbare et al.82 presented for the first time a design of a multichannel microfluidic chip embedded with a planar waveguide for evanescent excitation and fluorescence detection across multiple microchannels. A schematic of the device is shown in Figures 2a and 2b. The chip was composed of a PMMA cover plate with an embedded waveguide made of cyclic olefin copolymer and a PMMA substrate containing multiple microchannels (100 μm width × 30 μm depth). A monolithic prism was integrated into the waveguide in a single fabrication 14586



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Figure 2. Multichannel microfluidic chip with embedded waveguides for fluorescence spectroscopy. (a) Schematics of the chip with embedded single planar waveguide with a monolithic coupling prism: (i) diagonal view; (ii) frontal view; (iii) cross-sectional view. (b) Schematics of a portion of the device showing the multichannel architecture and the interconnected waveguide. (c) Fluorescence image acquired from 11 multiple microchannels filled with 100 nM AlexaFluor 647 when light was launched into the waveguide through the monolithic prism (top) and the corresponding fluorescence intensity signal (bottom). A clear distinction between microchannels showing fairly uniform intensity and the separating fins showing dark background could be seen. Reproduced with permission from ref 82. Copyright The Royal Society of Chemistry, 2010.

ribonuclease A (RNase A). The droplets encapsulated a fluorogenic substrate and RNase A that reacted with each other, where the intensity of fluorescence indicated the amount of product. This droplet-based microfluidic system was demonstrated to allow the extraction of kinetic information better than millisecond resolution. Recently, fluorescence correlation spectroscopy, which does not rely on the measurement of fluorescence intensity but on the spontaneous fluctuations of fluorescence intensity caused by deviations from a mean, has been applied for chemical kinetic study in microreactors.97 One review about the use of this technique for microreactor applications was given by Tian et al.98 Fluorescence spectroscopy usually requires the labeling of the target analyte to introduce fluorescence, which makes this detection technique somewhat limited to specific applications in microreactors such as the assessment of mixing quality or reaction kinetic studies using model fluorescent systems. For practical chemical synthesis in microreactors, it is less appealing as compared to other noninvasive spectroscopic techniques such as UV−vis and Raman spectroscopy. 2.2. Reaction Monitoring with UV−Vis Spectroscopy. 2.2.1. Integration Scheme. UV−vis absorption spectroscopy is a well-established detection technique, which is based on the measurement of the absorption of the incident light by certain functional groups in the sample molecule due to the induced electronic transitions. The resulting spectrum showing absorbance as a function of wavelength reveals peaks in absorption and can be utilized to identify the composition and concentration of the sample.41 Although essentially all organic molecules exhibit significant absorption in the low ultraviolet range (160−180 nm), standard UV spectrometers cannot handle this wavelength range due to absorbance of optics and air gases.32 Fortunately,

laminar flow through the microchannel, Ag (I) was extracted to the organic phase to form the Ag-HTCO-eosin Y complex. The μ-LIF signal via a fluorescence microscope on the Ag-HTCOeosin Y complex was then measured to elucidate the macroscopic kinetics of this extraction reaction in the microreactor. The extracting kinetics was shown to depend not only on the reaction between HTCO and Ag (I) cation occurring at the interface but also on the rates of diffusion of eosin Y, HTCO, and the complex in the proximity of liquid−liquid interface. However, the diffusion process of the Ag-HTCO-eosin Y complex was determined as the main rate-controlling step in Ag (I) extraction with HTCO. Kerby et al.94 carried out a kinetic study of enzymatic reactions in a glass microreactor. The alkaline phosphatase enzyme was immobilized on silica beads that were subsequently packed in the microreactor. The Michaelis−Menten kinetic parameters for this enzyme were measured by the dephosphorylation of 6,8-difluoro-4-methylumbelliferyl/phosphate to a fluorescent 6,8-difluoro-4-methylumbelliferone, where a mercury lamp was used in the system to excite the fluorescence signal indicating the reaction conversion. Jambovane et al.95 combined fluorescence detection with a microfluidic chip having 11 parallel architectures for determining kinetic parameters of an enzymatic reaction between galactosidase and resorufin-β-D-galactopyranoside. The parallel architectures on the chip generated a gradient of reagent concentrations, which enabled 11 parallel reactions to occur. By analyzing the acquired intensity of fluorescence emitted by the product (i.e., resorufin) in a multiple scan along the chip, the key parameters for the enzyme kinetics could be determined in one single experimental run. Song and Ismagilov96 performed fluorescence measurements in droplets moving through a microchannel to extract the kinetic activity of 14587



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One approach addressing this issue is by using the out-of-plane integration, for example, by interfacing the microreactor with a free-space microscope setup105 or by housing two optical fibers in perpendicular configuration with the plane of the microreactor which is UV or VIS transparent in order to guide the incident light through the microreactor and collect the transmitted light.106−108 As most materials used for the construction of microreactors (e.g., silicon, glass) cut off the wavelength below around 300−350 nm, spectroscopic detection in the visible range can be realized in such microreactors if the incident light has to transmit through the reactor material.106 To enable UV detection down to 200 nm, microreactors with quartz or fused silica windows are usually required. One solution was provided by Jackman et al.,106 who combined quartz with a photodefinable epoxy SU-8 to produce microreactors. Microchannels were defined in SU-8 and were capped on both sides with quartz to give UV transparent windows suitable for spectroscopy. The feasibility of performing online UV analysis in these hybrid microreactors was demonstrated with organic solvents such as benzene and acetone dissolved in hexane. Lu et al.107 coupled a detection chip according to the design of Jackman et al.106 with another microfabricated silicon/Pyrex reactor chip. The two chips were connected by a 10 cm long poly(ether ether ketone) (PEEK) tubing. To eliminate the delay for detection present in this two-chip integration scheme, a monolithic integration of the reaction and detection units in one chip was also demonstrated (see Figure 4b), which was realized by bonding quartz wafers to patterned silicon wafers using Teflon-like polymer. The chip was assembled between a plexiglass plate and a steel mount with an opening to house both the optical fibers for UV spectroscopy. The coupling of online UV−vis spectroscopy with microreactors under high pressure conditions (up to 600 bar) was demonstrated by Benito-Lopez et al.108 In their design, a fused silica capillary microreactor ran through a stainless steel cross that was connected with a fiber-optic system for UV−vis spectroscopy. The out-of-plane integration requires a delicate optical alignment process. A further improvement can be made by using inplane integration of spectroscopic detection. Park et al.109 described the combination of a microreactor with in-plane type optical source and detector for the development of an integrated microammonia analysis system. As illustrated in Figure 4c, the system is composed of mixing microchannels, reaction microchannels plus one UV−vis detection cell that were defined on silicon substrate. The detection cell has two silicon oxide windows of 2 μm thickness between each of the two optical fibers and the detection solution. A precise optical alignment was achieved by using tailor-made microchannels to embed optical fibers that were externally connected with optical source and optical detector. Ohlsson et al.110 designed a microchip based on oxidized silicon with integrated in-plane waveguides for simultaneous detection of UV absorbance and native UV-excited fluorescence. A 1 mm-long U-shaped detection cell following the separation channel was coupled to two integrated waveguides that guided the incident light to the detection cell and collected the transmitted light to the detector (cf. Figure 4d). The core of waveguides consisted of silicon oxide with etched air grooves as cladding on the sides, air as top cladding, and silicon substrate as bottom cladding. Both waveguides were connected with respective optical fibers via an integrated fiber coupler on the chip, thus ensuring perfectly optical alignment. This design was shown to be capable of detecting compounds in the micromolar range by UV

Figure 3. Visualization of transverse diffusive mixing in a microchannel by confocal fluorescence microscopy. (a) Schematics of the experiment used to generate fluorescence in the microchannel and the coordinate system. (b) A 100 × 250 μm2 yz slice of fluorescence image obtained 12 μm from the top of the microchannel. (c) A 90 × 90 μm2 xy slice of fluorescence image obtained 50 μm (top) and 200 μm (bottom) from the inlet Y junction. Reproduced with permission from ref 92. Copyright The American Institute of Physics, 2000.

functional groups in many organic molecules contain valence electrons of low excitation energy (e.g., -(CC)n-, -CO, -CN) and therefore will undergo electronic transitions under light radiation in the high UV (190−390 nm) or VIS (390−770 nm) range. Many inorganic compounds (e.g., transitional metal complex) also show charge-transfer absorption under these wavelength ranges and therefore can be detected by UV−vis spectroscopy. Under favorite optical configurations, UV−vis absorption spectroscopy could achieve a detection limit in the micromolar range.32 Online UV−vis detection has been combined with microreactors via different means. The simplest way of integration is to connect a flow-through transmission cell externally with the microreactor, as done by Ferstl and co-workers53,99 for the product quantification during an organic reaction (see Figure 4a) and many others for process monitoring (e.g., residence time distribution and mixing).100−102 These optical flow-through cells are usually commercially available.21 Tailor-made cells can be also devised for specific applications. For instance, a micromachined silicon/Pyrex detection cell for UV−vis measurement of indophenol blue and its hybird integration into the ammonia microanalysis system was demonstrated by Tiggelaar and coworkers.103,104 Although the arrangement of an external detection cell enables a faster data acquisition compared to off-line analysis, this is still not the ideal in situ integration in which spectroscopic measurements are focused inside the microreactor itself. 14588



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Figure 4. Various ways of coupling UV−vis spectroscopic detection into microchemical systems. (a) External UV−vis(-NIR) spectroscopic flowthrough cell (left) and a schematic of its inner configuration (right). Reproduced with permission from ref 99. Copyright Wiley, 2005. (b) Microreactor design with combined reaction-detection unit (top left) and photograph of the microreactor chip (bottom left). Two optical fibers for UV spectroscopy were coupled with the microreactor in an out-of-plane fashion (right). Reproduced with permission from ref 107. Copyright The Royal Society of Chemistry, 2001. (c) Integrated microammonia analysis system with microreactor and in-plane type optical source and detector and cross-sectional view of UV detection cell at the left and bottom side. Reproduced with permission from ref 109. Copyright Elsevier, 2006. (d) Electrophoresis microchip with an in-plane waveguide design (left) and scanning electron microscope image of the detection cell and waveguides (right). Reproduced with permission from ref 110. Copyright Wiley, 2009.

compounds within the reaction mixture has been demonstrated by Ferstl and co-workers for one-phase or two-phase nitration of toluene in a microreactor setup using pure HNO3 or HNO3/ H2SO4 mixed acid.53 UV−vis spectroscopy also allows the determination of size and concentration (number density) of nanoparticles produced in microreactors via the method of wet chemical synthesis, which is based on the measurement of surface plasmon resonance of nanoparticles.115 Microreactors with online UV−vis detection capabilities are shown to allow a better optimization and access to kinetic parameters of the synthetic processes of nanoparticles.116−119 The development of such integrated systems is expected to be the priority in the forthcoming years in this field.120 2.3. Reaction Monitoring with IR Spectroscopy. 2.3.1. Integration Scheme. Infrared spectroscopy is the measurement of absorption or emission of electromagnetic radiation in the infrared region as a consequence of vibrations within a molecule. The infrared region is further divided into three regions according to the working wavelength range: nearinfrared (NIR) region at approximately 770 nm−2.5 μm; midinfrared (MIR) region at approximately 2.5 μm−25 μm; farinfrared region at approximately 25 μm−1 mm.121 The classical use of IR spectroscopy is mainly limited to the mid-infrared region where the fundamental vibrations are found, which is very efficient in determining the structural groups in almost all organic compounds primarily because of the highly specific information content in the spectrum.122 As the vibrational energy levels are unique to each molecule, MIR spectroscopy is able to

absorbance measurement. Other cell designs allowing the coupling of in-plane waveguides have been proposed to increase the sensitivity of UV−vis absorbance detection in the area of μTAS, which can be in principle utilized for the real-time reaction monitoring in microreactors.111,112 2.2.2. Examples of Application. UV−vis spectroscopy is commonly used to monitor the progress of liquid-phase reactions in microreactors via concentration measurements of the reaction mixture.53,107,108,113,114 Lu et al.107 investigated the photochemical synthesis of pinacol from benzophenone in silicon/Pyrex microreactor chips, where online fiber-optic based UV detection was conducted either in an individually packed detection chip or in the detection unit monolithically integrated onto the same microreactor chip. The absorbance of reaction mixture measured in the two-chip integration scheme could be correlated to the on-chip conversion of benzophenone, as verified by off-line analysis. A high conversion of the reactant was observed at a longer residence time. Furthermore, UV spectra obtained in the monolithic integration scheme further confirmed the presence of a strongly absorbing short-lived intermediate that would interfere conversion measurements. Benito-Lopez et al.108 showed that with online UV measurements of product formation, the reaction rate constants at various pressures and the activate volume could be determined for a nucleophilic aromatic substitution reaction and an aza Diels−Alder reaction in a capillary microreactor. Absorbance detection in the visible range has been reported to be a suitable tool to follow the course of dye synthesis in microreactors.113,114 The ability of visible spectroscopy for online quantification of 14589



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Figure 5. Selected examples of integrating IR spectroscopic detection into microchemical systems. (a) Schematics of the setup for spectroscopic measurements in a silicon microreactor using a high resolution FTIR microscope. Reproduced with permission from ref 129. Copyright Elsevier, 2004. (b) Sketch of a miniaturized fiber-optic flow-through cell developed for FTIR spectroscopic measurements of the exit stream out of a silicon microreactor. Reproduced with permission from ref 133. Copyright The American Chemical Society, 1997. (c) Experimental setup for the ozonolysis in a falling film microreactor (left) and integrated flow-through cell with diamond ATR probe for online FTIR analysis (right). Reproduced with permission from ref 135. Copyright The American Chemical Society, 2009. (d) Schematic representation of a microfluidic ATR-FTIR imaging system. Reproduced with permission from ref 134. Copyright The Royal Society of Chemistry, 2009.

provide a ‘fingerprint’ of a particular molecule.123 NIR spectroscopy has recently received increased attention, which is based on overtones or combinations of the fundamental vibrations and is typically useful for measuring organic compounds containing O−H, N−H, and C−H bonds. However, the limit of detection with NIR spectroscopy is usually around 0.1% in concentration, which is greatly reduced as compared with MIR spectroscopy because the corresponding intensity of an absorption band is much smaller than that in the mid-infrared region (e.g., smaller by a factor of 10 to 100).122,124 Far-IR spectroscopy is not well developed till now due to technical difficulties although it is able to detect the stretching vibrations of molecules having heavy atoms such as inorganic molecules and metalorganic complexes.122 Online IR measurements in a microreactor are usually performed by placing the microreactor either in a sample compartment or in a microscope stage connected to a conventional Fourier transform infrared (FTIR) spectrometer system.125−130 The transparency of silicon to electromagnetic radiation in most of the mid-infrared region (i.e., at wavenumbers from 800 to 4000 cm−1) allowed Floyd et al.125 to develop silicon-based microreactors that enabled online FTIR analysis of homogeneous liquid-phase reactions in microchannels. The microreactors were placed in the sample compartment of a conventional FTIR

spectrometer, and a thin elastomer gasket sealing the top of the microreactors was used to limit the transmitted IR radiation to the region of interest. It should be mentioned that this IR monitoring region had a swallow thickness of 50 μm, which is necessary due to the strong absorption of water in the midinfrared region. Hinsmann and co-workers126,127 developed micromixers that were constructed using an epoxy negative photoresist resin material on MIR transmitting CaF2 discs. A custom built optical setup was used to focus the external IR beam from a commercial spectrometer on the main mixing channel of the micromixers and to collect the transmitted light on an infrared detector. Synchrotron MIR microscopic detection allowing measurements at sample spot sizes near the diffraction limit (μm range) was combined by Kaun et al.128 with a similar micromixer to the design of Hinsmann and co-workers.126 The micromixer was mounted into a specially fabrication PMMA support that could be fixed onto the microscope stage. Keoschkerjan et al.129 demonstrated the feasibility of performing MIR or NIR reflectance spectroscopic measurements in a silicon microreactor using FTIR microscope, as illustrated in Figure 5a. On the back side of the microreactor a gold layer was deposited as a reflecting layer. Herzig-Marx et al.130 further developed an on-chip FTIR detection scheme suited to not only the analysis of liquid-phase 14590



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By utilizing both a 64 × 64 pixel focal plane array (FPA) detector for FTIR analysis and a combined imaging and mapping strategy (i.e., by moving the ATR-crystal-microfluidic chip to acquire an array of subimages which can be reconstructed to generate the desired image), it was possible to obtain infrared spectra as a function of the location over an area of 10 × 14 mm on the chip with a spatial resolution of 40 μm. This integrated ATR-FTIR-microfluidic system is schematically shown in Figure 5d. The working principle of this system design was validated by a test with a model isotope exchange reaction. Most of the existing studies, however, are dealing with a relatively straightforward combination of microreactors with small volume infrared cells for spectroscopic measurements. To expand the capability of online IR detection in microreactors, truly integration using planar waveguides is desired. This would allow an ease of coupling with the IR source radiation and also allow a closer interrogation of chemical reaction in microreactors.37 2.3.2. Examples of Application. MIR spectroscopy has been combined with microreactors for the investigation of kinetics of liquid-phase reactions. For homogeneous reactions, this is usually done via the use of microchannel structures at the inlet of a microreactor to initiate the mixing of reactants followed by online MIR measurements to monitor the reaction progress along the reaction microchannel, which translates into the reaction conversion as a function of the reaction time.125−127,130,139−141 Floyd et al.125 performed the alkaline hydrolysis of methyl formate in two microreactors that involved either a simple T-shaped channel design or an advanced design with interleaving mixing channels. The reaction rate constant determined according to online FTIR spectroscopic analysis compared well with the literature. Herzig-Marx et al.130 investigated the acid-catalyzed hydrolysis of ethyl acetate in a microreactor with a troughlike channel, which serves as a test case for the use of IR spectroscopy to characterize organic reactions in microreactors. The initial mixing of reactants was carried out either in this simple microreactor optimized for detection only or by external vigorous stirring in a macroscopic sample. In both mixing situations, online FTIR measurements could be used to follow the depletion of ethyl acetate as a function of time and thus enabled the extraction of values of the first-order rate constant at different HCl concentrations. The measured kinetic data were shown to be consistent with earlier studies of this reaction in macroscopic reactors. For multiphase reactions, Jähnisch and co-workers135,136 employed ATR-FTIR spectroscopy for online monitoring the ozonolysis of 1-decene in a falling film microreactor (cf. Figure 5c). The fast spectroscopic measurements at reaction conditions were able to detect unstable and explosive intermediates such as ozonides and hydroperoxides that were not amenable by delayed off-line analysis at ambient temperatures, which provided a deeper insight into mechanistic aspects of this gas−liquid reaction. The integration of ATR-FTIR spectroscopy with a silicon-based microreactor platform by means of a ReactIR flow cell has been demonstrated recently by Kebyl and Jensen for kinetic analysis of rhodium catalyzed gas−liquid hydroformylation of 1-octence under high pressures.142 The reaction was performed under gas− liquid segmented flow and was assumed to fall into the kinetically controlled regime at the investigated experimental conditions. A gas−liquid membrane separator was added in the system to ensure the introduction of only liquid stream into the flow cell, which circumvented the fluctuations in the MIR signal intensity that would be present when a gas−liquid flow was analyzed in the

concentrations but also the detection of surface-bounded function groups in a silicon/Pyrex microreactor integrated with a multiple internal reflection geometry. IR measurements can also be performed via the attachment of an in-line flow-through cell to the microreactor, which represents a practical solution if one considers the lack of microreactor substrates that are transparent in the longer wavelength of the IR spectrum.37 Such an arrangement further adds to the system flexibility as the customized flow-through cell is replaceable and can be improved in its design without affecting the upstream microreactor performance. The flow-through cell can be simply a conventional IR transmission cell. For example, the cell suitable for MIR spectroscopy could consist of two CaF2 windows that were separated by a Teflon spacer, thus giving an optical path length below around 50 μm.131,132 Alternatively, the flowthrough cell can accommodate optical fibers to deliver the IR radiation and collect transmitted light. A miniaturized fiber optical flow-through cell for MIR spectroscopy has been developed by Lendl et al. (cf. Figure 5b).133 The cell consisted of two AgClxBr1‑x fiber tips that were coaxially mounted in a poly(tetrafluoroethane) (PTFE) block and thus produced an optical path length of 23 μm. The use of optical fibers further makes it in principle possible for a local separation between a bulky FTIR spectrometer containing the interferometer and the highly sensitive liquid nitrogen-cooled mercury cadmium telluride (MCT) detector. Ferstl and co-workers53,99 have developed flow-through cells for NIR spectroscopy that can be integrated with microreactors to allow online process analysis. Such NIR flow-through cells may be also utilized for UV−vis spectroscopy (see Figure 4a) and generally accommodate much longer optical path lengths (e.g., 2 mm) than those used for MIR spectroscopy given by the weak absorption from overtones.53,122 Attenuated total reflection (ATR) based flow-through cells can be used for applications requiring ATR-FTIR measurements. As compared with conventional FTIR detection in transmission or reflection mode, FTIR detection in ATR mode utilizes an IR transparent crystal to create an internal reflection of the light forming the evanescent wave which travels into the sample and is more suited to strongly MIR absorbing solvents like water due to its short probe distance up to several micrometers.134 The flow cell in this case does not need to contain MIR transparent windows due to the possibility of directly contacting the ATR probe with the liquid medium. A small ATR diamond fiber-optical probe mounted in a purpose-built microflow-through cell has been employed by Jähnisch and coworkers135,136 to analyze the reaction product exiting from upstream falling film microreactors. A typical microreactor setup with online ATR-FTIR monitoring used in their experiments is shown in Figure 5c. Carter et al.137 reported a ReactIR flow cell (Mettler-Toledo AutoChem, Maryland, U.S.A.) operated with ATR-FTIR spectroscopy as a convenient and versatile in-line analytical tool for microreactor flow processing. The flow cell comprises an integrated ATR gold sealed diamond sensor in direct contact with the stream, which allows the acquisition of almost full mid-infrared spectral region (650−4000 cm−1) except a weak absorbance in the diamond ‘blind spot’ from about 1950−2250 cm−1. A closer integration between ATR-FTIR spectroscopy and microreactors can be made by using the surface of an ATR crystal as the base of microreactors.134,138 An example according to this design was given by Chan et al.,134 who developed a microfluidic chip with channels molded in a PDMS elastomeric substrate and sealed by an ATR prism of ZnSe. 14591



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flow cell. Real-time FTIR analysis, in combination with off-line analysis by a gas chromatography (GC), enabled the determination of kinetic parameters as required in the formulation of the reactor and mechanistic models. The observed activation energy and reaction orders with respect to the reactant species or the homogeneous catalyst were found to agree with the literature results. The capability of MIR or NIR spectroscopy for online reaction monitoring can be also used for the optimization of reaction processes in microreactors. FTIR microscopy has been applied by Antes et al.143 to evaluate the performance of a silicon microreactor consisting of nine parallel reaction channels for carrying out nitration of dialkyl-substituted thioureas. Different nitrating agents including fuming HNO3, HNO3/ H2SO4 mixed acid, and N2O5 dissolved in CH2Cl2 were used in the experiments. By focusing the MIR beam consecutively on different positions inside the microreactor, spatially resolved measurements were realized to indicate the localized concentrations of the reactants, intermediates, and products. N,N′Dialkyl-N-nitroso-urea was identified as the key intermediate, and a quantitative FTIR analysis of the target product, N,N′dialkyl-N,N′-dinitro-ureas, specified the reaction yield along individual microchannels, which made it further possible to rapidly determine the desired flow rates for the best reactor performance. ATR-FTIR analysis has been applied during the preparation of Grignard reagents in a PTFE capillary microreactor144 and asymmetric hydrogenations of benzoxazines, quinolines, quinoxalines, and 3H-indoles in a glass microreactor,145 where online reaction monitoring was achieved by a ReactIR flow cell that was attached to the microreactor. Fast process analysis and reaction optimization in microreactors were demonstrated thanks to the integration of online FTIR spectroscopic detection. NIR spectroscopy has been attempted by Ferstl et al.53 for the real-time qualification of the main product during toluene nitration reaction in a silicon-based microreactor with 11 parallel channels. The reaction experiment was carried out at various parameter levels specified by a statistical experimental design tool. The product mixture was quantified by spectroscopy based on a multivariate calibration method. For the one-phase reaction system using pure HNO3 as the nitration agent, the calibration for NIR spectroscopy was found less precise than for Raman spectroscopy that was also under investigation. Thus a more robust calibration method is still required. 2.4. Reaction Monitoring with Raman Spectroscopy. 2.4.1. Integration Scheme. Raman spectroscopy relies on the measurement of inelastic scattering of an incident monochromatic light (usually from a laser source with radiation in the ultraviolet, visible, or near-infrared range) upon interaction with a sample molecule. The spectrum of the scattered light exhibits bands with different frequency differences, which provides information about characteristic fundamental vibrations within the molecule and thereby is able to provide a unique ‘fingerprint’ for each molecule. Raman spectroscopy is suitable for the identification and quantification of not only organic compounds but also inorganic compounds given by the fact that it can reveal bands well below 400 cm−1.122 However, the sensitivity with conventional Raman spectroscopy (in absence of enhancement techniques) is usually low due to the weak scattering effect. For example, a limit of detection around 1 μM in solution is hardly accessible by this detection method.146 Raman spectroscopy and MIR spectroscopy are complementary techniques, where generally Raman spectroscopy works best at symmetric

vibrations of nonpolar groups and MIR spectroscopy works best at asymmetric vibrations of polar groups.122 Another notable feature with Raman spectroscopy is its applicability to aqueous solutions. In comparison, the measurement with MIR spectroscopy is usually obscured by the intense absorption of water except in the ATR mode. Raman spectroscopy is an excellently suitable technique to perform vibrational spectroscopy in microreactors. This compatibility is due to the fact that Raman spectroscopy works conveniently with Raman lasers at a wavelength in the visible light range, while microreactors can be made out of optically transparent materials with ease, most particularly out of glass. In contrast, UV and IR measurements in transmission mode are normally impractical in glass microreactors due to high extinction coefficients of glass in the UV and IR regions of the electromagnetic spectrum.55 At this moment, Raman spectroscopic studies in microreactors typically utilize a conventional Raman (microscopy) system and are analyzing fluids in the device.53−55,147−153 A simple combination between Raman spectroscopy and microreactors was provided by Ferstl et al.53 Their setup is shown in Figure 6a. A small flow-through Raman cell was integrated right after the microreactor unit and was plugged to a Raman spectrometer via an optical fiber. Urakawa et al.54 utilized Raman spectroscopy for reaction study in a silicon/glass microreactor chip, where the Raman fiber optical probe was placed in front of the microreactor. Fletcher et al.147 took a step further by switching to confocal Raman microspectroscopy, where a Raman microscope spectrometer is used instead of a standard Raman spectrometer. This arrangement allowed them to determine the localized concentrations of each reactant and product species inside a glass microreactor at a horizontal spatial resolution around 20 μm (cf. Figure 6b). Raman spectroscopy and microspectroscopy were also used by many others to monitor chemical reactions in microreactors under single-phase or multiphase flow conditions.148−153 For the detection of chemicals at very low concentrations in the liquid phase (e.g., in the nanomolar or picomolar range), signals obtained using conventional Raman spectroscopy are considerably weak. The advanced technique of surface-enhanced Raman scattering (SERS) turns to be promising, where the target molecule’s Raman cross section can be significantly increased when it is absorbed to a metallic nanoparticle that is smaller than the wavelength of the incident laser light.154,155 The feasibility of combining this technique with microfluidics toward the development of microfluidic based-SERS chips has been demonstrated by many authors.156−162 For SERS measurements under static conditions, the sensitivity can be further improved by using capillary forces to generate an increased local density of nanoparticle/target.157 More reproducible Raman measurements were achieved under flowing conditions, where a rapid and highly sensitive detection was made possible either by the use of in situ synthesized colloidal nanoprticles158 or by enhancing the mixing of the analyte with nanoparticles.159−162 All these studies are dedicated to SERS measurements in the homogeneous mode in which the target analyte is absorbed onto metallic nanoparticles in a solution acting as the Raman enhancer. Alternatively, SERS measurements can be performed in the heterogeneous mode in which the analyte in a solution interacts with SERS-active substrates such as roughened metallic electrodes.48 More detailed information on the recent development of coupling SERS with microfluidic analytical systems can be found in several reviews.43,45,48,50 However, the way of 14592



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Figure 6. Selected examples of coupling Raman spectroscopy with microreactors for online reaction monitoring. (a) Microreactor with integrated external flow-through Raman cell (left) and typical Raman spectra of main products obtained for the toluene nitration using pure HNO3 in onephase system (right). Reproduced with permission from ref 53. Copyright Wiley, 2007. (b) Schematics of a T-type microreactor that was coupled with a confocal Raman microscope spectrometer (left) and 3-D plots of Raman intensity in the T-junction region at 893 cm−1 for acetic acid (middle) and 882 cm−1 for ethanol during the synthesis of ethyl acetate (right). Reproduced with permission from ref 147. Copyright Wiley, 2003. (c) Schematic of a microreactor integrated with waveguide confined Raman spectroscopy (the inset detailing the signal collection region) (left) and a comparison of the Raman spectra of pure analytes with that of the product mixture for the acid catalyzed esterification of ethanol and acetic anhydride (right). Reproduced with permission from ref 164. Copyright The Royal Society of Chemistry, 2011.

inherent limitations of this ‘direct but incompact’ combination are strong background from the substrate and the lack of portability.164 One way of improvement is to optimize the Raman probe optics, as done by Mozharov et al.165 They found that for analysis of liquids in a glass microreactor, a miniature aspheric lens could achieve higher sensitivity and reduced glass background as compared to a commercial Raman microscope objective. A future improvement would be the switch to the usage of on-chip embedded waveguides for the transport of light to and from a desired measurement location in a microchannel,166 which are linked to an optical fiber Raman system toward the development of a fully functionalized and compact

integration between SERS detection and microreactors for online reaction monitoring is not very clear. In this regard, SERS detection in the homogeneous mode might not be suitable for characterizing the microreactor performance due to the presence of the added solution-phase Raman enhancers which would contaminate the reaction mixture. In comparison, SERS detection in the heterogeneous mode can be more conveniently applied for analyses of the reaction mixture but requires complicated reactor or detection chip fabrication.163 Up to now, the application of Raman spectroscopy in microchemical systems mostly involves the combination of bulk freespace optics with microreactors or microfluidic devices. The 14593



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compared with an estimated time scale of 48 h when extracting the equivalent from macroscale batch reactors. Online Raman spectroscopic detection can be further employed for deriving kinetic information of liquid-phase reactions in microreators, as demonstrated by Mozharov et al. for the base-catalyzed Knoevenagel condensation reaction between ethyl cyanoacetate and benzaldehyde.150 In their study, noninvasive Raman measurements were performed at the end of the reaction channel inside a Y-type glass microreactor chip. By adopting a novel procedure based on a step change from a low to a high combined flow rate of both reactants, location-specific concentration information at this prescribed low flow rate could be inferred from the Raman spectra collected over the period during which the high flow rate pushed the profile of reactants and products along the reaction channel to the detector. A 5-fold reduction in the time required for collecting kinetic data was achieved compared to when one set of kinetic data corresponding to a specific residence time was obtained by separate experiment. The derived reaction constants in both procedures, however, were shown to be consistent with each other. 2.5. Reaction Monitoring with X-ray Spectroscopy. 2.5.1. Integration Scheme. X-ray spectroscopy is a powerful detection technique for determining the structure and composition of the material by using X-ray excitation. When a beam of monochromatic X-ray radiation hits a sample of the material, the radiation will be either absorbed or scattered as a result of its interaction with electrons bound in the atom of the sample. For example, electronic transitions or elastic collision between electrons and X-ray photons may happen. Each of these electronic transitions is also accompanied by the emission of an X-ray photon (fluorescence X-rays). These phenomena form the bases of three important X-ray methods: the absorption technique; the scattering effect; and the fluorescence effect. These methods allow the quantification of almost all elements in the periodic table. For instance, X-ray fluorescence analysis is in principle able to detect elements from B (atomic number 5) upward, and, in many cases, elements can be analyzed down to the ppm level.168 Up to now, the integration of X-ray spectroscopic detection into microchemical systems is mostly realized by accommodating individual microreactors into the sample compartment of conventional X-ray spectrometers, where a monochromatic X-ray beam usually from an external synchrotron source is focused into the microreator channel and the spectra are obtained based on the absorption, emission, or scattering properties of Xray radiation.169−180 Microreactors suitable for X-ray analysis are often seen to include inspection windows made of materials such as PMMA and polymides which have relatively low absorption of X-rays and are relatively resistant to the high intensities of X-ray beam generated by synchrotrons. In comparison, other common materials used to manufacture microreactors such as silicon, glass, and PDMS exhibit high radiation absorption, which usually requires the presence of extremely thin inspection windows if online X-ray measurements are to be performed in such devices.176,181 The integration of X-ray absorption spectroscopy (XAS) for online monitoring of nanoparticle synthesis in microreactors has been reported by several authors,169−172 where the spectra were usually recorded in fluorescence mode. A representative system design can be found in the work of Chan et al.,173 who employed XAS to probe a nanocrystal cation exchange reaction in a silicon-based microreactor. Monochromated X-rays were focused through a silicon nitride window into the reaction

microreactor system. Some pioneer work has been seen in this direction. Ashok et al.164 have proposed the use of waveguideconfined Raman spectroscopy (WCRS) to couple with microreactors. A schematic of the PDMS microreactor chip combined with WCRS is shown in Figure 6c. A pair of multimode optical fibers were embedded in the microreactor chip through two fiber insertion channels to enable excitation and collection of the Raman signal from close vicinity of the ends of the waveguides. This resulted in an alignment-free architecture and would maximize the collecting efficiency of the signal without any background interference. This design was shown to function well with regard to the detection of analytes within a miscible liquid−liquid flow and in droplets within a segmented flow. The integration of liquid-core waveguides to excite the Raman signal (e.g., in the case where light and fluid are transported in the same volume) within microfluidic platforms has also been reported.167 2.4.2. Examples of Application. Online integration of Raman spectroscopy has been applied for monitoring and optimizing chemical reactions in microreactors. Ferstl et al.53 found that Raman spectroscopy provided the highest precision in the quantification of the main product during the nitration of toluene using pure HNO3 in a microreactor when compared with online NIR and VIS spectroscopy (see Figure 6a for their microreactor setup and typical Raman spectra). Urakawa et al.54 demonstrated that critical information on phase behavior and reaction performance of the cyclohexene hydrogenation reaction over a Pd catalyst in supercritical CO2 in a silicon/glass microreactor could be interrogated using online Raman spectroscopy, thus facilitating very fast optimization and diagnostics of the process. Fletcher et al.147 showed the potential of confocal Raman microspectroscopy in imaging the localized concentration field for a model reaction of the synthesis of ethyl acetate from ethanol and acetic acid inside a glass microreactor. Raman microscopy was also found by Lee et al.148 as a sensitive detection technique with a high spatial resolution to monitor the reaction between benzaldehyde and aniline to produce benzylideneaniline in a glass microreactor. The formation of diazonium salts in anhydrous conditions and their subsequent in situ chlorination in a microreactor chip made of glass was presented by Fortt et al.55 By utilizing Raman microspectroscopy for a direct on-chip detection of reagents, the optimization of the reaction process could be speeded up. The optimum residence time could be determined in about 10 min using Raman spectroscopy whereas residence time optimization by off-line GC analysis usually required hours to complete. Waveguide-confined Raman spectroscopy was employed by Ashok et al.164 for monitoring the progress during the acid catalyzed esterification of ethanol and acetic anhydride to ethyl acetate in a PDMS microreactor chip (cf. Figure 6c). Even though it was not possible to achieve spatial mapping in the reaction channel, the reaction dynamics could be followed easily by varying the reactant flow rate. The benefit of confocal Raman microscopy for providing a rapid online analysis and reaction condition screening was demonstrated by Leung et al.149 for the catalytic oxidation of isopropyl alcohol to acetone using the tetra-N-propylammonium perruthenate/N-methylmorpholine N-oxide system in a glass microreactor based on a radial interdigitated mixer design. The real-time monitoring of the reaction progress using Raman spectroscopy, further assisted by the fast response of the microreactor to varying reaction conditions, allowed serial reaction analyses to be performed in a matter of minutes, which 14594



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Figure 7. In situ EXAFS measurements for investigating the nucleation process of CdSe nanocrystals in a microreactor. (a) Schematics of the microreactor setup. Flows 1−3 correspond to three stock solutions of trioctylphosphine-Se (TOP-Se), Cd, and dodecylamine, respectively. (b) Four different positions along the microchannel and the corresponding temperatures measured in the EXAFS experiment. (c) Fourier transform magnitudes of the k2-weighed Se K-edge EXAFS functions χ(k) at positions A-D and those of TOP-Se solution and CdSe powder measured at room temperature. Reproduced with permission from ref 170. Copyright The American Institute of Physics, 2009.

channel at a 45° angle to the direction of flow. X-ray fluorescence monitored at a 90° angle to the incident radiation was used to measure absorption, which was based on the following considerations: the intensity of the emitted fluorescence radiation had a better signal-to-noise ratio than that of X-ray transmission; it was also in principle linear with absorption at the short path lengths and dilute concentrations used in their experiment. Thus the recorded fluorescence spectra were equivalent to conventional absorption spectra. Small angle X-ray scattering (SAXS) has also been integrated with microreactors to assist in process analysis and kinetic investigation.174−180 Spectroscopic measurements therein were taken by focusing X-ray beam either into the microreactor or into an externally connected flow-through cell embedded in the SAXS instrument. A future development on the integration of X-ray analysis into microreactor processes is envisaged in the miniaturization of the coupled analytical system. Greave and Manz181 reviewed the recent advances in the availability of compact X-ray sources and miniature radioactive sources as promising alternatives to the traditional heavy and bulky X-ray generator. The possibility of generating X-rays of significant intensity directly on the microchip was further demonstrated experimentally under reduced air pressure. 2.5.2. Examples of Application. X-ray absorption spectroscopy is reported to be a valuable method for characterizing the formation/reaction process of nanoparticles in microreactors.169−172 Zinoveva et al.169 developed a PMMA microreactor for the synthesis of cobalt nanoparticles in water with cobalt acetate tetrahydrate as a precursor and sodium borohydrate as a reducing agent. Monochromatic radiation was focused onto the microchannel at different axial locations and the spectra of Co K-edge X-ray absorption near edge structure (XANES) were obtained in fluorescence mode. In situ time-resolved measurements at better than 2 ms time resolution were realized, and different stages of the synthesis could be observed along the microchannel. A similar study, but on the wet chemical synthesis of CdSe nanocrystals, was reported by Uehara and co-workers.170−172 In a capillary microreactor made of Kapton (a commercial polymide) they performed Se K-edge extended X-ray absorption fine structure (EXAFS) measurements along the microchannel to track the early stages of the nucleation process of CdSe nanocrystals, as schematically shown in Figure 7a.170 The obtained EXAFS spectra indicated a rapid increase in

the reaction yield to CdSe nuclei along the microchannel (cf. Figure 7b-c). Chan et al.173 described the use of XAS for probing the kinetics of the CdSe-to-Ag2Se nanocrystal cation exchange reaction in a silicon-based microreactor. Time-resolved Se K-edge absorption spectra clearly revealed the cation exchange of the particles from CdSe to Ag2Se over the course of 100 ms without the presence of long-lived intermediates. The integration of small-angle X-ray scattering opens another possibility of studying nanoparticle formation in microreactors.174−176 This has, for example, been demonstrated by Polte et al.174 for the preparation of gold nanoparticles by the reduction of tetrachloroauric acid using sodium borohydride in aqueous media in a microreactor system. The synthesis was carried out in a microstructured static mixer that was interconnected with a laboratory SAXS instrument via a PTFE capillary. The variable reaction time was achieved by adjusting the length and diameter of the PTFE capillary to enable SAXS analysis at different stages of the reaction. In combination with XANES investigation, the obtained data revealed an initial rapid conversion of the ionic gold precursor into metallic gold nuclei within less than 100 ms, followed by particle growth via coalescence of small nuclei. The combination of SAXS analysis for investigating other types of chemical reactions such as protein folding in microfluidic reactors has also been reported but is mainly in the interest of biological applications.177−179 However, the use of X-ray analysis for monitoring organic synthesis in microreactors will be hampered by the low value of the energy of X-rays emitted by most organic molecules containing light elements.181 2.6. Reaction Monitoring with NMR Spectroscopy. 2.6.1. Integration Scheme. NMR spectroscopy is based on the measurement of absorption or emission of an oscillating radio frequency (RF) radiation that interacts with a collection of atomic nuclei immersed in a strong external magnetic field.182 The superior site selectivity of NMR spectroscopy makes it probably the most powerful tool for determining molecular structure of the majority of organic and inorganic compounds, for example, providing structural information and also data on both intermolecular and intramolecular dynamics.183 However, the main limitation in NMR spectroscopy is the fact that the low energy scales involved would lead to a rather low sensitivity as compared to other spectroscopic methods like fluorescence and UV−vis spectroscopy in terms of the minimum sample amount for an analysis.36,183 Over recent years, this adverse 14595



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Figure 8. Selected examples of integrating NMR spectroscopic detection into microchemical systems. (a). Microreactor interfaced with a conventional NMR sample tube for the real-time monitoring of the reaction progress: (left) a schematic diagram of the experimental setup; (right) an illustration of the microreactor design. Reproduced with permission from ref 184. Copyright The Japan Society for Analytical Chemistry, 2007. (b) Micromixer externally connected with a solenoidal-type microcoil NMR cell suitable for the investigation of liquid phase kinetics. A, B, C, and D refer to syringe pumping system, micromixer, microcoil NMR cell, and outlet reservoir, respectively. Arrows indicate the flow direction. Reproduced with permission from ref 188. Copyright The American Chemical Society, 2003. (c) Photograph of a glass microreactor chip with integrated planar NMR microcoils on top of the chip (left) and a schematic cross-section of the microcoil with three windings (right). Reproduced with permission from ref 192. Copyright The Royal Society of Chemistry, 2005. (d) Photograph of the two halves of a microfluidic NMR detection chip based on the stripline design showing the copper construction of the λ/2 resonator on one-half and the microchannel on the other half. A constricted strip section was constructed by removal of narrow copper lines shown in this figure. The two halves were bonded together in the final device. Online NMR analysis can be accomplished by fluidically connecting the chip to a microreactor. Reproduced with permission from ref 195. Copyright The American Institute of Physics, 2008.

effect has been successfully circumvented mainly through the development of strong magnet and more sensitive NMR probes.51 A simple way to interface a microreactor with a NMR instrument is realized by fitting the microreactor into a conventional NMR flow probe. Takahashi et al.184 presented such a design for the purpose of the real-time reaction monitoring in a microreactor. As schematically shown in Figure 8a, the developed microreactor is composed of a plastic part and a glass part that is in fluidic interconnection with each other. The plastic part has three inlet ports for reagent solutions and one outlet port for the exhaust. The glass part, which was made from laminated three Pyrex glass plates, consists of two replications of one Y-shaped microchannel for mixing plus one serpentine microchannel for reaction. Another serpentine microchannel for NMR detection was arranged at the end section before it led to the outlet port. The laminated glass substrates were cut into a rod shape so as to be inserted into a conventional 5-mm NMR sample tube. Deuterated solvents were added into the gap between the microreactor and the sample tube acting as a NMR lock. They found that NMR signal intensity with this design was lower than that obtained with a standard sample tube, but the system performance was already sufficient for the real-time monitoring of the intermediates or products in some organic reactions.

NMR spectroscopy is relatively insensitive compared to other spectroscopic methods mentioned above. An effective approach to improve the mass sensitivity of NMR detection is to use microcoil flow probes.51 The detection limits in the low nanomole range can be reached for probes equipped with solenoidal-type microcoils having an active detection volume well below 1 μL, which far exceeds the mass sensitivity of conventional macroscopic NMR probes.185−187 Microreactors can be also connected with microcoil flow probes to realize online NMR detection. An example of such integration was given by Kakuta et al.,188 who developed a micromixer-based time-resolved NMR to follow protein-folding kinetics. A sketch of their experimental setup is shown in Figure 8b. A solenoid RF microcoil wrapped around a bubble-type NMR flow cell with an observation volume of about 800 nL was connected to a micromixer via a capillary of fixed length. The microcoil was immersed in a perfluorinated organic liquid for magnetic susceptibility matching purposes. Time-dependent structure change in protein conformation could then be inferred from the obtained NMR spectra. Multiple NMR microcoil flow probes can be connected with a microreactor in a series fashion to enable multiple measurements, as reported by Ciobanu et al.189 In their design, two reactant solutions are mixed in a micromixer, and the resulting homogeneous mixture passes through a capillary around which are wound multiple, physically distinct 14596



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configuration, which bypasses the trade-off between resolution and sensitivity of on-chip NMR spectroscopic detection.193−196 In this new design, the RF current is carried by a thin metal strip in close contact with the sample in a microchannel (cf. Figure 8d). Ground planes made from the same material as the strip are applied on both backsides of the microfluidic chip to confine the RF radiation.193 A silicon-based microfluidic chip integrated with a planar stripline probe for NMR analysis was shown to bring the resolution in ethanol (line width of

Integration of Microreactors with Spectroscopic Detection for Online

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