Financial support provided by National Science Foundation (CAREER award CHE-0847262. We thank Vapourtec Ltd and Enwave Optronics for equipment support, and Daniel Daleb of the University of Connecticut for his assistance in construction of the flow cell apparatus.

1. Find Suitable Signals for Reaction Monitoring
<ol>
	<li>Obtain Raman spectra for all starting materials and the product.</li>
	<li>Overlay spectra and identify an intense band that is unique to the product.</li>
	<li>Use this Raman band to monitor the progress of the reaction. A band at 1,608&#160;cm-1 was selected in this case.</li>
</ol>
2. Set up the Flow Cell
<ol>
	<li>Obtain a suitable flow cell. Here use one with the following dimensions: width of 6.5 mm, height of 20 mm, and a path length of 5 mm (Figure 2A).</li>
	<li>Place the flow cell in a container that provides an environment free of ambient light.</li>
	<li>Connect tubing to the inlet and outlet of the flow cell (in this case 1 mm I.D. PFA tubing).</li>
</ol>
3. Interface the Raman Spectrometer with the Flow Cell
<ol>
	<li>Obtain a suitable Raman spectrometer with a flexible optical assembly that can be placed in close proximity to the flow cell.</li>
	<li>Place the optical assembly through a suitably sized aperture in the box containing the flow cell assembly (Figure 2B).</li>
	<li>Slide the optical assembly until it touches the flow cell and then pull it back leaving a gap of ~ 2 mm.</li>
	<li>Fill the flow cell with 100% acetone.</li>
	<li>Turn on the Raman spectrometer and acquire spectra in continuous-scan mode.</li>
	<li>Focus the laser by gently moving the light pipe a fraction at a time. Keep moving the light pipe until the signal is at its greatest intensity and the peaks are sharp and well defined.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52393/52393fig2.jpg" /> 
Figure 2. (A) Flow cell and (B) Raman interface used. <a href="https://www.jove.com/files/ftp_upload/52393/52393fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Prepare Reagent and Solvent Solutions
<ol>
	<li>Add salicylaldehyde (6.106 g, 50 mmol, 1 equiv) and ethyl acetoacetate (6.507 g, 50 mmol, 1 equiv) to a 50 ml volumetric flask.</li>
	<li>Add ethyl acetate to a total volume of 50 ml and then thoroughly mix the contents.</li>
	<li>Transfer a 10 ml aliquot of the stock solution to a 20 ml glass vial containing a magnetic stir bar. Label this vial &#8220;reagent.&#8221;</li>
	<li>In a 100 ml bottle place 90 ml of ethyl acetate. Label this bottle &#8220;solvent&#8221;. In a 100 ml bottle place 90 ml of acetone. Label this bottle &#8220;solvent intercept&#8221;.</li>
</ol>
5. Prepare the Flow Apparatus
<ol>
	<li>Ensure that the flow unit has at least two pumps and label them &#8220;P1&#8221; and &#8220;P2&#8221;. Identify solvent and reagent inlet lines for each pump. Place the exit lines from the &#8220;collect&#8221; and &#8220;waste&#8221; lines into two individual 100 ml bottles labeled &#8220;product&#8221; and &#8220;waste&#8221;, respectively.</li>
	<li>As a reactor, use a 10 ml capacity PFA coil capable of being heated.</li>
	<li>Connect the tube exiting P1 to the inlet of the PFA reactor coil.</li>
	<li>Install a three-port polyether ether ketone (PEEK) tee-mixer after the reactor coil.</li>
	<li>Connect the tube exiting P2 to the tee-mixer, 180&#176; from the reactor coil exit tubing. Connect a piece of tubing to the third port of the Tee-mixer. On the other end of this tube place a back-pressure regulator.</li>
	<li>Connect a line from the output of the back-pressure regulator to the input of the flow cell. Connect a line from the output of the flow cell to the &#8220;waste/collect&#8221; switch.</li>
	<li>Prime the solvent lines for both P1 and P2 as well as the reagent line for P1 with solvent. Move the reagent line for P1 from the solvent bottle to the reagent bottle.</li>
	<li>Using P1, pass ethyl acetate through the reactor coil at 2 ml/min until it is filled. Pass acetone through P2 at a flow rate of 2 ml/min for 2 min.</li>
	<li>Adjust the solvent flow rates for both P1 and P2 to 1 ml/min. Set the back-pressure regulator to a pressure of 7 bar. Set the reactor coil temperature to the desired temperature.</li>
	<li>Double-check the equipment is configured as shown in the schematic in Figure 3.</li>
	<li>Once the system reaches constant temperature and pressure, check for leaks and then run the reaction.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52393/52393fig3.jpg" /> 
Figure 3. Schematic of the equipment configuration used for reaction monitoring experiments. <a href="https://www.jove.com/files/ftp_upload/52393/52393fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Monitor the Reaction
<ol>
	<li>Take a background scan of the ethyl acetate/acetone solvent system as it passed through the flow cell. This will be automatically subtracted from all subsequent scans.</li>
	<li>Configure the spectrometer to take scans every 15 sec (in this case the Raman spectrometer was set to a 10 sec integration time, boxcar = 3, and average = 1).</li>
	<li>Inject piperidine (0.05 ml, 0.05 mmol, 0.1 equiv) all at once into the glass vial labeled &#8220;reagent&#8221;.</li>
	<li>After thoroughly mixing, switch P1 from &#8220;solvent&#8221; to &#8220;reagent.&#8221; Set the exit stream to &#8220;collect.&#8221;</li>
	<li>When all material is completely loaded, switch P1 from &#8220;reagent&#8221; back to &#8220;solvent.&#8221; Continue flowing solvent through the reactor coil for another 30 min. Once this time has elapsed, turn off the heating.</li>
	<li>Turn pumps P1 and P2 off when the reactor coil temperature has cooled to below 50 &#176;C.</li>
</ol>
7. Analyze the Data
<ol>
	<li>Export the Raman spectrometer data to a spreadsheet and plot Raman intensity at 1,608 cm-1 vs. time.</li>
	<li>To optimize conditions, perform the reaction across a number of flow rates and reactor temperatures in an iterative manner.</li>
	<li>Overlay plots of Raman intensity at 1,608 cm-1 vs. time. 
	Note: Higher Raman intensity correlates with higher product conversion.</li>
</ol>
8. Run the Reaction Using Optimized Conditions
<ol>
	<li>Having screened various conditions (varying flow rates / reactor temperatures), run the reaction using the optimized conditions to afford the highest product conversion.&#160;</li>
</ol>
9. Isolate the Product
<ol>
	<li>Take the contents of the product flask and pour it into a beaker containing 100 ml of ice and 20 ml of 2 M HCl.</li>
	<li>Rinse the product flask with a minimal amount of ethyl acetate (2 ml) and transfer to the beaker.</li>
	<li>Stir the icy mixture until all the ice is completely melted.</li>
	<li>Set up a filtration system with a Hirsch funnel, side-arm flask, rubber collar and a length of rubber vacuum tubing.</li>
	<li>Filter the resulting precipitate under vacuum, rinse with cold diethyl ether (10 ml) and allow it to dry under a heat lamp (2-3 hr) or dry O/N under vacuum.</li>
	<li>Confirm the identity of the product by 1H nuclear magnetic resonance (NMR) spectroscopy using CDCl3 as the solvent. For a 500 MHz NMR spectrometer, the 1H NMR data of 3-acetylcoumarin is as follows: &#948; = 2.73 (s, 3 H) 7.31 - 7.40 (m, 2 H) 7.65 (ddd, J = 7.53, 4.37, 2.60 Hz, 2 H) 8.51 (s, 1 H) ppm, 13C NMR data: &#948; = 30.84 (CH3) 117.00 (CH) 118.56 (C) 124.86 (CH) 125.27 (CH) 130.51 (CH) 134.68 (C) 147.74 (CH) 155.64 (C) 159.52 (C) 195.77 (C) ppm.</li>
</ol>

<ol>
	<li>Wiles, C., Watts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Micro Reaction Technology in Organic Synthesis. , (2011).</li><li>van den Broek, S. A. M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Flow+Production+of+Thermally+Unstable+Intermediates+in+a+Microreactor+with+Inline+IR-Analysis:+Controlled+Vilsmeier&#8211;Haack+Formylation+of+Electron-Rich+Arenes.">Continuous Flow Production of Thermally Unstable Intermediates in a Microreactor with Inline IR-Analysis: Controlled Vilsmeier&#8211;Haack Formylation of Electron-Rich Arenes.</a> Org. Process Res. Dev. 16 (5), 934-938 (2012).</li><li>Baxendale, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integration+of+flow+reactors+into+synthetic+organic+chemistry.">The integration of flow reactors into synthetic organic chemistry.</a> J. Chem. Technol. Biotechnol. 88 (4), 519-552 (2013).</li><li>Malet-Sanz, L., Susanne, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Flow+Synthesis.">Continuous Flow Synthesis.</a> A Pharma Perspective J. Med. Chem. 55 (9), 4062-4098 (2012).</li><li>Hartwig, J., Metternich, J. B., Nikzad, N., Kirschning, A., Ley, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+flow+chemistry:+a+discovery+tool+for+new+chemical+reactivity+patterns.">Continuous flow chemistry: a discovery tool for new chemical reactivity patterns.</a> Org. Biomol. Chem. 12 (22), 3611-3615 (2014).</li><li>De Beer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+and+Raman+spectroscopy+for+the+in-process+monitoring+of+pharmaceutical+production+processes.">Near infrared and Raman spectroscopy for the in-process monitoring of pharmaceutical production processes.</a> Int. J. Pharm. 417, 32-47 (2001).</li><li>McMullen, J. P., Jensen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+microreactors+for+reaction+automation:+New+approaches+to+reaction+development.">Integrated microreactors for reaction automation: New approaches to reaction development.</a> Annu. Rev. Anal. Chem. 3, 19-42 (2010).</li><li>Moore, J. S., Jensen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+multitrajectory+method+for+reaction+optimization+in+a+microfluidic+system+using+online+IR+analysis.">Automated multitrajectory method for reaction optimization in a microfluidic system using online IR analysis.</a> Org. Process Res. Dev. 16 (8), 1409-1415 (2012).</li><li>Carter, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ReactIR+Flow+Cell:+A+New+Analytical+Tool+for+Continuous+Flow+Chemical+Processing.">ReactIR Flow Cell: A New Analytical Tool for Continuous Flow Chemical Processing.</a> Org. Process Res. Dev. 14 (2), 393-404 (2010).</li><li>Ferstl, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inline+Analysis+in+Microreaction+Technology:+A+Suitable+Tool+for+Process+Screening+and+Optimization.">Inline Analysis in Microreaction Technology: A Suitable Tool for Process Screening and Optimization.</a> Chem. Eng. Technol. 30 (3), 370-378 (2007).</li><li>Benito-Lopez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+fiber-based+on-line+UV/Vis+spectroscopic+monitoring+of+chemical+reaction+kinetics+under+high+pressure+in+a+capillary+microreactor.">Optical fiber-based on-line UV/Vis spectroscopic monitoring of chemical reaction kinetics under high pressure in a capillary microreactor.</a> Chem. Commun. (22), 2857-2859 (2005).</li><li>Gökay, O., Albert, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+single+to+multiple+microcoil+flow+probe+NMR+and+related+capillary+techniques:+a+review.">From single to multiple microcoil flow probe NMR and related capillary techniques: a review.</a> Anal. Bioanal. Chem. 402 (2), 647-669 (2012).</li><li>Jones, C. J., Larive, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Could+smaller+really+be+better?+Current+and+future+trends+in+high-resolution+microcoil+NMR+spectroscopy.">Could smaller really be better? Current and future trends in high-resolution microcoil NMR spectroscopy.</a> Anal. Bioanal. Chem. 402 (1), 61-68 (2012).</li><li>Mozharov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Method+for+Kinetic+Studies+in+Microreactors+Using+Flow+Manipulation+and+Noninvasive+Raman+Spectrometry.">Improved Method for Kinetic Studies in Microreactors Using Flow Manipulation and Noninvasive Raman Spectrometry.</a> J. Am. Chem. Soc. 133 (10), 3601-3608 (2011).</li><li>Chaplain, G., Haswell, S. J., Fletcher, P. D. I., Kelly, S. M., Mansfield, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+Raman+flow+cell+for+monitoring+continuous+flow+reactions.">Development and evaluation of a Raman flow cell for monitoring continuous flow reactions.</a> Aust. J. Chem. 66 (2), 208-212 (2013).</li><li>Browne, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+flow+reaction+monitoring+using+an+on-line+miniature+mass+spectrometer.">Continuous flow reaction monitoring using an on-line miniature mass spectrometer.</a> Rapid Comm. Mass. Spectrosc. 26 (17), (1999).</li><li>Koster, S., Verpoorte, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+microfluidic+analysis+coupled+with+electrospray+mass+spectrometry:+An+overview.">A decade of microfluidic analysis coupled with electrospray mass spectrometry: An overview.</a> Lab Chip. 7 (11), 1394-1412 (2007).</li><li>Leadbeater, N. E., Schmink, J. R., Hamlin, T. A., de la Hoz, A., Loupy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+Monitoring+Reactions+Performed+Using+Microwave+Heating.">Tools for Monitoring Reactions Performed Using Microwave Heating.</a> Microwaves in Organic Synthesis. 1, 327-376 (2012).</li><li>Leadbeater, N. E., Schmink, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Raman+spectroscopy+as+a+tool+for+in+situ.+monitoring+of+microwave-promoted+reactions.">Use of Raman spectroscopy as a tool for in situ. monitoring of microwave-promoted reactions.</a> Nature Protoc. 3 (1), 1-7 (2008).</li><li>Schmink, J. R., Holcomb, J. L., Leadbeater, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Raman+spectroscopy+as+an+In+Situ.+Tool+to+Obtain+Kinetic+Data+for+Organic+Transformations.">Use of Raman spectroscopy as an In Situ. Tool to Obtain Kinetic Data for Organic Transformations.</a> Chem. Eur. J. 14 (32), 9943-9950 (2008).</li><li>Hamlin, T. A., Leadbeater, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+spectroscopy+as+a+tool+for+monitoring+mesoscale+continuous-flow+organic+synthesis:+Equipment+interface+and+assessment+in+four+medicinally-relevant+reactions.">Raman spectroscopy as a tool for monitoring mesoscale continuous-flow organic synthesis: Equipment interface and assessment in four medicinally-relevant reactions.</a> Beilstein J. Org. Chem. 9, 1843-1852 (2013).</li><li>Kelly, C. B., Lee, C., Leadbeater, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+for+continuous-flow+processing+of+reactions+that+involve+the+in+situ.+formation+of+organic+products.">An approach for continuous-flow processing of reactions that involve the in situ. formation of organic products.</a> Tetrahedron Lett. 52 (2), 263-265 (2011).</li><li>Wren, S. N., Donaldson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glancing-angle+Raman+spectroscopic+probe+for+reaction+kinetics+at+water+surfaces.">Glancing-angle Raman spectroscopic probe for reaction kinetics at water surfaces.</a> Phys. Chem. Chem. Phys. 12, 2648-2654 (2010).</li><li>Leadbeater, N. E., Smith, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Monitoring+of+Microwave-Promoted+Suzuki+Coupling+Reactions+Using+in+Situ.+Raman+Spectroscopy.">Real-Time Monitoring of Microwave-Promoted Suzuki Coupling Reactions Using in Situ. Raman Spectroscopy.</a> Org. Lett. 8 (20), 4589-4591 (2006).</li></ol>

The authors have nothing to disclose.

The ease in which the Raman spectrometer can be interfaced with the flow unit makes this inline technique valuable for reaction monitoring. A number of reaction variables can be probed in an expedited manner, allowing the user to arrive at optimized reaction conditions faster than when using offline methods. Application of the techniques described herein also allows for monitoring of the formation of side products, assuming a suitable band can be found. Conditions can be screened and selected, which allow both for the hi...

By using continuous-flow processing, chemists are finding that they can perform a range of chemical reactions safely, effectively, and with ease1,2. As a result, flow chemistry equipment is becoming an integral tool for running reactions both in industrial settings as well as research labs in academic institutions. A wide variety of synthetic chemistry transformations have been carried out in flow reactors3,4. In select cases, reactions that do not work in batch have been shown to proceed smoothly under continuous-flow conditions5. For both reaction optimization and quality control, incorporation of in-line reaction monitoring with flow processing offers significant advantages. In-line monitoring provides continuous analysis with real-time response to actual sample conditions. This is faster and, in some cases, more reliable than comparable off-line techniques. A number of in-line analytical techniques have been interfaced with flow reactors7. Examples include infrared8,9, UV-visible10,11, NMR12,13, Raman spectroscopy14,15, and mass spectrometry16,17.
Our research group has interfaced a Raman spectrometer with a scientific microwave unit18. Using this, a range of reactions have been monitored from both a qualitative19 and quantitative20 standpoint. Building on this success, we have recently interfaced our Raman spectrometer with one of our continuous-flow units and employed it for in-line reaction monitoring of a number of key medicinally-relevant organic transformations.21 In each case it was possible to monitor the reactions and also in one example, by means of a calibration curve, we could determine product conversion from Raman spectral data. In Here we describe how to set up the apparatus and use it to monitor reactions. We use the piperidine-catalyzed synthesis of 3-acetylcoumarin (1) from salicylaldehyde with ethyl acetoacetate (Figure 1) as the model reaction here.
<img alt="Figure 1" src="/files/ftp_upload/52393/52393fig1.jpg" /> 
Figure 1. Base catalyzed condensation reaction between salicylaldehyde and ethyl acetoacetate to yield 3-acetylcoumarin (1). <a href="https://www.jove.com/files/ftp_upload/52393/52393fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Salicylaldehyde</td><td>Sigma-Aldrich</td><td>S356</td><td>Reagent Grade, 98%</td></tr><tr><td>Ethyl acetoacetate</td><td>Acros Organics</td><td>117970010</td><td>99%</td></tr><tr><td>Piperidine</td><td>Sigma-Aldrich</td><td>104094</td><td>Reagent Plus, 99%</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>320331</td><td>ACS Reagent, 37%</td></tr><tr><td>Ethyl acetate</td><td>Sigma-Aldrich</td><td>34858</td><td>CHROMASOLV, for HPLC, &gt;99.7%</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>650501</td><td>CHROMASOLV, for HPLC, &gt;99.9%</td></tr><tr><td>Flow cell</td><td>Starna Cells</td><td></td><td>583.65.65-Q-5/Z20</td></tr><tr><td>Flow unit</td><td>Vapourtec</td><td></td><td>E-series system</td></tr><tr><td>Raman spectrometer</td><td>Enwave Optronics Inc</td><td></td><td>Model EZRaman-L</td></tr></tbody></table>

real-time monitoring of reactions performed using continuous-flow processing: the preparation of 3-acetylcoumarin as an example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

The continuous-flow preparation of 3-acetylcoumarin was chosen as a representative reaction for in-line monitoring. In batch, the reaction proceeds well when using ethyl acetate as the solvent. However, the product (1) is not completely soluble at RT. To prevent potential clogging of the back-pressure regulator, as well as mitigate the risk of having solid particles in the flow cell which would perturb signal acquisition, we used a technique we developed previously for this and other reactions22</su...

Watch this Scientific Journal Video about Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example at JoVE.com

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

real-time-monitoring-reactions-performed-using-continuous-flow

Research

JoVE Journal

Chemistry

9.7K Views.  University of Connecticut.  Real-time monitoring zorgt voor een snelle optimalisatie van de reacties uitgevoerd met behulp van een continue flow verwerking. Hier is de bereiding van 3-acetylcumarine wordt als voorbeeld gebruikt. De inrichting voor het uitvoeren van in situ-Raman bewaking wordt beschreven, evenals de stappen die nodig zijn om de reactie te optimaliseren. 

Video: Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

Biochemistry

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

We describe the use of a stopped-flow instrument to investigate both the reductive and oxidative half-reactions of Aspergillus fumigatus siderophore A (SidA), a flavin-dependent monooxygenase. We then show the spectra corresponding to the species in the reaction of SidA and we calculate the rate constants for their formation.

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

Bioengineering

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Temperature-programmed Deoxygenation of Acetic Acid on Molybdenum Carbide Catalysts

Temperature programmed reaction (TPRxn) is a simple yet powerful tool for screening solid catalyst performance at a variety of conditions. A TPRxn system includes a reactor, furnace, gas and vapor sources, flow control, instrumentation to quantify reaction products (e.g., gas chromatograph), and instrumentation to monitor the reaction in real time (e.g., mass spectrometer). Here, we apply the TPRxn methodology to study molybdenum carbide catalysts for the deoxygenation of acetic acid, an important reaction among many in the upgrading/stabilization of biomass pyrolysis vapors. TPRxn is used to evaluate catalyst activity and selectivity and to test hypothetical reaction pathways (e.g., decarbonylation, ketonization, and hydrogenation). The results of the TPRxn study of acetic acid deoxygenation show that molybdenum carbide is an active catalyst for this reaction at temperatures above ca. 300 &#176;C and that the reaction favors deoxygenation (i.e., C-O bond-breaking) products at temperatures below ca. 400 &#176;C and decarbonylation (i.e., C-C bond-breaking) products at temperatures above ca. 400 &#176;C.

Presented here is a protocol for the operation of a micro-scale temperature-programmed reactor for evaluating the catalytic performance of molybdenum carbide during acetic acid deoxygenation.

Temperature programmed reaction (TPRxn) is a simple yet powerful tool for screening solid catalyst performance at a variety of conditions. A TPRxn system ...

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described. Triplicate runs of 48 parallel experiments are reported, varying concentration, solvent and the excess of some of the reagents. The isolation of the product was achieved by a simple filtration and wash procedure. The highest yield obtained was 66% from 0.4 M methanol with 1.2 eq. of imine. This is significantly above the 49% yield obtained from the initial reaction under equimolar concentration at 0.4 M in methanol. Methanol solutions with reagent concentrations of 0.4M or 0.2M gave superior yields while all solvent systems at 0.07M performed poorly. At 0.2M, methanol and ethanol/methanol (60/40) mixtures were statistically equally good while THF/methanol (60/40) was poor and acetonitrile/methanol (60/40) was intermediate. Good reproducibility of the precipitate yields was obtained in these replicate experiments, allowing for subtle interaction effects to be positively identified.

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

Biology

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Utilization of Stop-flow Micro-tubing Reactors for the Development of Organic Transformations

A new reaction screening technology for organic synthesis was recently demonstrated by combining elements from both continuous micro-flow and conventional batch reactors, coined stop-flow micro-tubing (SFMT) reactors. In SFMT, chemical reactions that require high pressure can be screened in parallel through a safer and convenient way. Cross-contamination, which is a common problem in reaction screening for continuous flow reactors, is avoided in SFMT. Moreover, the commercially available light-permeable micro-tubing can be incorporated into SFMT, serving as an excellent choice for light-mediated reactions due to a more effective uniform light exposure, compared to batch reactors. Overall, the SFMT reactor system is similar to continuous flow reactors and more superior than batch reactors for reactions that incorporate gas reagents and/or require light-illumination, which enables a simple but highly efficient reaction screening system. Furthermore, any successfully developed reaction in the SFMT reactor system can be conveniently translated to continuous-flow synthesis for large scale production.

A protocol for organic reaction screening using stop-flow micro-tubing (SFMT) reactors employing gaseous reactants and/or visible-light mediated reactions is presented.

A new reaction screening technology for organic synthesis was recently demonstrated by combining elements from both continuous micro-flow and conventional ...

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

Samenvatting

Meer video's verkennen