Introduction

Microwave-promoted synthesis is an area of increasing interest in both academic and industrial laboratories as it can enhance the rate of reactions and in many cases improve product yields^{1, 2, 3, 4, 5, 6, 7}. A problem with performing reactions using microwave apparatus is that monitoring its progress generally requires stopping it, allowing the reaction mixture to cool and then using standard analysis techniques, such as IR and NMR spectroscopy. Therefore, optimization of reaction conditions such as time and temperature is often a matter of trial and error. With conventional heating, the reaction can be slow and aliquots removed and analyzed over time, but with microwave heating, the reaction may be complete within a matter of minutes and accessing a sealed vessel during a reaction is not possible. There have been some attempts to monitor reactions under microwave irradiation. Neutron and X-ray scattering have been used for studying the preparation of inorganic materials^{8, 9}. Recently, Pivonka and Empfield have reported the use of Raman spectroscopy as a tool for monitoring organic transformations¹⁰. Raman spectroscopy is a particularly useful technique for in situ spectroscopy because it relies on light scattering and hence no mechanical interaction with the sample. Building on this work, we have recently reported an apparatus for the monitoring of reactions under microwave irradiation using in situ Raman spectroscopy (Fig. 1)¹¹. We have interfaced a commercially available Raman spectrometer (Fig. 2) with a commercially available scientific microwave unit. The apparatus proves to be not only a very valuable tool for monitoring reactions while they run but also for rapid optimization of reaction conditions^{12, 13, 14}.

Figure 1: Apparatus for the monitoring of reactions under microwave irradiation using in situ Raman spectroscopy.

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Figure 2: Raman module.

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The basis behind Raman spectroscopy is the idea that when an incident photon strikes a molecule, an electron in that molecule may either absorb or impart some energy upon that photon (Fig. 3). This energetically altered photon is then detected as a different wavelength. Most photons strike a molecule elastically, not altering their wavelength. This is termed Rayleigh scattering. Most photons (>99.9%) simply bounce back, unaltered, and give no information regarding the molecule. When a photon imparts some of its energy onto an electron in a low-energy virtual state, the detected wavelength of the returning photon is longer. This is termed the Stokes shift. When a photon absorbs some energy from an electron that is in a high-energy virtual state, the detected wavelength is shorter. This is termed anti-Stokes shift.

Figure 3: Fundamentals of Raman spectroscopy.

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In this protocol, we outline the practicalities of interfacing a Raman spectrometer with the microwave unit and then how the apparatus can be used for monitoring a reaction. We have chosen the preparation of a coumarin derivative as our case study here. Coumarins are a group of important natural compounds, and have been found to have multi-biological activities such as anti-HIV, antitumor, antihypertension, antiarrhythmia, antiosteoporosis, pain relief, preventing asthma, and antisepsis^{15, 16, 17}. Natural products like esculetin, fraxetin and daphnetin are recognized as inhibitors not only of the lipoxygenase and cycloxygenase enzymic systems, but also of the neutrophil-dependent superoxide anion generation¹⁸. Synthetic derivatives of coumarin have found uses as anticoagulants (warfarin) and rodenticides (brodifacoum). Coumarins can be prepared using a number of synthetic routes¹⁹ including the Pechmann condensation (reaction of phenols with -keto esters), Perkin reaction (aldol condensation of aromatic aldehydes and acid anhydrides) and Knovenagel condensation (base-catalyzed reaction of an aldehyde or ketone with a dicarbonyl compound). Microwave heating has been used as a tool for the synthesis of coumarins^{20, 21, 22, 23, 24}. As our model reaction, we chose the reaction of salicylaldehyde with ethylacetoacetate to yield 3-acetylcoumarin in a method similar to that first reported by Bogdal (Fig. 4)²⁵. We use a small volume of piperidine as the catalyst and ethyl acetate as the solvent. When using the in situ Raman monitoring tool, it is essential that all the reagents and the products of the reaction under investigation are completely soluble in the solvent of choice. As Raman spectroscopy works on the basis of light scattering, any heterogeneity in the reaction mixture will severely attenuate the signal. Another important concept to account for while monitoring reactions using Raman spectroscopy is the notion that the Stokes shift (which is being monitored) is inversely proportional to temperature. Therefore, product formation based simply on peak disappearance cannot be assumed. This is due to the fundamental manner in which Raman spectroscopy probes a molecule; that is it excites a molecule in the lowest-energy state. As temperature increases, there is a smaller population of molecules in that ground state to be excited, and so the signal intensity drops. Thus, monitoring growth of the signals due to product formation is the best way of monitoring reactions. As organic chemists are generally less familiar with Raman spectroscopy as compared with other analytical techniques, it is worth noting that the Raman spectra of a wide range of compounds are available on the web from a number of sources. (Raman spectra for a wide range of commercially available organics can be found at www.sigmaaldrich.com. For other organic compounds, Raman spectra may be found (free of charge) at the Spectral Database for Organic Compounds (SDBS), http://riodb01.ibase.aist.go.jp/sdbs/cgi-bin/cre_index.cgi?lang=eng.)

Figure 4: Reaction chosen for illustration of the in situ monitoring technique.

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When preparing to study a reaction using the in situ monitoring tool, it is prudent to first obtain the Raman spectra of the starting materials and products. It is then possible to find a clearly defined signal that either grows in (as product is formed) or out (as starting materials are consumed) as a function of time. Upon inspection of the Raman spectra of salicylaldehyde, ethylacetoacetate and 3-acetylcoumarin, we found that there are two clearly defined signals in the product (1,596 cm^{- 1}, 1,643 cm^{- 1}) that are not present in the starting materials so it is these that we decided to follow in our reaction. We heated the reaction mixture to 130 °C in our microwave unit and held it at this temperature until a total time of 20 min had elapsed. The composite temperature, pressure and microwave power profiles for the reaction are shown in Figure 5. We started the Raman spectrometer at the same time as the microwave unit and recorded spectra every 6–8 s. Clearly, any signals owing to starting materials or product would be masked by those due to the solvent. To overcome this problem, we subtracted the time = 0 spectrum from subsequent spectra of the series. As a result, features such as solvent and functional groups that are not impacted by the reaction do not appear in the profile. This is similar to taking a background scan when performing a conventional IR or Raman spectrum. We can monitor in real time the growth of the signals due to product formation. Shown in Figure 6 are the screenshots of the real-time monitoring at the start of the reaction (Fig. 6a), when the reaction mixture reaches 130 °C (Fig. 6b) and after 5 min (Fig. 6c). The signals at 1,596 cm^{- 1} and 1,643 cm^{- 1} are clearly seen in the last. Shown in Figure 7 are the first 24 Raman scans recorded, focusing on the signals at 1,596 cm^{- 1} and 1,643 cm^{- 1}. In Figure 8, the intensity of the signal at 1,643 cm^{- 1} is plotted as a function of time and shows that the reaction reaches completion after a total time of 250 s.

Figure 5: Composite temperature, pressure and microwave power profiles for the reaction.

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Figure 6: Screenshots of the real time Raman monitoring.

(a) At the start of the reaction; (b) when the reaction mixture reaches 130 °C; (c) after 5 min.

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Figure 7: First 24 Raman scans recorded during the course of the reaction.

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Figure 8: Intensity of the signal at 1,643 cm^{- 1} is plotted as a function of time.

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This example shows how, using the apparatus presented here, it is possible to watch a microwave promoted reaction spectroscopically, determine when it reaches completion and thus use it as a tool for rapid reaction optimization. A glance through the literature shows that most microwave promoted reactions are run for 5–20 min. From our experience using the in situ monitoring, in many cases the reactions are complete in far shorter times. Indeed many reactions are complete by the time they reach the target temperature. Although reaction products are often thermally stable, in cases where they are not then decomposition can be a significant problem. If, using the in situ monitoring, it is possible to see in real time when the reaction is complete, it would be possible to stop it before the onset of decomposition, this being another advantage of the technique. Finally, it can be used in more fundamental studies into the effect of microwave power on the rate and outcome of a reaction, something we in our group are actively pursuing.

Materials

Reagents

• Cyclohexane (Aldrich, cat. no. 227048)
  Caution Highly flammable, harmful, dangerous for the environment.
• Salicylaldehyde (Aldrich, cat. no. S356)
  Caution Harmful.
• Ethylacetoacetate (Aldrich, cat. no. 537349)
  Caution Flammable and toxic.
• Piperidine (99%; Aldrich, cat. no. 104094)
  Caution Harmful.
• Ethyl acetate (Mallinckrodt, cat. no. 4992)
  Caution Flammable and harmful.

Equipment

• Discover S-Class microwave unit (CEM Corporation, cat. no. 925475)
• Raman module (EZRaman-MAS-InSitu; Enwave Optronics)
• Quartz light pipe (Enwave Optronics)
• 10-ml Reaction vessels (CEM Corporation, cat. no. 908035)
• Caps for reaction vessels (CEM Corporation, cat. no. 908140)
• Teflon-coated magnetic stir bar (1 cm; CEM Corporation, cat. no.162810)
• Spill cup with a side hole (CEM Corporation, cat. no. 169210)
• Disposable glass pipettes (53/4"; Fisher, cat. no.3-678 20A)
• Büchner funnel (56 mm; Fisher, cat.no.10-356B)
• Filter paper (Whatman, cat. no. 1001 055)
• PC running Windows XP or Vista
• Synergy software (CEM Corporation)
• EZ Raman software (Enwave Optronics)

Procedure

1. Points from here (point 1) up to and including point 10 are related to Interface of Raman apparatus with CEM Discover S-ClassRemove the attenuator on the microwave unit and then the protective spill cup found inside the cavity (Fig. 9a).

   Figure 9: Interfacing the Raman and microwave modules.

   

   (a) Attenuator and spill cup removed from microwave unit. (b) Top of microwave unit opened to access side port and capping nut. (c) Teflon insert removed by pushing into microwave cavity. (d) Attachment of quartz light-pipe into the Raman laser unit. (e) Quartz light-pipe placed into the side port of the microwave cavity.

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2. Carefully remove the top of the Discover S-Class to access the side port (Fig. 9b).
3. Remove the capping nut on the outside left side of the cavity (Fig. 9b).
4. Remove the Teflon insert by pushing it into the cavity (Fig. 9c).
5. Replace the cover to the Discover and insert the spill cup with a side hole, ensuring that the hole lines up with that of the access port on the Discover S-Class.
6. On the Raman system, connect the 6" quartz light pipe to the laser by gently tightening the provided locking nut (Fig. 9d).
7. Gently slide the light pipe into the Discover S-Class through the access port, ensuring that the spill cup is still properly aligned (Fig. 9e).
8. Once the light pipe is in place, replace the attenuator on the microwave and insert an empty 10-ml glass microwave tube to act as a guide.
9. Continue sliding the light pipe into the microwave until it touches the tube, then pull it back until there is a space of 2–5 mm between the light pipe and the microwave tube.
10. Connect the Raman system to the desktop computer using a USB 2.0 cable. Turn the Raman unit on by turning the key to the 'ON' position. This must be done before opening the Enwave software or the software will not recognize the Raman laser and display an error message.Troubleshooting
11. Points from here (point 11) up to and including point 24 are related to Focusing the Raman laserOpen the EZ Raman software (V3.5.4 MAS) (Fig. 10).

    Figure 10: Setting up the EZ Raman software.

    

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12. On the main screen, set Integration to 4, Average to 1 and Boxcar to 3.
13. Place cyclohexane (3 ml) in a 10-ml glass microwave tube.
14. Place the tube containing cyclohexane into the microwave cavity.
15. Return to the EZ Raman software.
16. Click on the Continuous Scan icon.
17. At the prompt 'Turn on laser' click OK.
    Caution The laser is very powerful (500 mW), and all necessary precautions should be taken to avoid putting oneself in its path.
18. Watch the spectrum appear in the window.
19. Focus the laser by gently moving the light pipe, a fraction at a time.
20. Keep moving the light pipe until the signal is at its greatest intensity and the peaks are sharp and well defined.Troubleshooting
21. Once focusing is complete, click the Stop Scan icon.
22. At the prompt 'Stop continuous scans?' click YES.
23. At the prompt, 'Save all data as a txt file in your application directory?' click NO.
24. At the prompt, 'Save data' click NO.
25. Points from here (point 25) up to and including point 58 are related to Performing an experimentOpen the EZ Raman software (V3.5.4 MAS).
26. Turn on Discover S-Class microwave unit, open the Synergy software and log in.Troubleshooting
27. In the Synergy software under the Method tab, select New Method.
28. Enter a method name. Select the Dynamic method type from the icons on the left.
29. Set temperature to 130 °C, hold time to 20 min, pressure and power settings to 250 psi and 200 W, respectively. Select PowerMax, 'Off', and Stirring, 'High' (Fig. 11).

    Figure 11: Setting up the Synergy software.

    

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30. Enter a 15 s pre-stirring time.
31. Click on the OK. At the prompt, 'Do you want to save this method to the database', click YES.
32. Return to the Enwave software.
33. On the main screen, set Integration to 4, Average to 1 and Boxcar to 3.
34. From the Time Chart pull-down tab, select Time Trend (Fig. 12).

    Figure 12: Time trend function within the EZ Raman software.

    

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35. In the 'Cycle Time' field enter 4.
36. Return to the Synergy software.
37. Prepare the reaction mixture by adding 244.2 mg (2 mmol) salicylaldehyde, 273.3 mg (2.1) mmol ethylacetoacetate and 1.5 ml ethyl acetate to a 10-ml Discover tube. Add one drop of piperidine (8 mol%) and a Teflon-coated stir-bar.
38. Seal the 10-ml tube with a septum and place into the Discover S-Class microwave unit.
39. Click on the 'play' button in the Synergy software. The Discover S-Class will automatically close and seal the 10-ml tube, at which point the 15 s of pre-stirring will begin.
40. Return to the Enwave software. The 'Time Trend' window should still be open.
41. Click on the icon labeled Dark Scan.
42. At the prompt 'Please use same integration, average, and boxcar parameters for dark subtraction', click OK.
43. At the prompt, 'Turn on laser and save a reference spectrum for subtraction', click YES. This should be done while the reaction is still in the 'pre-stirring' stage in the Discover microwave.
44. Click OK at the 'Dark Spectrum Saved' prompt.
45. Click the icon labeled Continuous Scan.
46. Click OK at the 'Start scans without saving files?' prompt.
47. Click OK at the 'Turn on the laser?' prompt. Do this when the Discover has 2–3 s remaining in the 'pre-stirring' stage. The first scan with subtracted starting materials will be the t = 0 spectrum and is essentially baseline noise.
    Caution The laser is very powerful (500 mW) and all necessary precautions should be taken to avoid putting oneself in its path.
48. While the Discover S-Class is heating the reaction and the Raman software is monitoring the reaction's progress, the operator should check that both are running continuously.
49. After the Discover S-Class has held the reaction at 130 °C for 20 min, it will automatically begin the cool-down sequence, passing compressed air (50 psi) over the sealed tube.
    Caution The solvent is heated above its boiling point, so appropriate precautions must be taken to ensure user safety. Only vessels designed to withstand the elevated temperature and pressure generated should be used. In addition, the reaction must be cooled below the boiling point of the solvent before removing the septum and opening to the atmosphere.
50. Returning to the Enwave software, click the icon labeled Stop Scan.
51. At the prompt 'Stop continuous scans?' click YES.
52. At the prompt, 'Save all data as a txt file in your application directory?' click NO.
53. Click the icon labeled Save Data. At the 'Do you want to export all data to Excel?' click YES. Click NO at the prompt, 'Do you want to save all RAW data?'
54. Once the cool-down sequence has completed, remove the reaction flask, remove the septum and pour the contents into a beaker containing 10 ml crushed ice and 1 ml of 2 M HCl.
55. Rinse the tube with an additional 0.5 ml ethyl acetate and transfer to the beaker.
56. Stir this mixture until the ice completely melts.
57. Filter the precipitate under vacuum and allow it to dry for 1–2 h.
58. Rinse with 5 ml cold diethyl ether and allow it to dry.

Timing

Steps 1–24: 2 h; Steps 25–36: 15 min; Steps 37 and 38: 15 min; Steps 39–49: 25 min; Steps 50–53: 5 min; Steps 54–58: 3–4 h

Troubleshooting

Troubleshooting advice can be found in Table 1.

Table 1: Troubleshooting table.

Full table

Anticipated results

As the reaction runs, it is possible to monitor in real time the growth of the signals due to product formation as shown in Figure 2 and discussed already. The reaction is essentially complete when no further growth of the signals is observed.

Analytical data