NMR Flow Tube for Online NMR Reaction Monitoring - Analytical

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An NMR flow-tube for online NMR reaction monitoring David Anthony Foley, Eckhard Bez, Anna Codina, Kimberly L. Colson, Michael Fey, Robert Krull, Don Piroli, Mark T Zell, and Brian L. Marquez Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac502300q • Publication Date (Web): 06 Nov 2014 Downloaded from http://pubs.acs.org on November 10, 2014

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Analytical Chemistry

An NMR flow-tube for online NMR reaction monitoring David A. Foley,a Eckhard Bez,b Anna Codina,c Kimberly L. Colson,b Michael Fey,b Robert Krull,b Don Piroli b Mark T. Zell,a and Brian L. Marqueza*† a

Analytical Research and Development, Pfizer Worldwide Research and Development, 445 Eastern Point Road., Groton, CT 06340, USA b

c

Bruker BioSpin, 15 Fortune Drive, Billerica MA 01821, USA

Bruker UK Limited, Banner Lane, Coventry CV4 9GH, United Kingdom

Corresponding Author Brian L. Marquez, Bruker BioSpin, 15 Fortune Drive, Billerica MA 01821. Email: [email protected]

ABSTRACT: This article describes the development of a 5 mm NMR flow-tube that can be used in a standard 5 mm NMR probe, enabling the user to conduct experiments on flowing samples; or more specifically, on flowing reaction mixtures. This enables reaction monitoring or kinetic experiments to be conducted by flowing reaction mixtures from a reaction vessel to detection in the coil area of the NMR, without the need for a specialized flow NMR probe. One of the key benefits of this flow-tube is that it provides flexibility to be used across a range of available

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spectrometers of varying magnetic field strengths with a standard 5 mm probe setup. The applicability of this flow-tube to reaction monitoring is demonstrated using the reaction of pphenylenediamine and isobutyraldehyde to form the diimine product.

Nuclear magnetic resonance (NMR) spectroscopy has proven to be an invaluable technique to chemists in the area of structural elucidation and verification over the past number of decades. It provides key structural information for both small and large molecules in the areas of natural product discovery and synthetic organic chemistry, amongst many others. The quantitative nature of this technique has also found applicability in providing accurate potency evaluation of samples in solution.1 The structural and quantitative information that can be mined from the NMR spectroscopic analysis of species in solution means that the analysis of reaction mixtures by NMR spectroscopy can provide a comprehensive insight into chemical reactions being investigated for the sake of mechanistic or process development understanding. While many chemical transformations have been analyzed in regular NMR tubes, physical and chemical limits exist to the kinds of reactions to which this form of reaction monitoring is applicable. The lack of sufficient mixing, particularly for heterogeneous processes can have significant effects on the rate of a reaction.2 In order to circumvent these issues and allow the application of the advantageous features of NMR spectroscopy to reaction monitoring and reaction kinetics, a number of technological and experimental set ups have been outlined in the literature.3 These designs have attempted to address the challenges of preserving the valuable information that NMR spectroscopy can provide, while expanding its scope to the analysis of dynamic and complex reaction systems. Two basic strategies have been employed to achieve this outcome; the first (static) centers on a reaction taking place in an NMR tube within the probe area of the magnet, while the second 2 ACS Paragon Plus Environment

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(online) focuses on flowing a stream of reaction mixture from a reaction vessel to a cell in the spectrometer. The use of modified NMR tubes as static rapid injection reactors has been published.4 In these systems, the introduction of reagents and subsequent reaction occurs within the NMR tube, inserted in the probe. While introducing reactants into a tube contained in the NMR probe allows reasonably fast chemical dynamics to be captured, this approach lacks the control of reaction conditions that occurs in a reaction vessel or reactor. Many reports of online NMR reaction monitoring have taken advantage of flow cell technology developed for LC-NMR spectroscopy.3b This approach was originally designed to analyze individual components separated by a chromatographic technique, which were flowed through a microliter flow cell within the coil area of the probe.5 The use of flow probes typically requires a dedicated probe, which can be restrictive in terms of the variety of nuclei available for analysis, and problematic if particulates are present in the reaction mixture. As an alternative to flow probe technology, a number of customized NMR tube designs have been reported as low-cost alternatives to the use of flow probes.6 These tube type flow cells typically allow for greater flexibility, as they are designed to be used in any regular NMR probe of suitable coil diameter. Both approaches have advantages and disadvantages in terms of reaction monitoring. A reaction vessel connected to a flow-tube has the proper thermodynamic properties but the NMR detection lags behind, which makes it difficult to detect fast reactions. In addition, temperature changes in the connection lines may influence the kinetics of the reaction being analyzed. In this report, we outline the design and development of a novel flow-tube that combines the advantages of both approaches but eliminates many of the common disadvantages. Finally we demonstrate its implementation as a flow cell for online NMR reaction monitoring.7



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EXPERIMENTAL SECTION Flow system design: The key to the flow-tube design was four fold: 1.) Allow versatility for a broad application to NMR spectroscopy (no specially built probe required); 2.) Maintain reaction conditions and characteristics throughout the system; 3.) Minimize the delay between the sample leaving the reaction vessel and being detected in the cell; 4.) Improve the temperature control of the reaction sample. To achieve point 1 the flow-tube was designed to mimic a regular 5 mm NMR tube and rotor that is used in standard solution NMR spectroscopy experiments. This is fundamental, as it gives the advantage of increased flexibility of use over conventional flow cells used in commercial flow probes. Conventional flow cells also suffer from a lack of temperature control and poor chemical compatibility with the wide range of organic solvents used in synthetic chemistry, which can result in leaking of material at the connections to the cell. The flow-tube can be easily inserted and removed from a standard 5 mm NMR probe, allowing its use across a multitude of available spectrometers and probes. The modification of the rotor to enable coupling with the 5 mm glass tube, as well as inlet and outlet capillary tubing, facilitates cyclic sample transfer between a remotely located reaction vessel and the NMR spectrometer. Flow can be controlled via a pump to allow continuous or stopped flow NMR spectroscopy experiments to be conducted employing the same equipment. Point 2 is achieved by flowing the reaction mixture from the reaction vessel to the flow-tube. The flow-tube consists of four major components (Figure 1): Coaxial fluid transfer lines (A), cylindrical body (B), coupling section (C) and flow-tube (D).7 Transfer and temperature control


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of the reaction mixture from the reaction vessel to the flow-tube and its return following detection is achieved by means of four concentric tubes (A), of increasing diameter from inner to outer, as displayed in Figure 2 and described in greater detail below. The tubing used in the studies presented in this paper is described in Table 1. The body (B) of the flow-tube is designed to couple to the coaxial transfer fluid lines (D), as well as to provide rigidity and stability of the flow-tube once inserted in the bore of the magnet. The body (B) consists of a hollow tube formed of a PCTFE material (4), which is connected to concentric inlet and outlet transport capillaries (1) (shown in Figure 2 and described in detail below) via Swagelok® fittings (2) and (3). Its hollow nature allows temperature regulated transfer fluid to fill the cavity (5), maintaining a consistent temperature throughout the system, before returning to the circulator. The flow-tube (D) is made up of two parts; an interchangeable 5 mm O.D. NMR tube (6) designed to minimize the cell volume and to place the sample at the correct height within the radio frequency (RF) coils, and the second, a modified ceramic rotor (7) that contains a projection (8), over which the glass tube (6) is fitted. The flow-tube is made leak resistant using Viton® o-rings (9) and is fixed to the ceramic projection. The flow-tube (D) is designed to mimic a regular NMR tube and rotor, so that they can fit into a standard 5 mm NMR probe. The flow-tube (D) and the body (B) are mated using a coupling (C), comprising of two halves (10) and (11), which are fixed together by four non-magnetic screws (12). Regarding point 3, there is limited flexibility to move a reaction vessel next to the NMR magnet or vice versa, usually due to facilities infrastructure. Furthermore, the maximum flow rate within the flow system is limited by maximum pressure and also by the residence time of the fluid inside the NMR probe. Both conditions together limit the minimum time the fluid takes to go from the reaction vessel to the NMR probe. In this approach we allow for different flow rates



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with i) a “fast loop” that circulates between the reaction vessel and a “control box” placed close to the magnet, and ii) a “slow loop” between the control box and the NMR probe. The control box allows a reasonably fast flow rate (fast loop) to be achieved between the reactor and this box which is situated close to the magnet.3(b) This minimizes the transfer time, ensuring that the flowing reaction mixture is representative of the chemical composition in the reaction vessel. Within the control box there is a flow control valve that allows the flow rate to be adjusted to allow for optimal flow through the flow-tube in the NMR, optimizing NMR sensitivity resulting in only minor time delay. This slow loop, which has a volume of 6 mL, is fed back into the fast loop via the control box.7 The circulation time is not typically considered significant relative to the kinetics being measured. As outlined above and shown in Figure 2, fluid transfer is achieved by means of four concentric tubes of increasing diameter between the control box and the flow-tube. The innermost tube (13) (PTFE), which transferred the reaction mixture from the reaction vessel to the bottom of the glass tube (6), was contained within the sample return tubing (14) (PTFE). The return tubing (14) proceeded as far as the head of the ceramic rotor (7), where it was secured by a nut (15) within the coupling (C). This allows fluid to return to the reaction vessel through the coaxial cavity between the outer wall of tubing (13) and the inner wall of (14). Also inside the coupling (C), the other end of the tubing (14) was fixed to the body (B) using a second nut (16) which provided a seal to isolate the heating transfer fluid contained within the body from the remainder of the flow-tube. The sample return tubing (14) passed through the cavity (5) and into a third tube (17) (PTFE), which was used to bring the temperature regulated transfer fluid from the temperature circulator, terminating inside the cavity of the cylindrical body (B). The outer most tubing (1) (PTFE) facilitated return of the temperature fluid upon exiting the cylindrical


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body, and enabled union with the flow-tube system through Swagelok connections (2) and (3). Finally, the entire coaxial fluid transport system was enclosed in foam insulation to minimize temperature losses to the atmosphere. The control box has two functions as it first effectively moves the reaction vessel next to the NMR system, reducing the transfer time between the reaction vessel and the NMR; and also decouples the four concentric connections to the flow-tube into double concentric input/output lines. Both input and output lines have the reaction sample as the inner capillary and the heating transfer liquid on the outside. With reasonable flow rates on the heating fluid, the temperature difference on the reaction mixture can be limited.

Figure 1. Flow-tube design as described in US Patent 20120092013 A1, 2012. Diimine synthesis:



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Anhydrous acetonitrile (45 mL, DriSolv) was charged to a 50 mL reactor vessel and pphenylenediamine 1 (0.50 g, 4.6 mmol, Sigma) was added in one portion. The temperature was set to 20 °C throughout the system and the reaction mixture was pumped through the system for 10 min to allow the solution to equilibrate in terms of temperature and concentration. A 1H NMR spectrum of the flowing solution was recorded to establish the initial concentration of pphenylenediamine 1. Isobutyraldehyde 2 (0.84 mL, 9.2 mmol, Aldrich) was added and the reaction mixture was stirred for 47 h, while monitoring by 1H NMR spectroscopy. NMR spectra were recorded on Bruker AVANCE III 400 MHz spectrometer, equipped with a 5 mm BBFO probe. The probe temperature was set at 20 °C. The probe was tuned and matched prior to the start of the reaction and were then kept constant. 1H NMR spectra were recorded at regular intervals over the course of the reaction (2 scans, 4 s acquisition time, 10 s relaxation delay, 67 receiver gain, and 90° pulse). 1H NMR spectra were recorded at 1.5 min intervals between acquisitions for the first 15 min. This interval time was then increased to 10.5 min, and then 20.5 min for the remainder of the reaction monitoring. A total of 245 spectra were recorded over the course of the experiment which resulted in a total of 47 h monitoring period.

RESULTS AND DISCUSSION The NMR flow-tube design described here has a number of advantages over commercial flow NMR probes, particularly for reaction monitoring purposes. Primarily, the economic value of such an apparatus is large, as the cost of a dedicated flow probe may prove prohibitive to many laboratories interested in investigating flowing solutions. As highlighted previously, the flowtube mimics the design of a traditional 5 mm tube and rotor. This allows the set up to be used on multiple spectrometers of various field strengths, as well as taking advantage of multinuclear experiments by using it in multiple probes. Microliter cells contained within flow probes tend to 8 ACS Paragon Plus Environment

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have very narrow capillary pathways, which are particularly constricted at the union with capillary transfer tubing, often leading to clogging of the cell by particulates. In turn this may lead to back pressure build up and ultimately leaks. In their primary use in LC-NMR spectroscopy, this issue was not encountered as the sample was typically homogeneous; however the complex nature of reaction mixtures increases the possibility of blockages. The exclusion of solids from the sample loop can be maximized by placing a filter at the end of the inlet tube in the reaction vessel; however, this may not always be sufficient, especially with changing solubility of components in the mixture over the course of the reaction. In the current design the flow path is not constricted at any point, and this allows some small particles to move through the system unobstructed. To date, with 3 years of use, no clogging of the flow path or leaks have been observed. However the flow-tube can be removed if cleaning is required. A range of solvent and reaction matrices have been extensively tested on the flow-tube system. Under the reaction conditions described here, no compatibility issues were observed. Solvents such as methanol, DMSO (dimethylsulfoxide), THF (tetrahydrofuran), DMF (N,Ndimethylformamide, toluene and dichloromethane have been used to date. Care was taken when using ethereal solvents, as some components have limited compatibility with these solvents. The system was rinsed with methanol after each use, and checked for leaks before each use. Bruker’s TopShim proton shimming routine was used to obtain acceptable lineshape for all of the reaction solvents described above. Optimum flow rates for the system were tested by conducting a flow rate test, where a 20% v/v solution of t-butanol in water was circulated around the flow loop system. The slow loop was bypassed so that inlet line was connected directly to the pump, allowing the flow rate to be determined. The flow rate was varied and the effect of the flow rate on the t-butanol signal was



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evaluated. At 2 mL/min, the signal was found to be 98% of that recorded for a static sample. At 4mL/min the signal was 92% of that obtained without flow. The residence time distribution (RTD) function of the flow tube was also measured using a step tracer experiment and ‫ݐ‬res was calculated to be 3.2 min.3(b) Results from these experiments are included in the Supplemental Information. The flow-tube system was pressure tested to 10 Bar, which was found to be much greater than the back-pressure observed in the system under normal use. The temperature range of the system is dictated by the NMR probe that is used for the analysis. In this system, a 5 mm BBFO probe which has a temperature range of -150 to 150 °C was used, however the temperature range used to date for the reactions conducted on this system cover the range of -40 to 120 °C. The flow-tube described here has been designed to be used by NMR specialists and nonspecialists. With some operational training any user capable of acquiring 1D NMR spectra from standard NMR samples should be able to use the flow-tube to harvest kinetic data from their reactions. To ensure longevity of the system care needs to be taken, which includes cleaning the system thoroughly with methanol after each use, and storing with methanol in the transfer lines when not in use. It is recommended to replace the o-rings on the rotor once per month, to minimize the opportunity for leaks to occur.


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Figure 2. Diagram of reaction mixture transfer and temperature regulation lines as described in US Patent 20120092013 A1, 2012.

Material Composition

Tubing Purpose

Outer Diameter (O.D.) in inches

PTFE

Sample delivery

0.048″

PTFE

Sample return

0.125″

PTFE

Heat transfer fluid delivery

0.250″

PTFE

Heat transfer fluid return

0.375″

Table 1. Table describing the tubing material, the intended use and the outer diameter (O.D.) of the tubing in inches. All tubing used is of a “standard wall thickness”.



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The insertion of the flexible PTFE inlet transfer capillary into the 5 mm cell was tested for disturbances to the 1H line shape by a series of line shape tests. The line shape results achieved with the flow-tube were compared to those obtained with a line shape standard sample in a standard 5 mm NMR tube. As summarized in Table , the presence of the PTFE capillary inlet in the flow-tube resulted in a loss of line shape when compared to the line shape standard (test A and D); however this is not detrimental when using a signal for tracking purposes. Test B was conducted on a static mixture of 1% chloroform in acetone (protonated), and the line shape was optimized using TopShim (Bruker BioSpin). A slight increase in the full width at half-height value was observed once the flow rate was increased to 4 mL/min. Overall the results obtained from these tests establish that the presence of the PTFE capillary tube inserted in the flow-tube does not significantly impact the ability of this flow-tube to be used for on flow NMR reaction monitoring. Test

50%

0.55%

0.11%

A) Standard line shape sample in 5 mm NMR tube (1% CHCl3 in acetone-d6)

0.56 Hz

8.0 Hz

14.5 Hz

B) Flow-tube, static (1% CHCl3 in acetone)

1.60 Hz

13.8 Hz

23.2 Hz

C) Flow-tube, flowing @ 4 mL/min (1% CHCl3 in acetone)

2.16 Hz

14.4 Hz

22.6 Hz

D) Flow-tube, static (1% CHCl3 in acetone-d6)

1.30 Hz

11.7 Hz

20.6 Hz

Table 2. Line shape analysis of flow-tube with PTFE tubing inserted in flow-tube. The reaction monitoring capabilities of the flow-tube were demonstrated using the reaction of p-phenylenediamine 1 and isobutyraldehyde 2 to form the diimine 4 (Scheme 1). The reaction


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was conducted in fully protonated acetonitrile, therefore 1H NMR spectra were acquired unlocked and the methyl resonance of acetonitrile was suppressed using the standard Bruker WET pulse sequence with shape pulse wetsupress.8 Spectra were acquired with 2 scans, a 90° flip and relaxation delay of 10 seconds between each scan. Therefore, each spectrum had a total acquisition time of 30 seconds. The flow rate in the cell was maintained at a rate less than 4 mL/min to ensure maximum detection of the species in solution, while minimizing the sample transfer time. The flow rate was determined prior to beginning the reaction, using acetonitrile. The return line from the slow loop was uncoupled from the junction with the fast loop and the flow rate calculated by collecting the solvent stream into a graduated cylinder over a set time. The flow rate during the reaction monitoring experiment was not monitored, as an inline flow meter could not be found with compatibility with the broad temperature and range of reaction conditions that we typically encounter. The flow rate will be solvent dependent because of the passive split between the fast and slow loops and should therefore be determined for each solvent used. The split can be crudely regulated by the needle valve to ensure the desired flow rate into the flowtube.

Scheme 1. Reaction scheme.



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Two equivalents of isobutyraldehyde 2 were added to a solution of p-phenylenediamine 1 (104 mmol/L, 1 equivalent) in acetonitrile at 20 °C. A 2 µm stainless steel frit was attached to the outlet tube to ensure that no solids were drawn into the sample loop, which may result in blocking of the flow path, or inhomogeneity in the flow-tube. Spectra were recorded at 1.5 min intervals for the first 15 min. This interval time was then increased to 10.5 min, and subsequently 20.5 min as the kinetic profiles slowed. A total of 245 spectra were recorded over the course of the experiment. 1H NMR spectra were processed with TopSpin 3.2.pl6 and analyzed with Dynamics Center 2.2 (Bruker BioSpin).9 A stack plot of the 245 spectra acquired during the experiment is shown in Figure 3. Each spectrum was referenced to acetonitrile at δH 1.96 ppm and direct integration of characteristic resonances were used to track the evolution of reaction species over time. The resonance at δH 6.45 ppm was used to track p-phenylenediamine starting material 1, while monoimine intermediate 3 was monitored using the doublet at δH 6.58 ppm. The diimine product 4 could be tracked by integration of its characteristic resonance at δH 6.97 ppm.


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Figure 3. 1H NMR stacked plot, acquired at 400 MHz on a 5 mm BBFO probe. Resonances used to monitor the progress of reaction mixture components are labeled 1, 3 and 4. Traces at the beginning, middle and at the end of the reaction are highlighted in red. Note that the Y axis is spectrum number, with spectrum 1 (time = 0 h) at the bottom and spectrum number 245 (time = 47 h) at the top. Data were acquired with three different intervals: 1.5, 10.5 and 20.5 min. Highlighted in red are traces where the interval changes. The reaction profile of this transformation is shown in Figure 4. Consumption of the diamine 1 starting material began immediately following addition of the aldehyde 2. This coincided with the formation of the monoimine 3, via reaction with one molecule of isobutyraldehyde 2. Figure 4 demonstrates the intermediacy of this species as it reacts with a second aldehyde molecule to produce the diimine 4. The equilibrium between diimine formation and reversion to imine was established after 47 h. 15 ACS Paragon Plus Environment


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Figure 4. Expansion of a 400 MHz 1H NMR spectrum, showing the monitored species (left). Reaction profile generated using the 1H NMR data (right).

CONCLUSION The design and implementation of a novel flow-tube for the monitoring of reactions is described in this work. The sample reaction outlined demonstrates the utility of such device in monitoring the progress of a multi-component reaction using online NMR spectroscopy. The flow-tube is designed to be used across a range of existing spectrometers and probes, which may already be available to an individual user. This technology opens the possibility of conducting online NMR spectroscopy experiments to those interested in examining mechanistic and kinetic aspects of reactions, without requiring a specialized flow probe.

AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected] Present Addresses † Bruker BioSpin, 15 Fortune Drive, Billerica MA 01821 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

SUPPORTING INFORMATION AVAILABLE This information is available free of charge via the Internet at http://pubs.acs.org/.

REFERENCES (1) (a) Do, N. M.; Olivier, M. A.; Salisbury, J. J.; Wager, C. B., Anal. Chem. 2011, 83 (22), 8766-8771. (b) Holzgrabe, U., Prog. Nucl. Magn. Reson. Spectrosc. 2010, 57 (2), 229-240. (2) Rabinovitz, M.; Cohen, Y.; Halpern, M., Angew. Chem. Int. Ed. Engl. 1986, 98 (11), 958968. (3) (a) Foley, D. A.; Zell, M. T.; Marquez, B. L.; Kaerner, A., Pharm. Technol. 2011, (Suppl.), S19-S21. (b) Maiwald, M.; Fischer, H. H.; Kim, Y.-K.; Albert, K.; Hasse, H., J. Magn. Reson. 2004, 166 (2), 135-146. (c) Bernstein, M. A.; Stefinovic, M.; Sleigh, C. J., Magn. Reson. Chem. 2007, 45 (7), 564-571. (d) Buser, J. Y., McFarland, A. D., Chem. Comm. 2014, 50 (32) 42344237.



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(4) (a) McGarrity, J. F.; Prodolliet, J., J. Org. Chem. 1984, 49 (23), 4465-4470. (b) Mix, A.; Jutzi, P.; Rummel, B.; Hagedorn, K., Organometallics 2010, 29 (2), 442-447. (c) Yushmanov, P. V.; Furo, I., J. Magn. Reson. 2005, 175 (2), 264-270. (d) Denmark, S. E.; Williams, B. J.; Eklov, B. M.; Pham, S. M.; Beutner, G. L., J. Org. Chem. 2010, 75 (16), 5558-5572. (e) Christianson, M. D., Tan, E. H. P., Landis, C. R., J. Am. Chem. Soc. 2010, 132 (33), 11461-11463. (5) (a) Keifer, P. A., Mod. Magn. Reson. 2006, 2, 1195-1201. (b) Foley, D. A.; Doecke, C. W.; Buser, J. Y.; Merritt, J. M.; Murphy, L.; Kissane, M.; Collins, S. G.; Maguire, A. R.; Kaerner, A., J. Org. Chem. 2011, 76 (23), 9630-9640. (6) (a) Green, D. B.; Lane, J.; Wing, R. M., Appl. Spectrosc. 1987, 41 (5), 847-851. (b) Khajeh, M.; Bernstein, M. A.; Morris, G. A., Magn. Reson. Chem. 2010, 48 (7), 516-522. (7) Marquez, B.; Fey, M.; Colson, K.; Krull, R.; Bez, E.; Piroli, D.; Maas, W. NMR flow cell. US 20120092013 A1, 2012. (8) Smallcombe, S. H.; Patt, S. L.; Keifer, P. A., J. Magn. Rson., Ser. A 1995, 117 (2), 295-303. (9) http://www.bruker.com/products/mr/nmr/nmr-software/software/dynamicscenter/overview.html 06 May 2014.


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