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Suppression of Protonated Organic Solvents in NMR Spectroscopy using a DISPEL pulse sequence Peter Howe Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00621 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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Analytical Chemistry

Suppression of Protonated Organic Solvents in NMR Spectroscopy using a DISPEL Pulse Sequence Peter W.A. Howe, Syngenta, Jealott's Hill Research Centre, Bracknell, Berkshire. RG42 6EY. UK. Tel. +44 1344 424701 fax +44 1344 455629 E-mail: [email protected]

Abstract Proton NMR spectra are usually acquired using deuterated solvents but in many cases it is necessary to obtain spectra on samples in protonated solvents. In these cases, the intense resonances of the protonated solvents need to be suppressed to maximise sensitivity and spectral quality. A wide range of highly effective solvent suppression methods have been developed, but additional measures are needed to suppress the 13C satellites of the solvent. Because the satellites represent 1.1% of the original solvent signal, they remain problematic if unsuppressed. The recently proposed DISPEL pulse sequences suppress 13C satellites extremely effectively and this letter demonstrates that combining DISPEL and presaturation results in exceptionally effective solvent suppression. An important element in the effectiveness is volume selection, which is inherent within the DISPEL sequence. Spectra acquired in protonated Dimethlysulfoxide and Tetrahydrofuran show that optimum results are obtained by modifying the phase cycle, cycling the pulse-field gradients and using broadband 13C inversion pulses to reduce the effects of radiofrequency offset and inhomogeneity.

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In a recent Letter in Analytical Chemistry, Moutzouri et al 1 describe two highly effective NMR pulse sequences that suppress resonances from protons attached to 13C nuclei. They show that the sequences, which they name Destruction of Interfering Satellites by Perfect Echo Low-pass filtration or 'DISPEL', simplify the detection of trace level impurities by NMR spectroscopy, because 13C satellites of major components no longer complicate the spectra. The sequences are based on a combination of isotope editing and the Perfect Echo sequence, and have the advantages that they are simple to implement and relatively undemanding of NMR spectrometer hardware. In the final sentence of their Letter, Moutzouri et al note that DISPEL sequences have potential application for suppression of protonated organic solvents. This Note aims to highlight practical and theoretical aspects of solvent suppression using DISPEL sequences which are not mentioned by Moutzouri et al. In typical applications of NMR, solvents are present at concentrations of >1000× that of the solutes of interest. Although NMR has reasonable dynamic range, intense protonated solvent resonances obscure parts of NMR spectra and distort the spectral baseline making quantitative analysis difficult. NMR spectra are usually acquired using deuterated solvents but this can be extremely expensive with unusual solvents, such as some used in chemical synthesis, or in cases where a subsample of a larger volume of solution is taken for NMR analysis, such as quality control of chemical libraries. If protonated solvents need to be used, a range of solvent suppression methods are available 2,3. The most effective are those based on WATERGATE 4 and Excitation Sculpting 5,6, but these suppress resonances across broad regions of NMR spectra (typically several tens of hertz). The most selective suppression method, and the longest established, is presaturation 2,7,8. This can achieve good suppression of solvent signal(s) without hindering the observation of nearby resonances. However, this selectivity also means that 13C satellites of solvent resonances are not significantly suppressed so they remain as the largest resonances in proton NMR spectra. The combination of DISPEL and presaturation offers a simple method of suppressing solvent resonances and their associated 13C satellites. One important aspect of solvent suppression is 'volume selection', described in detail in the papers by Neuhaus et al 9 and McKay 8. Put briefly, NMR spectra require magnetic field homogeneity because regions which experience different magnetic fields resonate at different frequencies. These regions are normally detected with low sensitivity, but because solvent resonances are so intense these regions contribute significantly to the NMR spectrum and, because they resonate at different frequencies from the central homogenous regions of the sample, they broaden the area of the spectrum obscured by the solvent. Spectra are significantly improved if signal from inhomogeneous regions is removed. This can be achieved by cancelling or dephasing magnetization from regions of poor radiofrequency (RF) homogeneity because regions of poor field ACS Paragon Plus Environment

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homogeneity typically also have poor RF homogeneity. Volume selection in this way is usually achieved by π-pulses which are either sandwiched by Pulsed Field Gradient (PFG) pulses or phase cycled, so that any signal that is not refocussed/inverted is dephased or cancelled 9,10,11. A cursory inspection of the DISPEL sequences reveals that they inherently achieve Volume Selection through the presence of two refocussing pulses sandwiched by PFG pulses which are additionally phase cycled. This inherent property contributes significantly to the quality of spectra that the DISPEL sequences deliver. It does have the consequence of an increased offset dependence 11. As well as this important benefit, four practical aspects of the DISPEL sequences merit further consideration. First, Moutzouri et al propose two different pulse sequences. Their recommended sequence contains four separate filtering steps so will be referred to as 'DISPEL-4'. This yields exceptionally effective satellite suppression across a wide 1JCH range but requires a total refocussing delay of 16.2ms. Their alternative sequence contains two filtering steps (hence referred to as DISPEL-2), so yields less effective suppression across a wide 1JCH range but requires a shorter total refocussing delay of 8.4ms. In the case considered here of suppressing protonated solvents, there are usually only one or two narrow ranges of 1JCH to be suppressed, so the DISPEL-2 sequence is preferable owing to its shorter refocussing delay which minimises relaxation losses. The second practical aspect that merits consideration is the phase cycle proposed by Moutzouri et al which differs from those proposed in the original publications of the Perfect Echo 12,13. As they note in their Supporting Information, better results are obtained using the original phase cycle where the first proton excitation pulse is cycled independently of the second one; this suppresses artefacts caused by pulse imperfections and relaxation during the echo delay (see p33-34 of van Zijl et al 12). Using the original phase cycle reduces the need for the final z-filter because, in the case of weak coupling, the echo delay is very much shorter than 1/JHH so almost all magnetization is refocussed in-phase. For example, the echo delay in the two-stage DISPEL sequence is less than 8.2ms and during this delay, for the protons of an AX3 system with unusually high J-couplings of 20Hz, more than 99.95% of magnetization is refocussed in-phase 12,13 . In the case of strong coupling, transfer of magnetization is independent of the echo delay but the effect of the refocussing delay is minimal if it is short compared to the inverse of the frequency difference of the strongly coupled spins 14 . The shorter echo delay of DISPEL-2 is an advantage to minimise relaxation and coupling evolution, but it has the counter-balancing disadvantage that it achieves editing using 13C inversion pulses during echo delays. Moutzouri et al do not address the impact of the wide 13C spectral width on this sequence, even though it is well known that the 13C spectral width is ineffectively covered by simple inversion pulses 15. Resonance offset and pulse inhomogeneity have been shown to affect the efficiency of 13C editing 16,17 but effectiveness can be easily restored by using composite 15, ACS Paragon Plus Environment


adiabatic 18,19,20,21 or shaped pulses 17 which are less affected by offset and inhomogeneity. As an example, a simple 24µs inversion pulse with an RF field of 20.8kHz only inverts 90% of magnetization at a 5kHz offset while a basic 90x240y90x composite pulse with the same RF field inverts 99%. Therefore, the simple 13C inversion pulses of DISPEL-2 should be replaced by adiabatic, composite or shaped pulses to achieve its expected effectiveness. A final practical aspect of the sequences applies if they are implemented on a cryo-cooled NMR probe (Moutzouri et al show data acquired using a room-temperature probe). As has been recently noted by other authors with Excitation Sculpting 22 and PURGE 23 solvent suppression sequences, baseline artefacts and peak distortions are often observed on cryo-cooled probes. These are sufficiently severe to compromise the effectiveness of the sequences and their cause is unclear but they can be removed by summing two experiments where all PFG pulses are reversed in polarity. This solution can also be used effectively with the DISPEL-2 sequence, albeit with the consequence of doubling the number of transients required for a spectrum.

Experimental Section Two examples were used to demonstrate the effectiveness of the modified DISPEL-2 sequence. The first was a sample of 0.1mg Linalool 24 in 90% protonated/10% deuterated Dimethysulfoxide (DMSO), which mimics typical sample conditions used when verifying pharmaceutical or chemical libraries. The second was a sample of 1.4mg Chlorsulfuron 25 in 90% protonated tetrahydrofuran (THF) /10% deuterated acetonitrile, which mimics conditions used for reaction monitoring by NMR spectroscopy. Full details of sample preparation, pulse sequence delays and phase cycling, and spectrometer acquisition parameters, are given in Supporting Information (S1).

Results and Discussion Figure 1 shows the protonated DMSO peak of the Linalool sample without solvent suppression and with the DISPEL sequence preceded by presaturation. This demonstrates effective suppression of the main and satellite DMSO peaks. Figure 2 shows enlargements of spectra acquired using the DISPEL sequence with and without application of 13C pulses. This demonstrates that the DISPEL sequence attenuates the 13C satellite peaks 80× compared to the simple Pefect Echo and thus allows observation of the resonances close to the DMSO peaks. The improvement owing to PFG polarity inversion is shown in Supporting Information (S2). Solvent suppression is of slightly better quality than that obtained using NOESY-PRESAT (see Supporting Information S3 and S4), with the additional advantage that a higher receiver gain can be used resulting in slightly higher signal-tonoise. The residual negative satellites signals which are observed are unaffected by changes in the echo delays or by phase cycling, so are likely to originate from other mechanisms such as zeroACS Paragon Plus Environment

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quantum or a combination of radiation damping and the nuclear Overhauser effect. Figure 3 shows one alkene resonance of Linalool; despite the omission of the final z-filter, no phase distortions of the resonance are observed. The trans 3JHH coupling is 17Hz, at the high end of the range of protonproton couplings, suggesting phase distortion is unlikely in most common organic molecules. However, the intensity imbalance between the multiplet components of the resonance differs from that observed in a pulse-acquire spectrum; the components are more symmetric and less ‘roofed’ in the DISPEL sequence. This intensity distortion probably originates from strong coupling 26 because the inverse of the frequency difference of the strongly-coupled spins is not short compared to the echo delay (∆ν-1≈2.85ms, τ ≈1.82ms), which Aguilar et al 14 identify as being necessary for strong coupling effects to be small. The distortion can be reduced by reinstating the z-filter 27 or applying a π/2 pulse with phase y immediately before acquisition (see Supporting Information S5). Figure 4 shows analogous spectra for the Chlorsulfuron sample. Figure 3 shows the region of the THF solvent resonances without solvent suppression and with presaturation and DISPEL-2. Solvent suppression is good, even though the spectra were acquired using phase-modulated presaturation of the two solvent resonances; better results may have been obtained if separate spectrometer frequency channels had been available to irradiate both solvent resonances simultaneously. Figure 5 shows enlargements of the region between both solvent peaks acquired using DISPEL with and without application of 13C pulses (full spectra are shown in Supporting Information S6). Most of note are the resonances around 1.8ppm which originate from the explosive impurity THF peroxide 28; these are exceptionally difficult to discern without suppression of the satellite signals. The combination of DISPEL-2 and presaturation clearly delivers effective solvent suppression, but other methods are available. The first option is to combine NOESY-PRESAT with bi-level broadband 13C decoupling 29. As reported in the original publication of DISPEL, this approach causes sample heating which degrades the quality of NMR spectra. This disadvantage was also noted in the recent publication of Kew et al 30, who proposed the alternative approach of combining NOESY-PRESAT with selective 13C decoupling of all solvent 13C resonances during the presaturation period. Kew et al demonstrated automatic calibration of the selective 13C decoupling to minimise the power required and thus avoid spectral distortions caused by sample heating. Their results show this provides a viable alternative to the DISPEL approach. The advantage of their approach is that magnetization of interest is not transverse until observation, so relaxation and coupling evolution are minimised, while the DISPEL approach presented here has the advantage that accurate calibrations of 13C frequencies and decoupling power are unnecessary. Conclusions These results demonstrate that combining presaturation with a modified DISPEL-2 sequence ACS Paragon Plus Environment


provides robust and effective solvent suppression. The removal of 13C satellites reduces the risk that resonances of interest are obscured, and this benefit is obtained with an improvement in spectral quality compared to simple presaturation owing to volume selection. It provides a new benchmark for presaturation-based suppression when NMR is applied to samples in protonated organic solvents.

Acknowledgements I would like to thank the Manchester NMR Methodology Group for sharing their pulse sequence prior to publication and for several useful discussions about their methods. I am very grateful to Dr Phil Sidebottom (Syngenta Ltd) for comments on this manuscript.

References 1. Moutzouri, P.; Kiraly, P.; Phillips, A.R.; Coombes, S.R.; Nilsson, M.; Morris, G.A. Anal. Chem., 2017, 89, 11898–11901. 13

C Satellite-Free 1H NMR Spectra

2. Albert, K. in On-line LC-NMR and related techniques; Albert, K. ed.; J. Wiley & Sons: Chichester, 2002; pp 1-22. 3. Claridge, T. High Resolution NMR Techniques in Organic Chemistry, 3rd ed.; Elsevier, Oxford. 2016. 4. Piotto, M.; Saudek, V.; Sklenář, V. J. Biomol. NMR. 1992, 2, 661-665. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions 5. Hwang, T.-L.; Shaka, A.J. J. Magn. Reson. Ser. A. 1995, 112, 275-279. Water Suppression That Works. Excitation Sculpting Using Arbitrary Waveforms and Pulse Field Gradients 6. Adams, R.W.; Holroyd, C.M.; Aguilar, J.A.; Nilsson, M.; Morris, G.A. Chem. Commun. 2013, 49, 358-360. Perfect echo excitation sculpting “Perfecting” WATERGATE: clean proton NMR spectra from aqueous solution 7. Hoult, D.I. J. Magn. Reson. 1976, 21, 337-347. Solvent Peak Saturation with Single Phase and Quadrature Fourier Transformation 8. McKay, R.T. Concepts Magn. Reson., Part A 2011, 38A, 197–220. Volume selection review: How the 1D-NOESY suppresses solvent signal in metabonomics NMR spectroscopy: An examination of the pulse sequence components and evolution 9. Neuhaus, D.; Ismail, I.M.; Chung, C.-W. J. Magn. Reson. Ser. A. 1996, 118, 256-263. “FLIPSY” – A New Solvent-Suppression Sequence for Nonexchanging Solutes Offering ACS Paragon Plus Environment

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Improved Integral Accuracy Relative to 1D NOESY 10. Simpson, A.J.; Brown, S.A. J. Magn. Reson. 2005, 175, 340-346. Purge NMR: Effective and easy solvent suppression 11. Mo, H.; Raftery, D. J. Magn. Reson. 2008, 190, 1-6. Pre-SAT180, a simple and effective method for residual water suppression 12. van Zijl, P.C.M.; Moonen, C.T.W.; von Kienlin, M. J. Magn. Reson. 1990, 89, 28-40. Homonuclear J Refocusing in Echo Spectroscopy 13. Takegoshi, K.; Ogura, K.; Hikichi, K. J. Magn. Reson. 1989, 84, 611-615. A perfect spin echo in a weakly homonuclear J-coupled two spin-½ system 14. Aguilar, J.A.; Nilsson, M.; Bodenhausen, G.; Morris, G.A. Chem. Commun. 2012, 48, 811813. PROJECT: Spin echo NMR spectra without J modulation 15. Freeman, R.; Kempsell, S.P.; Levitt, M.H. J. Magn. Reson. 1980, 38, 453-479. Radiofrequency Pulse Sequences Which Compensate Their Own Imperfections 16. Jahnke, W. J. Magn. Reson. Ser. B. 1996, 113, 262-266. Spatial Aspects of Radiofrequency Inhomogeneity in High-Resolution NMR and Their Consideration in Improving Isotope-Editing Experiments 17. Hyre, D.E.; Spicer, L.D. J. Magn. Reson. Ser. B. 1995, 108, 12-21. Improved Excitation Pulse Bandwidths Using Shaped Pulses, with Application to Heteronuclear Half Filters in Macromolecular NMR. 18. Tannús, A.; Garwood, M. NMR Biomed. 1997, 10, 423-434. Adiabatic Pulses 19. Kupče, Ē.; Freeman, R. J. Magn. Reson. Ser. A. 1995, 115, 273-276. Adiabatic Pulses for Wideband Inversion and Broadband Decoupling 20. Skinner, T.E.; Kobzar, K.; Luy, B.; Bendall, M.R.; Bermel, W.; Khaneja, N. Glaser, S.J. J. Magn. Reson. 2006, 179, 241-249. Optimal control design of constant amplitude phase-modulated pulses: Application to calibration-free broadband excitiation 21. Hwang, T.-L.; van Zijl, P.C.M.; Garwood, M. J. Magn. Reson. 1998, 133, 200-203. Fast Broadband Inversion by Adiabatic Pulses 22. Aguilar, J. A.; Kenwright, S. J. Analyst 2016, 141, 236– 242 DOI: 10.1039/C5AN02121A. Robust NMR water signal suppression for demanding analytical applications 23. Le Guennec, A.; Tayyra, F.; Edison A.S. Anal. Chem. 2017, 89, 8582–8588. Alternatives to Nuclear Overhauser Enhancement Spectroscopy Presat and Carr–Purcell– Meiboom–Gill Presat for NMR-Based Metabolomics ACS Paragon Plus Environment


24. http://www.chemspider.com/Chemical-Structure.13849981.html (accessed 2nd Jan. 2018). 25. http://www.chemspider.com/Chemical-Structure.43209.html (accessed 2nd Jan. 2018). 26. Ernst, R.R.; Bodenhausen, G.; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions, 1st ed.; Oxford University Press, Oxford. 1987. 27. Thrippleton, M.J.; Keeler, J. Angew. Chem. Int. Ed. 2003, 42, 3938–3941 Elimination of Zero-Quantum Interference in Two-Dimensional NMR Spectra 28. http://www.chemspider.com/Chemical-Structure.13393661.html (accessed 2nd Jan. 2018) 29. Kupče, Ē.; Freeman, R.; Wider, G.; Wüthrich, K. J. Magn. Reson. Ser. A. 1996, 122, 81-84. Suppession of Cycling Sidebands Using Bi-level Adiabatic Decoupling 30. Kew, W. ; Bell, N.G.A.; Goodall, I.; Uhrín, D. Magn. Reson. Chem. 2017, 55, 785-796. Advanced solvent signal suppression for the acquisition of 1D and 2D NMR spectra of Scotch Whisky


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Figures

Figure 1. Part of NMR spectra of the sample of 0.1mg of Linalool in 90% protonated DMSO acquired using a) Pulse-acquire and b) DISPEL-2 with presaturation.

Figure 2. Sub-spectra of the Linalool sample acquired using DISPEL-2 with presaturation and a) without and b) with application of 13C pulses.

Figure 3. The alkene region of the spectrum of the Linalool sample acquired using DISPEL-2 with presaturation ACS Paragon Plus Environment


Figure 4. Sub-spectra of the sample of 1.4mg Chlorsulfuron in 90% protonated THF acquired using a) Pulse-acquire and b) DISPEL-2 with presaturation.

Figure 5. Sub-spectra of the Chlorsulfuron sample acquired using DISPEL-2 with presaturation a) without and b) with application of 13C pulses. Resonances marked ‘P’ originate from THF peroxide.


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