1H/19F Double ResonanceSpectroscopy – Simplifying SpectralInterpretation

AuthorJarrett Farias, Ph.D.NMR Applications ScientistResearch Products GroupSanta Clara, CA USA

Application Note

AbstractVnmrJ 3 software provides easy-to-use, interactive tools for setting up advancedexperiments. Simplified set-up procedures allow even novice users to get criticalinformation about their research samples using the most advanced NMRexperiments available. This application note is one of a series designed to providestep-by-step guidance for setting up sophisticated experiments to collect exactlythe data you need for your analyses.

IntroductionNuclear Magnetic Resonance (NMR) spectroscopy is the premier analyticaltechnique for providing information on the spatial relationships between nuclei in amolecule. Using a variety of experiments, through-bond and through-spacecorrelations between individual nuclei can be obtained. Sometimes, however, theresulting spectra may be complex or have overlapping patterns that makeinterpretation difficult. This is especially true for samples that are impure or thatare made up of complex mixtures, such as samples from a biological matrix. Inthese cases, if the molecule of interest contains any fluorine atoms, then19F spectroscopy can be used as a filter to select only the desired signals, or simplify the NMR spectra.

The most basic of these experiments is the 1H observed with 19F decouplingexperiment, HObs_FDec, which simplifies the 1-dimensional (1D) spectra byremoving the through-bond coupling interactions of fluorine nuclei from protonresonances. This experiment is particularly useful for molecules that containmultiple fluorine atoms or where the fluorine spin is highly coupled, creatingcomplex multiplets in the proton spectrum. Decoupling the fluorine can simplify thespectrum to make interpretation easier, and it also confirms which protons aremagnetically coupled to fluorine atoms.

The 1H/19F class of experiments, suchas 1H{19F} and 19F-1H HOESY, arevaluable tools for simplifying spectraand allowing exploration of spatialpositioning of atoms. Using bothexperiments, through-bond andthrough-space couplings between 1H and 19F can be identified andmeasured. In this application note,examples are shown for theseexperiments acquired using trans-2,5-difluorocinnamic acid as atest compound.

An Example of 1H/19F DoubleResonance SpectroscopyA sample of trans-2,5-difluorocinnamicacid (Figure 1) was used todemonstrate simplification of the1H NMR spectra of a compoundthrough the application of fluorinedecoupling. All data were collected onan Agilent 400-MR DD2 instrumentequipped with an ATB probe, with theprobe simultaneously tuned to 1H and19F on the same RF coil.

The converse of the HObs_FDec is theFObs_HDec experiment which, as thename implies, acquires a 1D 19Fspectrum with proton decoupling.Removing the proton coupling can bequite useful as the fluorine signals willtypically collapse to singlets,simplifying the spectrum andincreasing the sensitivity of theexperiment. In some cases, because ofthe naturally low background forfluorine signals (for example, fromsolvent, chemical noise, impurities, andso forth), the detection limit for theFObs_HDec experiment can be lowerthan for the comparable 1D protonexperiment. The high sensitivity,combined with the large chemical shiftrange of 19F, makes it a goodexperiment for working with impuresamples or mixtures of fluorinatedcompounds.

Another double resonance experiment,the 19F-1H heteronuclear Overhauserenhancement spectroscopy (HOESY), isa 2-dimensional (2D) experiment thatprovides data to show through-spacecorrelations for these two nuclei. This2D spectrum is simple to interpret, andcombined with the aforementionedheteronuclear decoupling experiments,can be used to rationalize the structureof 1H and 19F containing molecules.The H/F HOESY experiment isparticularly useful for assigning the19F resonances in molecules thatcontain multiple fluorine atoms, or fordetermining the regiochemistry of afluorine substitution.

2

10

9

8

O11

1

OH13

2

6

5

3

4

F12

F7

Figure 1. The chemical structure of trans-2,5-difluorocinnamic acid.

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Figure 2. One-dimensional 1H spectra of trans-2,5-difluorocinnamic acid without 19F decoupling (top) andwith 19F decoupling (bottom).

7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 ppm

H6 H9H3 H4 H8

The 19F coupled 1H spectrum (Figure 2,top) shows two complicated multipletsat 7.76 and 7.32 ppm, representing thethree aromatic protons, H3, H4, and H6.Because of the complexity of theresonance patterns, the substitutionpattern of the aromatic ring moiety isnot readily apparent. Applying19F decoupling while simultaneouslyacquiring 1H using the Hobs_FDecexperiments results in a 1H 1D spectrum(Figure 2, bottom). This provides a clear1H-1H coupling pattern for all threeprotons; H3 and H6 are doublets whileH4 is a doublet of doublets. Thecoupling constants of those protons canthen be easily measured, J4 = 3 Hz forH4-H6 and J3 = 9 Hz for H3-H4, clearlyconfirming the 1,2,5-trisubstitutedpattern.

The 19F-1H HOESY experiment can beused to confirm the positions of thefluorine atoms in the molecule bydetecting the through-spaceassociations between 1H and 19F nuclei.

Fluorine F7 has correlations to threeprotons, while F12 shows correlationsto two protons (Figure 3). Significantly,fluorine F7 correlates to protons H8and H9 while fluorine F12 correlatesonly to H6 confirming the spatialrelationships of the fluorine atoms.

Experimental MethodTo run the 1H/19F class of experiments,a probe simultaneously tuned to 1Hand 19F is needed and the probe fileshould be populated with 1H and 19Fpulse information obtained from theautomated calibration routine. TheHObs_FDec experiment requiresapproximate knowledge of the 19Fchemical shift(s) of interest, so asurvey 19F spectrum without decoupling is typically acquired first.

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F1(ppm)

6.6

F12 F7

6.8

7.0

7.2

7.4

7.6

7.8

8.0

H8

H4

H3

H9

H6

-118.0 -119.0 -120.0F2 (ppm)

-121.0 -122.0

Figure 3. The 19F-1H HOESY 2D spectrum of trans-2,5-difluorocinnamic acid with the 19F{1H} 1Dspectrum plotted on the top and 1H{19F} 1D spectrum plotted on the side. Correlations to F7 are indicated in blue arrows, with green arrows for F12.

To acquire an 1H{19F} spectrum in theStudy Queue:

1. Select the New Study bottom atthe bottom of the Study Queue.

2. In the Experiment Selector, selectthe Liquids tab, then selectHF_Expts and choose HObs_FDec.(Figure 4). An HObs_FDecexperiment will automatically beadded to the Study Queue. Thedefault settings are 16 ppm (14 to–2) for the 1H spectral width,8 scans, a relaxation delay of2 seconds, a pulse width of 90 °,and a F19 decoupler offset at–61.5 ppm.

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Figure 4. Adding the HObs_FDec protocol to the Study Queue. The HObs_FDec protocol is found in theHF_Expts drop down menu and is automatically added to the Study Queue when selected.

3. Right-click on the HObs_FDecprotocol in the Study Queue andselect Open Experiment. Theexperiment is retrieved, the pulsesequence displayed, and theDefaults panel is displayed(Figure 5). The typical fields whichmight be customized are Numberof scans and F19 Decoupler offset(ppm). The 19F decoupler offsetwas set to –120 ppm for thisinvestigation, appropriate for thechemical shift of the fluorineresonances of interest. The defaultdecoupler shape automaticallycreated is a WURST40ii with an8 kHz bandwidth, a maximumHF coupling of 60 Hz, and a dutycycle of 40 %. (Furthercustomizations can be found in thePulse Sequence panel.)

4. The experiment is now ready toacquire 1H{19F} data.

5. In the Experiment Selector, selectHF_Expts and choose FH_HOESY.(Figure 4). A FH_HOESYexperiment will automatically beadded to the Study Queue afterthe HObs_FDec protocol.

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Figure 5. Customizing the HObs_FDec experiment. The Defaults page contains parameters most oftenmodified, such as the Number of scans and the F19 Decoupler offset (ppm).

6. Right-click on the FH_HOESYprotocol in the Study Queue andselect Open Experiment. Theexperiment is retrieved, the pulsesequence displayed, and theDefaults panel is displayed(Figure 6). The typical fieldsmodified are Scans per t1increment, t1 increments, andHOESY mixing time. For theexample, the scans per t1increment was set to 16,t1 increments were set to 64, andHOESY mixing time was set to700 milliseconds for thisinvestigation. (Furthercustomizations can be found in thePulse Sequence panel.)

7. Use the Submit button in theStudy Queue to initiate data collection.

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Figure 6. Customizing the FH_HOESY experiment. The Defaults page contains the fields commonlymodified parameters, such as Scans per t1 increment, t1 increments, and HOESY mixing time.

www.agilent.com/chem/nmr

Information, descriptions, and specifications in this document are subject to change without notice.

© Agilent Technologies, Inc., 2011Published in the USA, October 27, 20115990-9229EN

ConclusionsThe 1H/19F class of experiments, suchas Hobs_FDec and FH_HOESY, arevaluable tools for simplifying spectraand allowing exploration of spatialpositioning of atoms. These double-resonance experiments are easy to setup and implement. As they areintegrated seamlessly into VnmrJ 3,they can be run as part of a StudyQueue in automation, or run manually.

References1. Bauer, W. Pulsed field gradient‘inverse’ HOESY applied to the isotopepairs 1H, 31P and 1H, 7Li. Mag. Reson. Chem., 1996, 34:532-537.

2. Kupce, E., and Freeman, R. Adiabaticpulses for wideband inversion andbroadband decoupling. J. Magn. Reson.,1995, 115A:273-276.