ASAPHMQC – QuickMultidimensional Spectra

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.

IntroductionThe Acceleration by Sharing Adjacent Polarization Heteronuclear Multiple QuantumCoherence (ASAPHMQC)1 experiment, similar to the commonly used HeteronuclearSingle Quantum Coherence (HSQC)2 experiment, provides information regardingone-bond (1H-13C) connectivity and heteronuclear chemical shifts for theprotonated carbons. ASAPHMQC, however, requires far less time than most two-dimensional (2D) experiments, and, as such, provides an elegant tool for thecollection of inspection or survey data.

In this application note, comparative ASAPHMQC and gradient heteronuclear singlequantum coherence (adiabatic version) (gHSQCAD) data were acquired with defaultsettings on 5 mg of clindamycin HCl. All 16 1H-13C correlations are clearlyidentifiable in the ASAPHMQC experiment in 56 seconds, whereas the gHSQCADrequired 8.25 minutes to complete. Additionally, with a few minor parameterchanges, a 10 mg sample of clindamycin HCl gives a complete ASAPHMQCspectrum in 19 seconds. These examples provide an illustration of the power of theASAPHMQC experiment.

Another use of the efficiency ofASAPHMQC is to obtainhigh-resolution (in F1) data whennecessary. For 2D 1H-13C correlations,the resolution in F2, determined by theacquisition time, is often limited by thecarbon-decoupling power requirements.Resolution in F1, however, requiresadditional t1 increments (ni), which canbe expensive in terms of time. Becauseof the rapid recycle time of theASAPHMQC experiment, ahigh-resolution 2D proton carboncorrelation (for example, ni = 2048) canbe obtained in approximately13 minutes for a sufficientlyconcentrated sample. This can beextremely useful for compounds withspectral overlap issues.

An ASAPHMQC exampleA sample of clindamycin (Figure 1) wasused to demonstrate the speed anddata quality obtained using theASAPHMQC experiment. All data wascollected on an Agilent 400-MR DD2instrument with a OneNMR probe.

The efficiency gain for ASAPHMQCcomes from a much shorter relaxationdelay as compared to an HSQC-typeexperiment (0.06 seconds versus1 second for the default). The loss oflongitudinal magnetization fromincomplete relaxation is compensatedfor by a magnetization transfer fromnearby 12C-attached protons (which arekept along z during the pulse sequence)to the observed 13C-attached protons.The default parameters give a totalexperiment time of 56 seconds, notmuch longer than a simple protonspectrum.

One way to exploit the efficiency ofASAPHMQC is to use the experiment,along with a one-dimensional (1D) 1Hspectrum, as an efficient and elegantstrategy for the investigation of amolecular structure at the initial stage.Typically, if the 1H spectrum does notprovide sufficient information, thesample is resubmitted with strategichomonuclear or heteronuclearcorrelation experiments. Addingheteronuclear information in theinspection stage can often solve thestructure and, if not, more targeted datacan be acquired, for example, selective1D or band-selective 2D experiments.This approach to structure elucidationwill often be much faster and minimizethe impact to spectrometer usage.

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Figure 1. The chemical structure of clindamycin.

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Figure 2. 1H-13C ASAPHMQC on 5 mg clindamycin dissolved in 600 µL DMSO-d6. The total experiment time was 56 seconds.

Figure 3. 1H-13C gHSQCAD on 5 mg clindamycin dissolved in 600 µL DMSO-d6. The total experiment timewas 8 minutes 18 seconds.

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The spectrum in Figure 2 is of a 1H-13CASAPHMQC experiment. Figure 3shows a 1H-13C gHSQCAD forcomparison. Using the unmodifieddefault parameters to set up bothexperiments, ASAPHMQC providedsignals for all 16 protonated carbonswith a significantly reduced experimenttime, 56 seconds as compared to8 minutes 18 seconds for thegHSQCAD. The intensities ofcorrelations 12 and 20 are smaller inthe ASAPHMQC experiment than othercorrelations due to the limited numberof 1H-13C pairs for magnetizationtransfer, and can be increased with alonger relaxation delay than the defaultof 60 milliseconds.

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Figure 4. 1H-13C ASAPHMQC on 10 mg clindamycin dissolved in 600 µL DMSO-d6. The total experimenttime was 19 s, using 56 increments, a spectral with of 100 ppm in F1, and a delay of 70 ms.

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Depending on the sensitivity of theprobe and the amount of samplepresent, the default number of scansper increment could be decreased toensure the rapid acquisition of anASAPHMQC experiment with minimalimpact to spectrometer time. InFigure 4, the concentration ofclindamycin was doubled, so fewerrepetitions are required to achieve anadequate signal-to-noise ratio. Therelaxation delay was increased to70 milliseconds to demonstrate theimpact to correlations 12 and 20, andthe number of repetitions perincrement was decreased to one. All16 protonated carbons are still clearlyidentifiable, with correlations 12 and 20more intense than in Figure 2. Thetotal time for this ASAPHMQCexperiment was only 19 seconds,which is similar to the time needed toacquire a short 1D 1H experiment.

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Experimental MethodThe ASAPHMQC experiment can berun within or outside the Study Queuemode without any modification. Toacquire an ASAPHMQC in the StudyQueue:

1. Select New Study at the bottom ofthe Study Queue.

2. In the Experiment Selector, selectthe Liquids tab, then selectJ1(CH)corr and chooseASAPHMQC. (Figure 5). A PROTONwill automatically add to the StudyQueue before the ASAPHMQC. Thedefault settings are 170 ppm (160 to –10) for the indirectdimension spectral width, arelaxation delay of 60 milliseconds,a mixing time of 25 milliseconds,96 increments, two repetitions perincrement, an acquisition time of64 milliseconds, and a 1H-13Ccoupling of 146 Hz.

3. To customize the ASAPHMQCexperiment, right-click on theASAPHMQC protocol in the StudyQueue and select Open Experiment.The experiment is retrieved, thepulse sequence displayed, and theDefaults panel is displayed(Figure 6). The typical fieldsmodified are Scans per t1Increment, t1 Increments, and C13Spectral Width (ppm).

Figure 5. Adding the ASAPHMQC protocol to the Study Queue. The ASAPHMQC protocol is found in theJ1(CH)corr drop down menu and will automatically add a PROTON protocol to the queue if no PROTONis already present.

Figure 6. Customizing the ASAPHMQC experiment. The Defaults page contains the fields mostcommonly modified, such as Scans per t1 Increment, t1 Increments, and C13 Spectral Width (ppm).

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4. To lengthen the relaxation delay,select the Acquisition page andenter a longer time in theRelaxation delay field (Figure 7).Lone 1H-13C pairs withoutneighboring protons may need alonger relaxation delay than thedefault of 60 milliseconds.

5. The experiment is now ready toacquire ASAPHMQC data. Use theSubmit button in the Study Queueto initiate data collection.

Figure 7. Customizing the ASAPHMQC experiment. The Acquisition page contains additionalparameters, such as the Relaxation delay, and provides information such as resolution in the indirect dimension.

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ConclusionsThe ASAPHMQC experiment is asuperb choice for obtaining quickprotonated carbon (or otherheteronuclear) information. Theefficiency gained from the cross-polarization period and shortening ofthe relaxation delay, means informationcan be obtained in the ASAPHMQCexperiment eight times faster than in agHSQCAD experiment. As such, theexperiment is an excellent choicecomplimentary experiment to becollected along with a 1D 1H spectrumto quickly provide additional structuralinformation with no special setuprequirements.

References1. Kupce, E., and Freeman, R. Fastmultidimensional NMR by polarizationsharing. Magn. Reson. Chem., 2007,45:2-4.

2. Bodenhausen, G., and Ruben, D. J.Natural abundance nitrogen-15 NMRby enhanced heteronuclearspectroscopy. Chem. Phys. Lett., 1980,69:185-189.

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

© Agilent Technologies, Inc., 2011Published in the USA, October 27, 20115990-9237EN