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    Bruker: 2D NMR Spectroscopy Overview

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    renatoporanga
    1. The document provides an overview of common 1H-detected 2D NMR pulse sequences including COSY, TOCSY, NOESY, ROESY, HMQC, HSQC, and HMBC. 2. It notes important experimental considerations for each sequence such as through-bond versus through-space connectivity, spin locking, and sensitivity. 3. Recommended Bruker parameter sets and pulse programs are provided for each sequence to help users set up 2D NMR experiments.

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    Bruker: 2D NMR Spectroscopy Overview

    Uploaded by

    renatoporanga
    91 views8 pages
    1. The document provides an overview of common 1H-detected 2D NMR pulse sequences including COSY, TOCSY, NOESY, ROESY, HMQC, HSQC, and HMBC. 2. It notes important experimental considerations for each sequence such as through-bond versus through-space connectivity, spin locking, and sensitivity. 3. Recommended Bruker parameter sets and pulse programs are provided for each sequence to help users set up 2D NMR experiments.

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    2d nmr

    Original Title

    Bruker2D_Oct2013

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    1. The document provides an overview of common 1H-detected 2D NMR pulse sequences including COSY, TOCSY, NOESY, ROESY, HMQC, HSQC, and HMBC. 2. It notes important experimental considerations for each sequence such as through-bond versus through-space connectivity, spin locking, and sensitivity. 3. Recommended Bruker parameter sets and pulse programs are provided for each sequence to help users set up 2D NMR experiments.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    91 views8 pages

    Bruker: 2D NMR Spectroscopy Overview

    Uploaded by

    renatoporanga
    1. The document provides an overview of common 1H-detected 2D NMR pulse sequences including COSY, TOCSY, NOESY, ROESY, HMQC, HSQC, and HMBC. 2. It notes important experimental considerations for each sequence such as through-bond versus through-space connectivity, spin locking, and sensitivity. 3. Recommended Bruker parameter sets and pulse programs are provided for each sequence to help users set up 2D NMR experiments.

    Copyright:

    © All Rights Reserved
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    of 8

    2D NMR Spectroscopy Overview

    BRUKER last edit 10/10/13

    PLEASE NOTE: This document assumes the user has a basic understanding of how to
    run 1H and 13C experiments on the Bruker 400 and 401.

    ‘Essential’ 1H-detected 2D Pulse Sequences:


    COSY – COrrelation SpectroscopY, good for determining basic connectivity via J-
    couplings (through-bond).
    TOCSY – TOtal Correlation SpectroscopY, same as COSY, but also able to generate
    cross peaks via intermediate spins (mix). Uses a spin lock that produces rf heating of
    the sample and hence requires many steady state scans (ds). (see separate training
    guide for TOCSY.
    NOESY – Nuclear Overhauser Effect SpectroscopY, allows one to see through-space
    effects, useful for investigating conformation and for determining proximity of adjacent
    spin systems. Not so useful for MWs in the 1 kDa range due to problems arising from
    the NMR correlation time.
    ROESY – Rotational Overhauser Effect SpectroscopY, same as NOESY, but works for
    all molecular weights. Has the disadvantage of producing more rf heating, hence it
    requires more steady state scans.
    HMQC – Heteronuclear Multiple Quantum Correlation, allows one to pair NH or CH
    resonances. Often uses X-nucleus decoupling and hence gives rise to rf heating,
    requires reasonably well calibrated pulses and many steady state scans.
    HSQC – Heteronuclear Single Quantum Correlation, provides the same information as
    HMQC, but gives narrower resonances for 1H-13C correlations. Also requires X-
    decoupling and hence a large number of steady state scans and is also more sensitive
    to pulse imperfections.
    HMBC – Heteronuclear Multiple Bond Correlation, a variant of the HMQC pulse
    sequence that allows one to correlate X-nucleus shifts that are typically 2-4 bonds away
    from a proton.

    PLEASE NOTE: If you want to run your VT experiment at a different temperature,


    please contact a DCIF staff member for VT training. Do not change the probe
    temperature unless you have been trained.
    Summary of Methodology

    In other words, what you need to do.

    1. Lock, tune, and shim.

    2. Acquire 1D 1H spectra, set: reference, sweep width, and transmitter frequency.

    3. Reacquire 1D 1H spectra with reduced sweep width, and then determine the
    number of scans required. Record parameter values.

    4. Repeat for 1D 13C spectra to run HSQC or HMBC (if needed).

    5. Load 2D parameter set.

    6. Check 2D pulse program.

    7. Load prosol parameters and setup the reference, sweep width, transmitter
    frequency, number of scans, and the number of points.

    8. Set receiver gain, acquire.

    9. Transform 2D data, phase and load projections.

    _______________________________________

    1. Lock, tune and shim.


    a. Check that the spinning is shut off.
    b. Shim the magnet: X, Y, Z1-Z5
    c. Make sure you tune (atma) each channel in use during the 2D experiment
    i. 1H ONLY: COSY, NOESY, TOCSY
    ii. 1H and 13C: HSQC and HMBC

    2. Collect a good 1D spectra

    Experiment 1 (EXPNO)

    a. Proton: Acquire a 1D and reference. Zoom in and display all proton


    signals leaving 0.5 ppm of baseline on each side. Type setsw (or click on
    the icon) to set the transmitter offset (o1p) and sweep width (sw).
    b. Reacquire “reduced-sweep width spectra” with the number of scans (ns)
    needed to get good signal to noise and phase. This dataset will become
    the 1H projection.

    Write down the following values:

    o1p:______________

    sw:______________

    sr:______________

    ns:______________

    **These values will be used in F2 (direct) dimension.**

    Type the parameter in the command line, hit enter, and TopSpin will display the value
    for you.

    Experiment 2

    c. X Nucleus (only if acquiring HMQC, HSQC, or HMBC), Acquire a 1D and


    reference. Zoom in and display all X signals leaving 0.5 ppm of baseline
    side. Type .setsw to set the transmitter offset (o1p) and sweep width
    (sw).
    d. Reacquire “reduced-sweep width spectra”. This dataset will become the X
    projection.

    Write down the following values:

    o1p:______________

    sw:______________

    sr:______________

    **These values will be used in F1 (indirect) dimension.**


    Experiment 100 (or any other new experiment)

    Type rpar on the command line or use Create Dataset.

    In the upper right corner of the Parameter Sets window, click the arrow next to Source,
    and make sure /opt/topspin3.1/exp/stan/nmr/par/ is selected. This will open the master
    list of all the Bruker standard parameter sets. Select the one you want, then click Set
    selected item in editor.

    If you check the Show Recommended box, this will display a list of Bruker’s most
    commonly used small molecule experiments.
    Suggested parameter sets and pulse programs

    PLEASE NOTE that these are suggestions only. These may or may not work for your
    particular sample.

    Experiment Parameter Set Pulse Program


    gCOSY COSYGPSW cosygpppqf
    gNOESY NOESYPHSW noesygpphpp
    ROESY ROESYPHSW roesyphpp.2
    gHSQC HSQCGPPH hsqcgpph
    gHMBC HMBCGP hmbcgplpndqf
    TOCSY MLEPHSW mlephpp

    COSY experiments

    COSYGPSW (cosygpppqf)– magnitude mode COSY (qf) with gradients (gp) and purge
    pulses (pp).

    COSYGPDFPHSW (cosygpmfphpp) – COSY with gradient pulses (gp), multiple


    quantum filter (mf), phase sensitive (ph), and purge pulses (pp). Difficult for a beginner
    to phase.

    NOESY experiment

    NOESYPHSW (noesygpphpp) – NOESY with gradient pulses during mixing time, phase
    sensitive (ph), and purge pulses (pp).

    ROESY experiment

    ROESYPHSW (roesyphpp.2) – ROESY sequence, phase sensitive (ph), and purge


    pulses (pp), using 180x-180x pulses for spin lock to suppress TOCSY artifacts (.2)

    HSQC experiments

    HSQCGPPH (hsqcgpph) – simple HSQC, phase sensitive (ph).

    HSQCETGP (hsqcetgp) – simple gradient HSQC, non-Edited.

    HSQCEDETGP (hsqcedetgp) - simple Multiplicity Edited gradient HSQC.

    HSQCEDETGPSISP_ADIA (hsqcedetgpsisp2.3) – Multiplicity Edited (ed), adiabatic


    pulses (sp), sensitivity improved (si). Gives you DEPT type information in addition to the
    1
    H-13C connectivity.

    HSQCETGPSISP_ADIA (hsqcetgpsisp2.2) – NOT Multiplicity Edited, adiabatic pulses


    (sp), sensitivity improved (si). Simple and all peaks are positive.
    HMBC experiments

    HMBCGP (hmbcqplpndqf) – Gradients for coherence selection (gp), low pass filter (lp),
    no decoupling during acquisition (nd), and magnitude mode (qf). Simple and no 180°
    pulses.

    HMBCETGPL3ND (hmbcetgpl3nd) – Echo Anti Echo (et), gradients for coherence


    selection (gp), 3rd order low pass filter (l3). More difficult to process (xfb + xf2m).

    TOCSY experiments

    MLEVPHSW (mlevphpp) – Homonuclear Hartman-Hahn using MLEV17 sequence,


    phase sensitive (ph), and purge pulses (pp).

    MLEVPHPR (mlevphpr.2) - Homonuclear Hartman-Hahn using MLEV17 sequence,


    phase sensitive (ph), and presat (pr).

    3. Check pulse program (in AcqPar tab) and make sure the correct pulse program
    (PULPROG) has be loaded (see above table). If you click the button with the
    three periods (…) next to the PULPROG window, it will open the list of available
    pulse programs.

    4. Load the prosol parameters by typing getprosol or by clicking the Acquire 


    Prosol button.

    5. Edit the basic parameters based on the information from the 1D experiments
    (the values you recorded in step #2).

    Homonuclear Experiments
     ns Number of Scans
     p1 Pulse width (us) (90 degree Pulse)
     o1p F2 Transmitter frequency (ppm)
     sw Sweepwidth (ppm). Enter the value for F2 and F1 dimensions.
     sr Reference (Hz). Enter the value for F2 and F1 dimensions.

    Inverse experiments ( 1H vs 13C) require additional parameters


     p2 Pulse width (us)
     o2p F1 Transmitter frequency (ppm)
     1 sw F1 Sweepwidth (ppm)
     1 sr F1 Reference(Hz)

    Check these parameters, loaded during getprosol. Adjust as needed (eg. if you
    measured the 90 o pulse).
     pl1 Power Level p1
     pl2 Power Level for p2
    6. Check Experiment Specific Parameters (listed below) and adjust as needed.

    7. Optional parameter changes


     2 td F2 Number of points
     1 td F1 Number of points

    8. Set receiver gain rga and acquire zg.


    Use multizg to start multiple incremental experiments (if desired).

    9. Data Processing

    TopSpin’s 2D display buttons. If you want to know what a button does, hover your
    mouse over the button, without pressing, and the software will tell you what the button
    does.

    a. Fourier Transform the data by typing xfb


    b. Phase the data (if needed).
    i. Type ph or hit the Adjust Phase button.
    ii. Choose manual phasing (if given the option).
    iii. Right click on two or three peaks that span your spectrum.
    iv. LBM on the “R” button to start phasing the rows.
    1. Click and drag on the “0” and “1” to adjust the zero and first
    order phasing.
    2. When finished, click save and return.
    v. LBM on the “C” button to start phasing the columns.
    1. Click and drag on the “0” and “1” to adjust the zero and first
    order phasing.
    2. When finished, click save and return.
    vi. Click return to go back to the spectra.
    c. Homonuclear experiments ONLY may be symmetrized to reduce the
    noise. Type sym to symmetrize.

    10. Load the 1D projections. Right click in the 2D spectrum window, at the top OR
    on the left side, and select External Projection. Fill in the name of the 1D
    experiment you want as that projection.

    11. To edit the contour lines, you can right click in the middle of the 2D spectrum
    and select Edit Contour Levels. Edit as desired. .

    12. When finished, remember to ro off, lock off, and eject (ej) your sample.
    Experiment Specific Parameters

    (step #9)
    COSY
    p1 90 degree pulse
    pl1 power level for p1 pulse

    NOESY
    d8 Mixing time. Default = 0.3 seconds. Recommend 400-500 ms for small
    molecules, 100-200 ms for larger

    ROESY
    p15 mixing time for dipolar or through-space interactions. Default = 200
    milliseconds.
    ds dummy scans to establish thermal equilibrium

    HSQC
    d1 Delay time
    CNST2 (average value for 1J(XH) will affect d24 1/(8 * 1J(XH))

    TOCSY
    d9 Mixing time. Default = 0.08 seconds.

    See the NMR Guide in TopSpin for more details.

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