uc davis nmr facility iconnmr user guide bruker 400 …

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UC DAVIS NMR FACILITY

ICONNMR USER GUIDE

BRUKER 400 – CHEM93

VERSION 2 August, 2016

TABLE OF CONTENTS

Disclaimer ...................................................................................... 2

Conventions ................................................................................... 2

Routine 1D NMR Experiments (1H, 13C, 19F, 31P, etc)

Procedure for acquiring 1D Proton spectrum ................................ 3

Procedure for acquiring routine 1D Carbon spectrum .................. 9

Other 1D X-Nuclei Experiments: 19F, 31P, 11B, etc ................... 12

Common 2D NMR Experiments (COSY, HSQC, HMBC, NOESY, etc)

2D COSY, HSQC, HMBC – Through Bond ................................. 15

2D NOESY, ROESY – Through Space ......................................... 20

Advanced 1D and 2D NMR Experiments

1D Carbon DEPT-135 and DEPT-90 ............................................ 25

2D Diffusion / DOSY NMR .......................................................... 28

Selective 1D-NOESY and 1D-TOCSY ......................................... 30

Using Non-Deuterated Solvents – Skip Lock and Shim ................ 34

Water Suppression Techniques (NOESYPR1D, WATERSUP) .... 36

Miscellaneous Topics

List and Brief Description of Important Commands ..................... 39

Date of last edit: August, 2016 Authors: Dr. Bennett Addison

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Brief IconNMR and Topspin 3.5 User Guide for Bruker NMR Spectrometers

Avance IIIHD 400MHz NMR with Autosampler in Chemistry room 93

DISCLAIMER

This document is intended to be a brief, bare-bones user’s guide for NMR data collection using Bruker’s IconNMR Automation software for the 400 MHz Avance-IIIHD Bruker NMR spectrometer located in Chem93 managed by the UC Davis NMR Facility. For detailed help with both routine and advanced NMR experiments, please consult Bruker’s User Guides, which can be found within the Resources tab on our website, nmr.ucdavis.edu.

CONVENSIONS

Some user control will be done in Bruker’s Topspin 3.5 interface. For Topspin interaction, keyboard input is shown as boldface type in this manual. Note that in TS the “enter” key must be used after the command is typed; this is assumed through-out this manual and “enter” key strokes are not given explicitly. Commands in TS are typed in on the TS command line near the bottom of the TS window; again this is assumed and will not generally be stated explicitly herein. LMB, MMB, and RMB are used to indicate actions of the left, middle, and right mouse button respectively. On a PC the mouse wheel acts as the MMB. Click, and double click refer to pressing the LMB.

GENERAL PROCEDURE

You will find that the general procedure for acquiring NMR data on all NMR spectrometers is essentially the same. IconNMR handles the majority of the tasks listed below. The general procedure is as follows:

1 Sample Preparation 2 Login and Startup 3 Setup Initial Parameters 4 Check/Edit Acquisition parameters 5 Insert Sample 6 Lock onto Solvent 7 Tune the Probe 8 Shim 9 Check Receiver Gain 10 Acquire 11 Process Data 12 Remove Sample 13 Logout, Sign Logbook

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Experiment: Routine Proton NMR Parameter Set: PROTON Instrument: Chem Bruker 400 using IconNMR

Sample Preparation

1. Dissolve your sample in an appropriate deuterated NMR solvent. Make sure there is no un-dissolved material. If there is, you will need to either centrifuge or filter your sample to remove crystals/debris.

2. Transfer about 550 - 600 uL of solvent into a clean NMR tube. We recommend high-quality NMR tubes - rated 600 MHz or higher, but economy tubes will be OK for routine work at lower fields (400 MHz and below). Your sample height should be about 4 - 5 cm.

Login and Startup

1. Log into your iconnmr user account. If a previous user is currently logged in, you will need to select Change User, then log into your account.

TIP: use a small amount of KimWipe in a Pasteur Pipette to quickly filter out any undissolved material

NOTE: If your IconNMR account has not yet been created, please reach out to Bennett Addison: [email protected]

mailto:[email protected]

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Set Initial Parameters 1. Identify an empty holder in the BACS120 autosampler. We strongly recommend you use

the holders in the inner ring: holders 1-60. 2. Place your sample into an available blue spinner, set depth using the depth gauge, and

drop your sample off in the appropriate holder. Do NOT drop off your sample while the carousel or sample-arm is moving.

3. Identify the matching holder in IconNMR menu and double-click to add an experiment. 4. Enter your details: Data Directory, Experiment Name, Experiment Number, Solvent,

Experiment/Parameter Set.

Directory: Your directory should be based on your PI. It is set automatically when you log in.

Experiment Name. Users often include initials, lab notebook page, date, and any additional info if desired. Note do not use spaces, special characters, etc.

Experiment Number. The default is increments of 10 (10, 20, 30…) for each sample/holder, or increments of 1 (10, 11, 12…) if you are collecting multiple experiments on the same sample/holder.

Solvent: Select your solvent from the drop-down menu

Experiment: Select your experiment from the dropdown list. If there is an experiment you would like to run but it is not on the list, contact Bennett.

Use inner ring: Inner ring is the lower numbers, outer ring higher numbers

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Check/Edit Acquisition Parameters 1. If desired, hit the equals button to edit standard Proton acquisition parameters

including number of scans, spectral width, acquisition time, offset frequency, etc.

2. Edit the title if desired. You can add any sample/experiment description in

this text box. You may find it useful later for your bookkeeping.

3. If using the nighttime queue (8pm – 8am) you may edit the Start Time if desired. The clock is set to 24 hr time. Note, if you set a start time between the hours of 8pm – 8am (night-time queue), please make a reservation on the online scheduler. Please avoid setting a start time during normal-use hours (8am – 8pm).

4. If you would like to also collect another experiment on this sample (e.g., C13CPD), you

can highlight the current experiment and then hit the Add button. Another experiment will appear below with experiment number 11 and an empty experiment type. Select the appropriate experiment and edit acquisition parameters if necessary. Details are provided in later pages.

Note: The default acquisition parameters are good, but feel free to change them as desired. If you would like to change any other parameters not accessible in this list, you can select Parameters – Edit All, which brings you to Topspin. You may need to do this for some 2D experiments.

11 pm Start Time

Note: This is red only because a parameter (ns) has been changed

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Submit your Experiment 1. When you are satisfied with all experiments and are ready to collect your data, highlight

the appropriate experiment or holder directory, and hit the Submit icon.

AUTOMATION PROCESSES: After hitting Submit, the autosampler will take over. The automation software will handle the following processes:

Insert Sample,

Tune the Probe,

Lock onto Solvent,

Shim,

Check Receiver Gain,

Acquire Data,

Process Data,

Remove Sample,

Backup Data to Google Drive.

Current Process

If Flashing: in Progress If Green: Completed If Clear: Skipped or Not Completed

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Data Processing 1. To look at your spectra, simply double-click on your completed experiment in the

Preceding Experiments list. If data acquisition and processing is complete, you will see a checkmark under Acq and Proc by your experiment. When you double click, you will be sent to Topspin for spectral viewing.

Spectral Processing Commands and Tips:

1. Navigate to the Process tab, and hit Proc. Spectrum button. This will perform Fourier transform (efp), autophase your data (apk), and perform an automatic baseline correction (abs).

2. Click and drag to zoom in 3. Double-click or hit the Full View button to zoom out. 4. Use middle mouse scroll wheel to zoom up and down 5. Click and hold the Hand icon to shift the baseline up or down or side to side

Identify your completed experiment and double-click. The list is ordered chronologically.

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Logout Procedure: 1. When the autosampler has returned your sample to the holder you can remove your

sample and spinner. Caution: do NOT remove your sample while the carousel is moving, and do NOT reach across the center where the sensor is located. This is especially problematic when there is a sample currently in the magnet. Tripping the sensor with your hand may result in shutdown of the autosampler. If this happens, be sure to call Bennett or an NMR TA.

2. When you are done and no longer using your chosen holder, return to IconNMR, select your completed holder, and hit Delete. The holder should become available.

3. Sign the log book, including Date, Name/PI, Nucleus, Drop-Off Time, Pickup Time, and check if you have deleted your completed run in IconNMR.

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Experiment: Routine 1D Carbon NMR Parameter Set: C13CPD Instrument: Chem Bruker 400 using IconNMR Set Initial Parameters

1. Identify an empty holder in the BACS120 autosampler. We strongly recommend you use the holders in the inner ring: holders 1-60.

2. Place your sample into an available blue spinner, set depth using the depth gauge, and drop your sample off in the appropriate holder. Do NOT drop off your sample while the carousel or sample-arm is moving.


Experiment/Parameter Set. If you are collecting both Proton and Carbon, you can hit the Add button and select both experiments from the drop-down list.

Check/Edit Acquisition Parameters

Directory: Your directory should be based on your PI. It is set automatically when you log in.

Experiment Name. Users often include initials, lab notebook page, date, and any additional info if desired. Note do not use spaces, special characters, etc.

Experiment Number. The default is increments of 10 (10, 20, 30…) for each sample/holder, or increments of 1 (10, 11, 12…) if you are collecting multiple experiments on the same sample/holder.

Solvent: Select your solvent from the drop-down menu

Experiment: Select your experiment from the dropdown list. If there is an experiment you would like to run but it is not on the list, contact Bennett.

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5. If desired, hit the equals button to edit standard Carbon acquisition parameters including number of scans, spectral width, acquisition time, offset frequency, etc.



7. Nighttime Queue or Set Start Time: To avoid holdups during peak usage, please submit long experiments (30 min or longer) during night hours. If using the nighttime queue (8pm – 8am) you may edit the Start Time if desired. The clock is set to 24 hr time. Note, if you set a start time between the hours of 8pm – 8am (night-time queue), please make a reservation on the online scheduler. Please avoid setting a start time during normal-use hours (8am – 8pm).

Submit your Experiment

8. When you are satisfied with all experiments and are ready to collect your data, highlight the appropriate experiment or holder directory, and hit the Submit icon.

Note: The default acquisition parameters for 13C are good, but if you have high sample concentration (>= 20mg) the default of 1024 scans may be overkill. Try changing to 256 scans for concentrated samples.

11 pm Start Time

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Preceding Experiments list. If data acquisition and processing is complete, you will see a checkmark under Acq and Proc by your experiment. When you double click, you will be sent to Topspin for spectral viewing.

Spectral Processing Commands and Tips:

6. Navigate to the Process tab, and hit Proc. Spectrum button. This will perform Fourier transform (efp), autophase your data (apk), and perform an automatic baseline correction (abs).

7. Click and drag to zoom in 8. Double-click or hit the Full View button to zoom out. 9. Use middle mouse scroll wheel to zoom up and down 10. Click and hold the Hand icon to shift the baseline up or down or side to side

Logout Procedure:

1. When the autosampler has returned your sample to the holder you can remove your sample and spinner.

Caution: do NOT remove your sample while the carousel is moving, and do NOT reach across the center where the sensor is located. This is especially problematic when there is a sample currently in the magnet. Tripping the sensor with your hand may result in shutdown of the autosampler. If this happens, be sure to call Bennett or an NMR TA.



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Experiment: 1D X-Nuclei NMR Experiments (19F, 31P, 11B, Si29, Al27, etc) Parameter Set: F19, F19CPD, P31, P31CPD, Si29IG, Al27ND, etc… Instrument: Chem Bruker 400 using IconNMR Set Initial Parameters

1. Identify an empty holder in the BACS120 autosampler. 2. Place your sample into an available blue spinner, set depth using the depth gauge, and








Select the relevant experiment from the drop down list. Parameter sets are typically named based on nucleus and the type of 1H decoupling used (ie, no decoupling, CPD for standard decoupling, IG for inverse-gated decoupling). Virtually any NMR-active X-nuclei can be tried on this instrument. If an experiment is missing please contact Bennett.

Note: For each routine 1D X-Nuclei experiments you should be able to edit all important acquisition parameters using the Equals icon. Be sure to set your center / offset O1P to the expected center of your spectrum. Also, be aware of acquisition time – try to set AQ based on the duration of signal in your FID. For example, if your FID is dead after 0.1 seconds then you only need 0.1 to 0.2 seconds of acquisition time. A wide variety of NMR-active X-Nuclei experiments can be done on the 400. Contact Bennett if you want to add a nucleus that is not listed.

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Acquisition Parameters – Most Common X-Nuclei Experiments, 19F, 31P, 11B, 29Si

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3. Nighttime Queue or Set Start Time: To avoid holdups during peak usage, please submit long experiments (30 min or longer) during night hours. If using the nighttime queue (8pm – 8am) you may edit the Start Time if desired. The clock is set to 24 hr. time. Note, if you set a start time between the hours of 8pm – 8am (night-time queue), please make a reservation on the online scheduler. Also, please avoid setting a start time during normal-use hours (8am – 8pm).



11 pm Start Time

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Experiment: Routine 2D Through-Bond Correlation NMR Experiments (COSY, HSQC, HMBC)

Parameter Sets: COSY.UCD, HSQC.UCD, HMBC.UCD Instrument: Chem Bruker 400 using IconNMR

1. Setup a new experiment and load a routine PROTON. You will always need to collect a

Proton experiment prior to any proton-detected 2D experiments. 2. After creating your initial PROTON experiment, hit Add and select the relevant 2D NMR

experiment or experiments. In this example, both routine 1H-1H COSY and 1H-13C HSQC experiments are selected. Note, there may be multiple variants of each experiment. Feel free to try other versions of these experiments. Ask NMR Staff for suggestions on which experiments will be best for your needs.

Caution: Be sure that your F2 reference has the same name and experiment number as your routine 1H experiment! Otherwise IconNMR will fail because it is looking for an F2 reference.

Note: many 2D experiments will optimize the 1H spectral width by taking an F2 reference spectrum. Be sure that the F2 reference of your 2D experiments are set correctly to your 1D Proton experiment. This will be done correctly by default, so be careful if you are changing the experiment name!

Experiment 10 is the reference 1H spectrum for both the COSY and the HSQC 2D experiment in this case.

Common 2D Experiments

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3. Using a Set Start Time, or using Nighttime Queue: Often you will want to submit multiple 1D and 2D experiments during the night queue (8pm – 8am). This helps avoid holdups during the day when many users are requesting short 1D experiments. Be sure to reserve your time on the online scheduler!

4. Edit Basic Acquisition Parameters (Optional but recommended): Select the equals

button to change basic parameters including number of scans, dummy scans, number of points, etc. Note, your spectral width SW is set automatically based on your F2 reference, so no need to change this. If you want to change the resolution (number of points) in the indirect (F1) dimension, you will need to follow directions in editing Advanced acquisition parameters. Check the tables on following pages for default and recommended acquisition parameters.

5. Edit Advanced / All Acquisition Parameters: Not all acquisition parameters are available for edit using the equals button – for example number of points in the indirect (F1) dimension - but you can edit them directly in Topspin. First highlight the 2D experiment in question, then select Parameters -> Edit All Acquisition Parameters. This will send you to Topspin. When you are done editing parameters, hit the flashing “Return to IconNMR” icon.

Suggested HSQC parameters are shown here for a well-concentrated sample (i.e. >= 15 mg/mL). The only parameters you might want to change in this menu are number of scans NS, the relaxation delay D1, and number of points TD. The spectral width SW for 1H dimension is set automatically based on your reference spectrum, so do not change SW.

Set a specific start time here. The format is in military (24:00) time. The Night queue starts at 8pm, or 20:00. Be sure to set your 1H Reference experiment to run BEFORE your any 2D experiment.

Highlight your 2D experiment, then select “Parameters -> Edit All”. Edit acquisition parameters as necessary, then hit “Return to IconNMR when done.

The default number of points in F1 (your indirect dimension) is 128, but this is often not enough. We recommend using 256 points in F1

Example: 1H-1H COSY

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Acquisition Parameters: COSY, HSQC, HMBC COSY – COSY.UCD

*Parameters in Red are only accessible through Parameters -> Edit All (step 5).

HSQC: - HSQC.UCD


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HMBC: - HMBC.UCD


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6. Submit your experiment: Return to IconNMR. Highlight ALL experiments you want to submit, or highlight the root directory, then hit Submit. Your experiments should now be added to the queue. If you chose to set a start time, be sure the 1H start time is before the start time for any 2D experiments that rely on the 1H reference.

Data Processing and Exit Procedure

1. Double click on your completed 2D experiment in the Preceding Experiments list. Your unprocessed spectrum should show up in the Topspin window.

2. Under the Process tab, select the Proc. Spectrum button. After auto-processing is

complete, you will see your 2D experiment. Most likely you will be processing this data offline using Mestrenova, so advanced 2D data processing is not discussed.

3. When you are done, return to Iconnmr. Remove your sample and delete your run, and

log out of your user account. 4. Sign the logbook. Be sure to note when you removed your sample and if you deleted

your run from the queue.

This example shows how to process a 2D HSQC spectrum, but it works equally well for any 2D experiment.

Hit the center of the Proc Spectrum button.

Highlight the root directory to submit all experiments in your data set. IconNMR will only submit the experiments that you select.

Example 2D HSQC showing 1H-13C 1-bond correlations. Dark blue are CH2’s, green are CH or CH3’s

Diastereotopic protons: 2x CH2 protons correlate to same 13C resonance

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Experiment: Routine 2D Through-Space Correlation NMR Experiments (2D-NOESY, 2D-ROESY)

Parameter Sets: NOESYPHSW, ROESYETGP.UCD Instrument: Chem Bruker 400 using IconNMR

1. Setup a new experiment and load a routine PROTON. You will always need to collect a

Proton experiment prior to any proton-detected 2D experiments. 2. After creating your initial PROTON experiment, hit Add and select the relevant 2D NMR

experiment or experiments. In this example, the 2D NOESY experiment is selected. Note, there may be multiple variants of each experiment. Feel free to try other versions of these experiments. Ask NMR Staff for suggestions on which experiments will be best for your needs.

3. Using a Set Start Time, or using Nighttime Queue: Often you will want to submit longer

1D and 2D experiments during the night queue (8pm – 8am). You can hit the moon icon to submit the experiments sometime in the night queue, or use the Set Start Time option to submit at a specific time. This helps avoid holdups during the day when many users are requesting short 1D experiments. Please do not use the set start time option for normal daytime experiments. If you set a start time be sure to reserve the timeslot on the online scheduler!

Caution: Be sure that your F2 reference has the same name and experiment number as your routine 1H experiment! Otherwise IconNMR will fail because it is looking for an F2 reference.

Set a specific start time here. The format is in military (24:00) time. The Night queue starts at 8pm, or 20:00. Be sure to set your 1H Reference experiment to run BEFORE your any 2D experiment.

Note: many 2D experiments will optimize the 1H spectral width by taking an F2 reference spectrum. Be sure that the F2 reference of your 2D experiments are set correctly to your 1D Proton experiment. This will be done correctly by default, so be careful if you are changing the experiment name!

In this case Experiment 12 is the Proton F2 reference for both NOESY and ROESY experiments

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4. Edit Basic Acquisition Parameters (Optional but recommended): Select the equals button to change basic parameters including number of scans, dummy scans, number of points, etc. Note, your spectral width SW is set automatically based on your F2 reference, so no need to change this. If you want to change the resolution (number of points) in the indirect (F1) dimension, you will need to follow directions in editing Advanced acquisition parameters. Check the tables on following pages for default and recommended acquisition parameters.

5. Edit Advanced / All Acquisition Parameters: Not all acquisition parameters are

available for edit using the equals button – for example number of points in the indirect (F1) dimension - but you can edit them directly in Topspin. First highlight the 2D experiment in question, then select Parameters -> Edit All Acquisition Parameters. This will send you to Topspin. When you are done editing parameters, hit the flashing “Return to IconNMR” icon.

Default NOESY parameters are shown here for a well-concentrated sample (i.e. >= 20 mg/mL). The only parameters you might want to change in this menu are number of scans NS, the relaxation delay D1, and number of points TD. The spectral width SW for 1H dimension is set automatically based on your reference spectrum, so do not change SW.

Highlight your 2D experiment, then select “Parameters -> Edit All”. Edit acquisition parameters as necessary, then hit “Return to IconNMR when done.

The default number of points in F1 (your indirect dimension) is 256. For a faster (but lower res) NOESY you can try 128. For more resolution try 400 or 512

Example: 1H-1H 2D NOESY

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Acquisition Parameters: NOESY, ROESY 2D NOESY - NOESYPHSW


ROESY: - ROESYETGP.UCD


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6. Check NOESY or ROESY mixing time Type d8 on the command line to edit the NOESY or p15 for ROESY mixing time. A dialogue box should pop up with the current value: 0.3 seconds for NOESY is default, 0.2 seconds (200,000 us) for ROESY. This is good for most cases, but you may want to edit.

7. Submit your experiment: Return to IconNMR. Highlight ALL experiments you want to submit, or highlight the root directory, then hit Submit. Your experiments should now be added to the queue. If you chose to set a start time, be sure the 1H start time is before the start time for any 2D experiments that rely on the 1H reference.


NOESY: The choice of d8 (mixing time) is usually based on molecule size. For small molecules (less than 500 Da), 0.5 to 0.3 seconds is good. For medium sized molecules, try 0.3 to 0.1 sec. For large molecules (1500 Da or grater) try 0.1 sec. ROESY: 0.2 seconds should be good in most cases. If you are hoping to be quantitative you may need to use a shorter spin-lock.

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Data Processing and Exit Procedure 1. Double click on your completed 2D experiment in the Preceding Experiments list. Your

unprocessed spectrum should show up in the Topspin window.

2. Under the Process tab, select the Proc. Spectrum button. After auto-processing is

complete, you will see your 2D experiment. Most likely you will be processing this data offline using Mestrenova, so advanced 2D data processing is not discussed.

3. When you are done, return to Iconnmr. Remove your sample and delete your run, and

log out of your user account. 4. Sign the logbook. Be sure to note when you removed your sample and if you deleted

your run from the queue.

This example shows how to process a 2D HSQC spectrum, but it works equally well for any 2D experiment.


2D NOESY experiment. Crosspeaks from NOE correlations should be phased opposite to the diagonal

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Experiment: 1D Carbon DEPT 135 and DEPT 90 Parameter Set: C13DEPT135, C13DEPT90 Instrument: Chem Bruker 400 using IconNMR Set Initial Parameters

5. Identify an empty holder in the BACS120 autosampler. 6. Place your sample into an available blue spinner, set depth using the depth gauge, and






*Parameters in Red are only accessible through Parameters -> Edit All

Note: The default acquisition parameters for 13C are good, but if you have high sample concentration (>= 20mg) the default of 1024 scans may be overkill. Try changing to 256 scans for concentrated samples.

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10. Edit the title if desired. You can add any sample/experiment description in this text box. You may find it useful later for your bookkeeping.

11. Nighttime Queue or Set Start Time: To avoid holdups during peak usage, please submit

long experiments (30 min or longer) during night hours. If using the nighttime queue (8pm – 8am) you may edit the Start Time if desired. The clock is set to 24 hr. time. Note, if you set a start time between the hours of 8pm – 8am (night-time queue), please make a reservation on the online scheduler. Please avoid setting a start time during normal-use hours (8am – 8pm).



11 pm Start Time

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Preceding Experiments list. 2. Under the Process tab, hit the center of the Proc. Spectrum button.

Logout Procedure:

1. When the autosampler has returned your sample to the holder you can remove your sample and spinner.

Caution: do NOT remove your sample while the carousel is moving, and do NOT reach across the center where the sensor is located. This is especially problematic when there is a sample currently in the magnet. Tripping the sensor with your hand may result in shutdown of the autosampler. If this happens, be sure to call Bennett or an NMR TA.



DEPT135: CH and CH3 phased Up, CH2’s phased down

DEPT90: Only CH should be visible.


Stacked Spectra comparing 13C DEPT135 (red) with DEPT90 (blue)

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Experiment: Diffusion Ordered Spectroscopy (DOSY) Parameter Set: DOSY.UCD

Instrument: Chem Bruker 400 using IconNMR

Set Initial Parameters

1. Select a routine PROTON experiment in iconnmr. Provide an experiment name, select your solvent, edit basic acquisition parameters and set a start time if desired.

2. Add another experiment and select DOSY.UCD as the parameter set. The default parameters are good for most small-molecule cases, but if you need better resolution in the diffusion dimension, you will need to edit the acquisition parameters.

Setup of DOSY Parameters in Topspin

1. Highlight the DOSY experiment so that the full experiment appears light blue, then select Parameters –> Edit All. This will take you to the AcquPars tab in Topspin.

2. Change number of points in F1 (your diffusion dimension) as desired. Note, changing

points in F1 from 16 to 32 will double the acquisition time. The default number of diffusion points is 16, which is a good starting point but may not give you the resolution you need in the diffusion dimension.

3. Check the parameters Big Delta d20 and little delta p30*2. An automated routine will

attempt to set these parameters for you, but typically you will see values near 50 ms for d20 and 1 ms for p30 (little delta = 2 ms). You may edit these values manually if you have a better initial guess. These values depend mainly on solute size and solvent

viscosity. For slower-diffusing compounds you may need to use longer delays. To check and/or change these delays, type the appropriate variable into the topspin command line, or find the parameter in the acquisition parameters.

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Execute dosy setup macro

1. Type the command dosy into the topspin command line. This runs a dosy setup macro: 2. Set the first (weak) gradient power. A good starting point is 5% - 10% 3. Set final (strong) gradient power. For best results try 90% - 95%

4. Indicate number of points in your DOSY dimension. This should match the number of acquired TD points in F1 – typically 16 or 32 depending on resolution need.

5. Indicate gradient ramp type. Default is linear, but feel free to try other types. 6. Topspin will ask if you would like to start data acquisition, but because acquisition is

controlled through iconnmr, this option will not work. Instead, hit Cancel. 7. When you are satisfied with all acquisition parameters, select the “Return to Iconnmr”

icon. You are now ready to start data acquisition.

8. Submit your experiment: Return to IconNMR. Highlight ALL experiments you want to

submit, or highlight the root directory, then hit Submit. Your experiments should now be added to the queue. If you chose to set a start time, be sure the 1H start time is before the start time for any 2D experiments that rely on the 1H reference.


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Experiment: Selective 1D Experiments: 1D NOESY, 1D TOCSY Parameter Set: SELNOGP, SELMLGP Instrument: Chem Bruker 400 using IconNMR

1. Setup and submit a routine 1H NMR experiment. 2. After the sample is in the magnet and collecting data, hit Pause, and select “Pause

leaving sample in Magnet”. This way the autosampler won’t eject your sample while you complete the setup of your 1D NOE experiment. Allow your 1D 1H spectrum to finish acquiring.

3. Double click on your experiment in the history list to load 1D 1H spectrum. Process with

efp, apk. Or hit Proc Spectrum button under Process tab.

4. Identify your target for your 1D NOESY or 1D TOCSY experiment, and zoom in using Click

and Drag in Topspin.

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5. Define your regions: a. Under Process tab, hit the Integration button. b. Delete all integrals if any are present c. With the integration curser enabled (yellow), click and

drag over your peak to define the region. d. Hit save as, save to region, then save and close

6. Return to iconnmr 7. Highlight your 1H experiment and hit Add experiment, then select the relevant

parameter set. Use SELNOGP for 1DNOESY and SELMLGP for 1D TOCSY. Make sure the F2 reference spectrum for your selective experiment is the 1H experiment you just collected (typically experiment number 10)

1: Delete all integrals

2: Enable integration (toggle yellow) then drag over your peak to define region

3: Select Save As, then Save Regions to ‘reg’. Then hit Save and Close.

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8. Check acquisition parameters including number of scans ns and mixing time.

Suggested Value

Parameter Parameter Name SELNOGP SELMLGP

ns Number of Scans 32, 64, 128… 32, 64, 128…

d1 Recycle Delay 3 seconds 3 seconds

d8 NOESY mixing time 0.3 - 0.5 sec N/A

d9 TOCSY mixing time N/A

0.0 sec (good for reference)

0.03 sec (common small value - 2-3 bond correlations)

0.08 sec (common long value - full spin system)

a. To edit number of scans, use the equals icon. The more scans the better. b. To edit NOESY or TOCSY mixing times, select your experiment and then select

Parameters / Edit All. This will take you to Topspin. Type d8 (NOESY) or d9 (TOCSY) on the command line, and change the value as desired.

c. For 1D TOCSY experiments, it’s a good idea to set up multiple experiments with varying mixing times. Try starting with d9 = 0.0 seconds as a reference, then d9 = 0.03 sec for short correlations and d9 = 0.08 sec for longer range correlations. Return to Iconnmr. Set these up as experiments 11, 12, and 13, respectively.

9. Highlight the experiments and hit submit 10. Now hit the “play” icon to resume automation. Your selective 1D

experiment will now begin.

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Data Processing 5. Double click on your completed Selective 1D experiment. Your spectrum should show

up in Topspin.

6. Under the Process tab, select the Proc. Spectrum button. You should see the peak you selectively irradiated phased up and any peaks with NOE contact phased down.

7. Use Multiple Displays to stack your 1D 1H spectrum and your 1D NOE or 1D TOCSY spectrum:

a. type the command .md on the topspin command line b. Now read in your 1D 1H spectrum with the command re 10 where re means

read, and 10 is the experiment number for your reference 1D 1H spectrum. Change the number for this reference spectrum experiment number if necessary.

8. When you are done, hit the Return icon and return to Iconnmr. Log out of your user account.

Selectively irradiated peak should be large, and phased either up or down, depending on convenience.

Your 1D 1H spectrum is displayed in Red to aid in analysis.

Look for peaks phased opposite to your irradiated peak: these peaks show 1D NOESY correlations to your irradiated peak.

Adjust the vertical scale if desired by selecting the colored spectrum and using the *2 or /2 icons

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Experiment: Non-Deuterated Solvents: Skip Locking and Shimming Parameter Set: 1D X-Nuclei Experiments, ie 19F, 31P, 11B, 27Al, etc Instrument: Chem Bruker 400 using IconNMR

Before You Start: There are some cases where you might want to use regular protonated solvents instead of deuterated solvents. This is not recommended for any 1H NMR experiments, but skipping locking/shimming can be extremely convenient if you are simply trying to observe the presence of and/or approximate chemical shift of X nuclei (31P, 19F, 27Al, etc), or perhaps that your other option is to use expensive deuterated solvents. You should only consider this as an option if you don’t have any overlapping peaks that need to be resolved, and if your sample concentration is high enough that resonances emerge within minutes. For this to work properly, be sure to prepare your sample so that the sample height is about 4.5 cm. You will be skipping locking and shimming, so your lineshape will depend on how your sample is prepared. For best lineshape results you should try to match the standard sample, which has a sample height of 4.5 cm.

9. Setup a standard 1D experiment. In this example we are collecting a 1D 27-Aluminum experiment of AlNO3 in H2O.

10. Set the solvent to the deuterated version of your actual solvent. This way your chemical

shift referencing will be close. Note, it will not be referenced according to publication standards, but it is often good enough for crude analysis.

11. Select the Lock/Tune icon to bring up options.

12. Change the Lock Program to “LOCK-OFF” and the Shim Program to “Skip Shimming”

13. Submit your experiment.

If your solvent is H2O, set lock solvent to D2O.

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Example: 27-Al NMR spectrum of AlNO3 in H2O/D2O with no locking and shimming (red) compared to a similar experiment but with locking and shimming (blue). The lineshape of the spectrum even without locking is excellent because of proper sample preparation. The chemical shift referencing is not perfect, but is close enough for crude analysis. If you are needing publication-quality data you should use a deuterated solvent and an internal or external standard.

No locking or shimming: Al-27 NMR of AlNO3 in H2O. Note, lineshape is good because sample height was 4.5 cm. Chemical shift referencing is close even without locking.

Lock and shim on D2O solvent Al-27 NMR of AlNO3 in D2O

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Experiment: Water Suppression Methods (solvent 90% H2O 10% D2O) Parameter Set: WATERSUPP, NOESYPR1D Instrument: Chem Bruker 400 using IconNMR

Before You Start: In order to see exchangeable protons in an exchangeable solvent (D2O, MeOD, etc) you will need to use solvent suppression techniques. In this case we assume your solvent is 90/10 H2O/D2O and you are hoping to suppress the massive water signal at 4.7 ppm. You may try simply loading the WATERSUPP or NOESYPR1D parameter sets, but often you will need to calibrate the presat frequency for good results. The following will guide you through the necessary calibration procedures using IconNMR.

1. Load a 1D watersupp-setup parameter set, or a routine 1D PROTON experiment a. If you use the 1D PROTON experiment, set the experiment to collect 1 scan and 0

dummy scans, and change your pulse width p1 to 1 microsecond (1 us). You can do this by selecting Parameters – Edit All, typing p1 in the command line and changing the value to 1. Then hit Return to Iconnmr. You need a low tip angle so you don’t saturate the detector with a strong 1H solvent signal.

b. Submit the experiment 2. After the sample is in the magnet and collecting data, hit Pause, and select “Pause

leaving sample in Magnet”. This way the autosampler won’t eject your sample while you complete the setup of your 1D NOE experiment. Allow your 1D 1H spectrum to finish acquiring.

3. Double click on your experiment in the history list to load 1D 1H spectrum. Process with

efp, apk. Or hit Proc Spectrum button under Process tab.

4. Zoom into your water peak near 4.7 ppm, and hit the Set Offset to Curser button or type

.seto1 on the command line. 5. Click on the center of your water peak. A dialogue box will show up with your selected

offset in Hz. For water at room temperature this will be near 1879 Hz. Copy or write down this number: this is your calibrated offset for your water suppression experiment.

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6. Select O1 or hit Cancel, and return to Iconnmr

7. Highlight your PROTON or watersup-setup experiment in IconNMR and hit Add, then select WATERSUPP or NOESYPR1D as the parameter set.

8. Highlight your WATERSUP or NOESYPR1D experiment and select Parameters – Edit All.

9. Type O1 on the topspin command line, and write in (or paste in) your calibrated offset

from step 6. Hit OK when done, then hit Return to IconNMR.

2: Copy or write down this value

1: Click cursor at center of water resonance

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10. Check acquisition parameters including number of scans ns and mixing time. It is recommended you use at least 32 scans and at least 4 dummy scans.

Suggested Value

Parameter Parameter Name WATERSUP NOESYPR1D

ns Number of Scans 32, 64, 128… 32, 64, 128…

d1 Recycle Delay ~3 seconds ~3 seconds

d8 NOESY mixing time N/A 0.1 sec

11. Highlight the experiment and hit submit 12. Now hit the “play” icon to resume automation. Your 1D solvent

suppression experiment will now begin.

Residual H2O peak after solvent suppression

H2O peak with no solvent suppression

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List and Brief Description of Important Commands Command Short description abs automatic baseline straighten apk automatic phasing of 1D spectra apk2d automatic phasing of 2D spectra ased displays data acquisition parameters (short list) atma automatic tuning and matching, automatic option atmm automatic tuning and matching, manual option. eda displays all data acquisition parameters edc create new dataset from old dataset edp displays all data processing parameters edte start-up and display temperature control window efp em + ft + pk em exponential multiplication expt check experiment time ft Fourier transform halt halts data acquisition (data saved) lb line broadening value (for em) lock displays lock solvent list and then locks on chosen solvent new create new dataset ns number of scans o1p set o1 offset (carrier frequency, center of spectrum) in ppm o2p set o2 carrier frequency (non-observe nucleus, ie 1H for decoupling, or 13C in HSQC) pk applies last phase correction pps pick peaks and display rg receiver gain rpar read parameter file rsh read shim file stop stops data acquisition (data not saved) or tuning sw sweepwidth in ppm td number of acquired data points wobb starts tune display xf2 Fourier transform of data in 1 dimension, intensity in the other dimension xfb two dimensional Fourier transform zg starts data acquisition .md enter multiple display mode .int enter integration mode .ph enter manual phasing mode .pp enter peak picking mode .all zoom out to display full spectrum .cal calibrate your 1D or 2D axis using the curser

uc davis nmr facility iconnmr user guide bruker 400 …

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