To become a NMR user of the facility, he/she needs to

  • complete the new account info form to open an NMR account.
  • agree to abide by the safety policy.

For self-operation

  • New users are strongly recommended to attend NMR courses (CHEM471, BCHM669) provided by the Department of Chemistry and Biochemistry
  • New user must demonstrate proficiency in practical NMR operation, or attend the NMR training provided by the facility.

NMR Trainings

  1. Basic NMR trainings at BioNMR Facility
    • Experiment set up (2 hours, mandatory to most of new users ):
      • Sample and Experiment Preparations
      • Basic 1D and 2D Experiment Setup
    • CP800 Operation Walk-through (30 mins)
      • File structure and permission differences in Linux system
      • NMR operation differences on CP800 comparing to 600MHz spectrometers at the facility
      • High temperature experiment
  2. Additional training sessions are available upon request.
    • 2D NMR Experiments: SOFAST-HMQC, HSQC, HMBC, 2D TOCSY, 2D NOESY
    • CARA (Computer Aided Resonance Assignment): Project Setup and Spectrum viewing, Sequential Assignment using CARA
    • DOSY Experiment
    • Saturation Transfer Difference Experiment
    • Relaxation Measurements
    • Triple Resonance Experiments for Protein Structure Determination

Data Processing Computer

A Data Processing computer is available in the lab. Data can be transfered from Spectrometer computers to this computer for further processing and viewing in TopSpin. This computer also has its own IP address to facilitate data transfer to another computer. Details of operation are posted next to it.

 

Sample Requirement

  1. Sample volume should be a minimum of 250 uL for a Shigemi tube, 180uL for a 3mm/5mm tube, or 500 uL for a regular NMR tube. Insufficient volume may cause bad shimming result, and consequently poor resolution of NMR spectrum.

  2. The NMR sample should be homogeneous, free of air bubbles or precipitate. Spin, filter, and/or sonicate the sample if necessary.

    High salt condition should always be avoided if possible, as well as high paramagnetic ion concentration.

  3. High quality NMR tubes should be used at BioNMR facility, in particular for the 800 MHz spectrometer.

  4. For volatile organic solvent, or long term use sample, sealing the sample NMR tube with material such as parafilm is highly recommended. NMR cap itself works OK to seal aqueous sample.

  5. For aqueous samples,

    • D2O concentration should be equal to or higher than 7%.

    • NaCN 0.02% is recommended for sample buffer to prevent bacterial growth.

    • For protein samples, minimum concentration of 1 mM is highly recommended for efficient NMR measurement.

  6. Labeling your NMR tube. All samples must be labeled with sample content, solvent, and user initials. Avoid writing directly on the NMR tube since the ink can be erased easily by mistake. Using a labeling tape/tag is recommended.

 

 

 

 

 

 

Experiment Preparation
  1. Insert sample
    • Center the sample in the spinner using the sample gauge.
    • Type "ej" or click “lift” button from the BSMS interface.
    • When placing the sample with the spinner, make sure it floats on the lift air.
    • Type "ij" or click “lift” again to lower the sample into the magnet.
  2. Adjust temperature
    • Type "edte" or go to temperature control window.
    • Set the target temperature and wait for temperature equilibrium. Do not change air flow rate.
    • To CP800, turn chiller power off if temperature is set high, and set chiller power to maximum if temperature is set low.
  3. Lock and shim
    • Type “lock” and choose the correct deuterated solvent that is with the sample.
    • Type "topshim gui ", choose "1D", click on "start ". Once topshim is done, you can check improvement in the Report page.
    • For shigemi NMR tube, you may use the command "topshim shigemi" to shim the tube.
    • Note that if topshim didn't give satisfied shim result, you may use "rsh" command to read the latest shim file, and then redo topshim.
  4. Tune and Match the probe
    • First go to a 1D zg data set; You may type "edasp" to check/set channels to be tuned.
    • For probe with ATM device, type "atmm". Adjust tune and match of each nucleus, then exit.
    • For probe without ATM device, type "wobb". Use the small driver to adjust corresponding match and tune knobs to current nucleus under the magnet. After one nucleus channel is finished, click on “SW” to switch to the next nucleus. At the end click "Stop" to finish the wobb procedure.
  5. Calibrate 1H 90° pulse
    • In zg experiment data set, check default pL1/pLW1 value and set p1=4us, ns=1, ds=1.
    • Then acquire FID by typing "zg". Fourier transform data by typing "ft". Phase the spectrum by typing “apk”.
    • Re-set p1 to 30us as a starting value, acquire FID by typing “zg”, and process the FID by typing “efp”.
    • Keep adjusting p1 until you see a signal null, which corresponds to a 360° proton pulse. 1H 90° pulse should be 1/4 of the final p1. Alternatively you may use "paropt" to determine 1H 90° pulse.
  6. Determine O1P offset (for aqueous samples)
    • Go to an 1D zgpr experiment (pulse program: zgpr). Or in an new experiment "rpar zgpr all".
    • Type "eda", in the acquisition parameter window check and adjust td, ns, ds, sw
    • Type "ased", in the pulse program parameter window, enter calibrated p1 and pL1 values, check pL9 (should be >=50dB) and d1 2s.
    • Type "gs"; in gs mode adjust O1P offset to achieve minimum FID area integral.
    • Start the experiment by rga and zg; Process the FID by typing “dfp”, and phase the spectrum when necessary.

 

Wobb Instruction for probes without ATM

Notes:

1. All the adjusting knobs are color coded for different nuclei.

2. If you don't see the dip in the wobb window, first you may try adjusting T or M. Second you may increase the sweep width in Wobb window to 10 or 20 MHz.

The following are general steps to set up a 2D experiment.

  1. Use command "edc" to generate a new experiment set from the current displayed experiment. Or, use "rpar" command if there is a stored paramenter set, such as "rpar HSQCETFPF3GP" to create a new 1H-15N HSQC experiment. If only the pulse program is available, then you will have to choose the desired pulse program and set parameters manually.
  2. Type "ased" to open the acquisition window; define/check the spectrum dimension and window position by setting parameters SW, O1P, O2P.
  3. Define spectrum resolution by setting parameters TD F1 and TD F2.
  4. Type "eda", and in the eda acquisition window set power level and pulse length,  including high power pulses, decouple pulses, shape pulses, and et al.  TopSpin sometimes complains when you type eda, and it most likely is caused by undefined parameters such as gradient files, composite pulse decoupling(CPD), various list such as vclist, vdlist...... Read the error message, find out which parameter is missing, and add the missing parameter(s).
    * A shortcut of setting pulse parameters is to use the command "getprosol 1H A B", in which A is the calibrated 90° proton pulse length, and B is the related power level. This command will set all pulse parameters for you based on the values provided in the command. Note that this command may not work for customized pulse programs if the pulse definitions differ from the Bruker ones.
  5. Check/adjust NS, DS, D1, mixing time if required; Set gradient files and percentages defined in the pulse program; For a 13C and 15N double labeled sample, ZGOPTNS should be set to "-DLABEL_CN" for decoupling purpose.
  6. Always check and adjust receiver gain using the command "rga" before starting the experiment, and the rg value should be set reservedly. Start the experiment using command "zg"
  7. Process the FID using command “xfb”, and phase the spectrum when necessary. Note that SI values (size of real spectrum) of both demensions should be reasonable (F2 =< 2K, F1=< 1K). Putting large numbers will increase the file size, causing slow data processing and spectrum viewing.


Cautions/Notes:
  1. NMR tube length limit. Position of the upper injection tube limits the length of NMR tubes in the SampleCase. A Shigemi tube should have less than 400uL sample. Use manual injection procedure below for special long NMR tubes.
  2. Available spinners. Two room-temperature spinners are designated for CP800. Please do not use them on other spectrometers. More spinners are available upon request for multiple sample experiments in automation mode.
  3. Quitting IconNMR Automation mode. To be able to set up an experiment manually in TopSpin after IconNMR automation, you must first click on "Stop" in IconNMR Automation window.
Single-Sample Loading Procedure
  1. Put the sample NMR tube with spinner in any sample position in the Carousel Unit.
  2. If the sample is in position 5, type the command "sx 5" in topspin to inject the sample. Alternatively you may press the blue Rotation button on the Carousel to move the sample to the injection position under the injection tube, then press the green Inject/Eject button to inject the sample.
  3. If you are ready to run another sample in position 8, simply type "sx 8". The previous sample will be ejected, and the new sample will be rotated and injected into the magnet.
  4. To eject a sample, use command "sx ej", or press the green Inject/Eject button on the Carousel.
  5. To remove the sample from the Carousel Unit, first press the blue Rotation button on the Carousel to rotate the sample away from sample injection tube. In this way, you will avoid accidently breaking the sample tube due to limited space between sample holder and sample injection tube. 
Multiple-Sample Experiments Setup

Please choose IconNMR from the left side panel for details

 
Manual injection procedure for long NMR tubes that won't fit in the SampleCase
  1. Type "ha" in TopSpin to open Hardware Address window.
  2. Click "open" in BSMS block to open BSMS Service Web.
  3. Choose Sample handling, then Sample Transporter Control page.
  4. Switch Lift Mode to Standard BSMS Lift, and then click on "set" button to confirm.
  5. Now you may manually load the sample on top of the magnet, and use command "ej" and "ij" to move the sample.
  6. After finishing your experiment, switch Lift Mode back to "SampleTransporter:PLC on TTY1 of ELCB1" from the same Sample Transporter Control page.

 

 

IconNMR Automation: Multiple sample experiments setup (CP800)

  1. Put samples with spinner in the carousel sampleholder, and record the corresponding positions of all samples.

  2. In TopSpin, type "iconnmr" to open IconNMR interface.

  3. Select "IconNMR Automation", then select user.

  4. Double click the holder number which has your first sample.

  5. Change/edit the following information if necessary

    (1) Disk and Name: By default, Disk is the current TopSpin address, something like Topspin3.5pl6, but you may want to change it to the address where you usually store NMR data, such as TopSpin 3.0. The name will be a data folder under your account

    (2) Experiment number: Check the data folder first. You may set any number but make sure it won't overwirte any existing important data..

    (3) Solvent: it should be the deteurated solvent in the sample.

    (4 )Experiment: Please read carefully the description of the experiment to make correct selection.

    (5) Priority: not necessary to set it if you submit the experiment in your desired order.

    (6) Parameters:

    (a) Acquiision parameters. You may click on icon to edit a set of specified parameters. Or go to the top menu bar and select /parameters/Edit all acquisition paramenters, which is open the AcquPars window in topspin. After editing, you can click on Return to IconNMR to go back to Automation window.
    Note that you should set temperature here (TE). When IconNMR automation starts, a window will pop up asking you whether you want temperature be regulated. If one temperature will be used for all samples/experiments, make sure set that temperature to all entries.

    (b) Locking, Shimming options. You may change the settings by clicking on . I recommend NOT to tune and match X-channels, if you will adjust it before starting the automation.

    (7) Title

    (9) Click on “Add” to add more experiments to this specific sample. Right click the experiment, you may also see "Iterate" option, which allow you to copy the same experiment including customized parameters.

  6.  

  7. The “Copy” option allows you to copy the already set-up experiment to the next holder number/sample, and then edit only what is necessary. Repeat this step to cover all the holder numbers that have samples. Use "Delete" option to remove entries that have no samples.

  8. Review all the experiments that have been set up. Highlight the holder number, then click “Edit” will allow you to modify that experiment.

  9. Highlight the holder number then click on “Submit” to add the experiment to the queue one by one. You may highlight multiple holder numbers at once.

  10. Click “Start” to run the queued experiments. You may still any experiment that has not been running yet.

  11. At the end, you must quit the automation mode by clicking on the stop icon “” . After that, you can set up single experiments in TopSpin window, which is the "manual mode". Note that there may be a bug in the iconnmr and you won't be able to quit/close iconnmr window or TopSpin, and you have to log out from your account to terminate the program.

 

Data transfer using Filezilla Client program

1. Open Filezilla, go to File/Site manager, choose New Site, put in information such as host name/IP, user name and password, and then select Connect or OK to finish the site setup.

2. In Filezilla main window, click on the icon "open the site manager", and choose the remote computer you want to access.

3. If all the information in the site-manager was correct, you should be able to see files from the remote computer.

4. To transfer files, go to the desired file directory on both local and remote sites, click and drag the target files from one site to the other.

Note that some directories may not have access permission in a Linux computer.

Possible Filzezilla related issue

Problem: NMR data transferred by FilleZilla can not be opened in TopSpin
Cause: The ser file may have been altered during transfer. If so, in the desination folder you will see that it has a different size.
Solution: Go to FileZilla Client menu, Edit/Settings/Transfers/File Types, change Default Transfer Type to "Binary", and uncheck Treat files without extension as SCII file. Then re-transfer the data.

 

You may use the following procedure to validate the setting temperature, ranging from 282K-330K. Temperature correction is routinely checked by the facility Note that temperature correction is usually being enabled which should not be altered by users.

  1. Load the 99.8% Methanol-d4 Bruker reference sample.
  2. Lock on Methanol-d4.
  3. Do a round of "topshim"
  4. create a zg30 experiment set; Click on "getprosol" icon to use default pulse length and power level; Set ds=0, ns=1, rg=1.
  5. Type "zg" to run the experiment, and then "apk" to process the data.
  6. type "calctemp D" to obtain the calculated probe temperature. Ideally the calculated temperature should be very close to the settting temperature.

calctemp

 

 

 

 

High Temperature Experiment (CP800 Spectrometer)

CP800 NMR Spectrometer is equipped with a special CPQCI probe which can run high temperature experiments. To set up a high temperature experiments, you need to know the followings:

  1. The CPQCI probe has operating temperature range from 5 °C-135 °C.

  2. Use a ceramic spinner for experimental temperature higher than 50 °C / 323 K.

  3. The maximum temperature should be at least 10 °C below boiling point of the sample solvent.

  4. Set BCU/Chiller power to OFF when you run high temperature experiments, and set it to Medium when lowering the probe temperature back to room temperature. Do not set BCU power to Maximum to cool a hot probe.

  5. Increase temperature in two steps. First set the temperature about 15°C below the target temperature, let it settle there and then step your way up to the end temperature.

Low Temperature Experiment (CP800 Spectrometer)

To run a low temperature experiment, such as at 5 °C, you may need to set BCU / Chiller power to MAXIMUM in temperature control window.

 

 

 

 

 

 

 

 

Non-Uniform Sampling (NUS) is available for CP800 spectrometer
(NUS Data processing can be done on either CP800 computer or BioNMR PC in the NMR room)

Non-uniform sampling (NUS) is a method of acquiring data using less time than the conventional NMR methods. The method can also be used to improve resolution if experiment time is limited. For details on how to use NUS, please read NUS manual from TopSpin 3.2 program. The following is a brief guild of NUS paramenter set up.

Parameter Setup

1. Click AcquPars, and change FnTYPE to non-uniform_sampling.

nus1

2. Go to NUS section, set NusAMOUNT[%]. The amount is usually 50% or less, and make sure NUSLIST is set automatic.

nus2

3. Run the experiment as usual.

4. To process the spectrum, go to ProcPars page, and at NUS section, set MDD_mod to either cs or mdd (cs may give better result). Then, process the data as usual.

nus3

3D  NUS Data phasing

(1) Using a third party software to process data if available, such as hmsIST or Mnova

(2) TopSpin method.

(a) It is highly recommended to acquire a 2D  plane first in conventional mode to determine the phase correction.

(b) You may read the first FID, "reser 1"; determine the phase for f3 from this 1D spectrcum. If the experiment is ET or Echo Anti-Echo, you need to add or subtract 90 degrees from the phc0 value determined from the 1D.

(c) The indirect dimonesions should not require a phase correction.

  1. CP800: Sample got stuck in the SampleMail system. Using TopSpin command "ej" or "ij" won't bring the sample down to the magnet or the sample injection compartment.
    The sample NMR tube is probably stuck on top of SampleMail vertical tube. You need to get on the platform, locate where the sample is. Note that the spinner can not be taken out from top of the delivery tube. You may carefully remove the sample NMR tube from the spinner, then unlock the mechanic switch (which holds the spinner) so the spinner can drop back to the injection compartment.

  2. CP600: Power outage ( during a thunder storm) caused Cryoprobe to warm up (emergency warmup mode); helium compressor stopped working.
    Once the system started to warm up, you may press cool-down button on one side of Cryp-Platform. If you does it quick enough, you may be able to force the system to cooldown instead of warmup, and the system will be ready for use in a few minutes. If it is too late and the system indicates that it needs to warm up first, then you will have to wait the system to warm up before you can cool it down. Overall it takes about  6 hours.
    In the emergency warmup mode, sample lift will also be activated, which can not be stopped until the system is completely warm or cold. Once the probe is warm or cold, you can uncheck the " Window locked" from the cryopanel windows, then close V13c (emergency lift).
    Emergency warmup mode may also switch off the heater unit. You should check/turn on the heater from Topspin temperature display window.

  3. NMR tube spinner appears too loose for the NMR tube, and/or good shimming is very difficult to achieve.
    It is possible that the NMR tube is not suitible for the 600 MHz or 800 MHz spectrometer. Please use brand name NMR tubes, which usually have labels telling what frequency magnet it is designed for. A temperary solution is to put a thin strip of Scotch tape on the NMR tube where the spinner will hold the NMR tube, and careful push the NMR tube through the spinner.

  4. CP800: Sample resolution poor at high temperature
    One reason is that the sample may have not equilibrated well. Do topshim again and run a quick 1D experiment once in a while and compare. Another reason could be air flow flucturation. If you see unstable air flow, +/- 5 L/h to the setting air flow, please report to the facility manager.

 

 

 

 

NMR Lab Safety Policy

Safety Policy Environment and Safety in Biomolecular Nuclear Magnetic Resonance Facility, University of Maryland, College Park: Responsibilities, Risks and Liabilities:

  1. Staff members, Teaching Assistants, and Users will expose themselves to strong magnetic fields (maximum of 14 Tesla) from the NMR superconducting magnets, while they are working around the spectrometers. To the best knowledge of the NMR community, no known health hazard has been reported when one is exposed to such field, in the past twenty years. However, there is no guarantee that future findings may show otherwise.

  2. Any person with metal implants, such as heart-pacers, should not work or enter the NMR labs. The strong field and the radio frequencies of NMR spectrometers are known to interfere with heart-pacers.

  3. Keep all loose metal objects at least ten-foot from the magnets. The strong magnetic field attracts any loose paramagnetic metal objects nearby. The collision force is so strong that it can induce a structural collapse of the magnet, which in turn causes spontaneous boil-off of the super-cold liquids in the magnet. A person around the collision (magnet) can be injured.

  4. Use proper procedure and obey safety policy when dealing with liquid nitrogen and liquid helium. Then superconducting magnet contains liquid nitrogen and liquid helium. Contact with these cold liquids will cause severe burns to the body. Safety policy must be complied with during transportation and refilling of the magnet with these liquids. During the refilling of liquid helium, the operator must be very careful and always maintain proper balance when stepping up to the platform (no more than 3 feet in height). Falling from the platform could lead to body injuries.

DISCLAIMER:

The prospective users should read ‘responsibility, risks and liabilities’ and fully understand the potential danger, implication and risks described. The staff members in the NMR facilities, the Department of Chemistry and Biochemistry or the University of Maryland is not responsible or liable for any possible misshape caused by any user negligence. By reading this, the users will behave responsibly and follow all the proper procedures as described in this form and the published NMR facility policy of the department.