Setup

Now that you have your sample ready, that meets the sample requirements and is in an NMR tube, you can insert it in the Bruker spinner and adjust the depth using the sample depth gauge as depicted below:

Source: Bruker - BSMS manual

In the present case, d should be at least 15 mm, ideally 20 mm. This corresponds to about 550 µl in a 5 mm tube. Do not hesitate to confirm with the NMR manager that your tube is properly positioned.

Never insert a sample tube without a spinner in the NMR magnet! The tube will break and cause expensive magnet bore and probe clean up!

Sample insertion

There is usually a dust cap on top of the sample bore of the magnet in order to prevent dust and debris from entering the bore. In order to insert your sample along with its spinner, you first need to remove it and put it aside, on the top of the magnet.

Then, in TopSpin, on the control computer, enter the command ej to activate the air lift and if it is the case, eject the sample already present in the magnet. Climb up the stairs and replace the previous sample in the bore (red turret) with yours. Make sure the air lift is activated so that your sample does not fall in the bore. The pneumatic system should be powerful enough to maintain your sample on top.

Come back to the control computer and type ij in TopSpin to inject your sample in the bore. Your sample will then slowly lower down into the core of the magnet and in the NMR probe.

Setting the temperature

The first thing to do following the insertion of your sample in the magnet, is to set the temperature, as most chemical shifts are temperature dependent, including the lock signal. Use the command edte to open the temperature window in TopSpin. The current temperature is written in the center of the window (25.0 °C), and is labeled as corrected (Corr.) as it was previously calibrated using 99.8% deuterated methanol (CD₃OD).

The temperature set on the computer is slightly different from the actual temperature in the magnet.

Lock

The lock is a separate NMR spectrometer within the spectrometer, which is specific to deuterium. In order to produce a high resolution NMR spectrum of a sample, it is key to keep a constant magnetic field strength over the duration of the experiment. This is achieved by "locking" the spectrometer to the frequency of deuterium, ie by centering the deuterium signal to a predefined frequency. This signal is constantly monitored and compensations for the external magnetic field (B₀) drift are made in order to keep the resonance frequency constant.

Modern NMR magnets do not drift much beyond 4 to 5 Hz per hour. Yet, this would be sufficient to lose spectral resolution (eg J-coupling) over a 1-hour experiment. As a reference, below are the current (as of February 2020) magnet drifts for our NMR spectrometers.

In order to lock onto a certain solvent, enter the command lock and choose the appropriate solvent. You could also include the solvent name in the command (eg lock h2o+d2o). Look for a change in the lock signal. Once the procedure is done, you should see lockn: done. Then adjust the power and gain in the BSMS panel (if it is not showing, you can open it with the bsmsdisp command) by clicking on the corresponding button in the AUTO section (Lock/Level tab). You may also want to make sure that the phase is optimized.

Alternatively, the AU program loopadj can be used to optimize the lock feedback loop. From the description of the lock process:

The AU program loopadj, automatically optimizes the lock phase, lock gain, loop gain, loop filter and loop time. Note that loopadj optimizes these parameters for best long-term stability, but not for best lineshape, resolution or homogeneity (for more information type edau loopadj and look at the header of the AU program).

Tune and match

Tuning of the probe to the observed nucleus frequency is done to adjust the resonance circuit of the probehead so that it is the same as the frequency of the transmitted pulse, whereas matching is a process to adjust the “efficiency” (impedance) of the resonance circuit. This is very important to do and will be affected by the composition of the buffer in which your sample is in.

First, make sure that the "virtual" routing is set properly by looking at the connections via the edasp command. If you will be running carbon and nitrogen experiments, you'll have to make sure they are enabled. This is how the edasp window looks like on our 700 MHz system (connections corresponding to the routing for our TXI probe).

Click on Save and Close when you're satisfied with the channel routing for your experiments. Then, to initiate the automatic tuning and matching procedure, use the command atma. This window will open:

You want to make sure that all the wobble curves peak at the desired frequency. Later, manual adjustments can be made using the manual tuning and matching procedure via atmm.

Source: ??

Shim

Shimming is an important process where adjustments are made in order to obtain a homogeneous magnetic field around the sample in the probe. This will result in better spectral resolution. It is necessary to shim each time a sample is inserted in the magnet. If not optimized, nuclei in one part of the tube will experience a different field than nuclei in another part of the sample tube.

Enter the command topshim gui to open the GUI.

Once the shimming procedure is done, the shim adjustments will be listed in the Report tab.

Expect a duration of about 30 seconds for 1D shimming, and 5-6 minutes for 3D shimming.

If you are using a Shigemi tube, make sure you include the shigemi mention in the parameters section.

Transmitter offset determination (O1)

Before recording a NMR spectrum, we need to set the frequency of the transmitted pulse at the centre of the desired observed resonances.

For biomolecules in aqueous solutions, this frequency is typically the frequency of the water proton, at around 4.7 ppm. Considering that our sample is typically in the micromolar (10-6) concentration range, the intensity of the signal coming from the water molecules will be about 5 orders of magnitude greater. We therefore need to use NMR "tricks" to suppress this particular water signal. This is done by setting the transmitter frequency on resonance with the water protons (offset value in Hz from the base frequency corresponding to the parameter O1), and can be determined experimentally using the AU program o1calib. Alternatively, one could use the popt program, array O1 around the estimated value and determine the frequency offset at which the water proton signal is at its minimum. More information on popt here.

90° pulse calibration (P1)

Another key parameter to optimize prior to recording your first NMR experiment is the duration of the 90° pulse for maximum signal strength and making sure the bulk magnetization is brought along the x-y plane. In TopSpin, this parameter is called P1 and can be optimized automatically (using a stroboscopic nutation method) using the AU program pulsecal. The optimal pulse length will be determined at the set power (PLW1) and getprosol will be executed at the end of the AU program in order for the other pulses to be calculated.

Alternatively, you can determine the optimal P1 manually by testing an array of values corresponding to 360° pulses (4x the duration of the 90° pulse), via the popt program and optimizing for the null (zero intensity). Then the value is divided by 4 to obtain the 90° value.

This value of P1 will be sample-dependent.

Automated O1 and P1 determination

Bruker has put together a calibration routine which (1) automates the determination of the optimal O1 and P1, and (2) records a 1D proton spectrum of the sample (using the zgesgp pulse program). The AU program to launch is called calibo1p1. The program will run

• o1calib
• pulsecal
• run a 1-minute excitation sculpting 1D proton experiment (zgesgp)

Below is a typical 1D proton spectrum of a small protein using excitation sculpting as water suppression technique.

Then the values of O1 and P1 can be retrieved by looking at the recording parameters in the resulting experiment (eda). Write them down and transfer them to the following experiments. You need to make sure you execute getprosol in the other experiments to calculate all the pulses based on P1.

getprosol 1H [P1 value in µs] [PLW1]W

For example: getprosol 1H 8.60 12.614W

Another advantage of the calibo1p1 program is that you can assess the quality of your sample by looking at the 1D proton spectrum it produces (see above). Well dispersed peaks, particularly in the amide protons region (6-10 ppm) is indicative of a diverse chemical environment around these hydrogen atoms, typically correlated with structural elements.

Data acquisition

Processing

References