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:

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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.

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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.

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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.

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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)

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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, if the P1 value was determined to be 8.60 µs, you can use the following command (assuming that its corresponding power level is 12.614W): 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) are indicative of a diverse chemical environment around these hydrogen atoms, which typically correlates with the presence of structural elements.

Data acquisition

Once everything is locked, tuned, matched, shimmed and calibrated for optimal signal intensity and quality, it is time to load a new experiment. In the case of a 1D proton experiment, the default acquisition parameters for a protein sample are saved as PROT_1DESNIG. To load them in a new experiment, first create a new experiment using the new command. In the dialog box, select the acquisition parameters PROT_1DESNIG s

Processing

References

Benjamin Görling (Bruker) - 1D Acquisition