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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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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
o1calibpulsecal- 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
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| Do not forget to specify the units for the power level (W for Watts)! |
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