The NMR Spectrometer
I will have to be a little vague at times in this section, as specifics will depend on your spectrometer vendor and age.
Very good temperature regulation of the sample in the probe is required for the best quality spectra. You should also allow ca. 5 minutes for your sample to temperature equilibrate in the probe before starting shimming and signal acquisition.
You will typically use the simplest pulse sequence â€“ repetitive pulse and acquire :
Relaxation delayÂ â€” PulseÂ â€”Â Acquire
The consideration is that all spins should be at their equilibrium position (fully relaxed) before the next pulse is applied. The sum of the acquisition time and relaxation delay is sometimes called the pulse repetition time, and this number needs to be carefully set up.
The consideration is that all nuclei have fully returned to equilibrium between pulses. If you are using 90Â° pulses, then this should be at least 5X the proton having the longest T1. If you are measuring the same material every time then it would be nice to determine this. It would be worthwhile determining the T1 of your reference material/solvent for your experimental conditions of temperature, etc. at least once so that you know this minimum pulse repetition time. You may be surprised with this figure â€“ it is often several 10s of seconds!
The issue of pulse repetition rate is often overlooked, and yet it is absolutely fundamental to a good experiment.
As mentioned, the interpulse delay is the sum of the acquisition time and relaxation time. But you should consider how you divide this time: a longer acquisition time than you normally use for routine 1H spectra will almost certainly be used.
You want to aim for an acquisition time that is long enough to collect the time-domain FID signal until it has decayed to the point where only noise is being digitised .Â If you use a shorter acquisition time then the signals will be truncated, and the effect in the time-domain spectrum will be for each peak to show â€œwigglesâ€ (truncation artifacts) at the base, and baseline artifacts. This will clearly affect the integration of each signal.
Spectrum digitisation and Peak Digitisation
The acquisition time and sweep width ultimately determine the number of Hz/pt in the final spectrum. One level of zero filling can be performed. But the digitisation in the spectrum is a very important consideration as it strongly influences the fidelity of the peak integration.
As a rule we suggest you aim to achieve >5 data points per peak if (a) GSD is to be successful, and (b) conventional integration is to be accurate. Ignore this rule at your peril â€“ it can really affect your reported integrals! The number of points you acquire will depend on the sweep-width, of course, but go for a much longer acquisition time than you usually use: perhaps 10s or longer. You will not be adding to the experiment time, and we all have big discs these days to store data!
Number of scans.
Time-averaging is often needed to build an adequate signal-to-noise ratio (SNR). So we need to ensure that the SNR is adequate to support good integration. You should target a SNR of 250:1 or better for any peak that will be integrated for qNMR purposes.
As with any acquisition, the receiver gain should be set so the NMR signal fills the digitiser (ADC) as best as is possible whilst avoiding early time points being â€œclippedâ€. Too small a receiver gain is undesirable, too. Note that automatic setting of the receiver gain must be done using experimental conditions, and may take some time if the interpulse delay is required to be long!
Choose a sensible sweep width: this would be ca. 20% larger than the separation between the signals at the extremes of the spectrum.
Number of data points.
This will be dictated by the required sweep-width and acquisition time. However, we need to consider the final digitisation of each signal in the spectrum: getting this right is crucial to accurate integration.Â I will come back to this topic in the processing section, but in extreme cases it may be necessary to collect the spectrum in parts. For typical 1H NMR spectra you should collect something like 64K data points.
I have saved the best for last! For good quantitation we need good lineshape, and shimming will play a critical role in this .Â Itâ€™s easy to get used to gradient shimming to do the work for us these days, and it works exceptionally well in the majority of cases. But if you only do â€œon axisâ€ gradient shimming, then remember to check and optimise â€œoff axisâ€ shim gradients on a regular basis.
A poorly shimmed sample can often result in an unsymmetrical lineshape, and that will cause significant errors for all line fitting methods, including GSD.
Broadband 13C decoupling.
With qNMR you should in principle also integrate the part of the 1H signal away from the central lines. These arise from the 1-bond coupling between the proton and attached 13C. This accounts for 1.1% of the 1H signal area, and the coupling is 135-200 Hz. Collapsing 13C satellites into the central peak has a number of additional benefits because it simplifies the spectrum. Some spectrometers will make this experiment quite easy to perform. But you must be aware that broadband 13C decoupling itself may raise the temperature in the probe quite significantly, and that would affect the lineshape and therefore be undesirable. You can decrease the acquisition time to minimise this effect. Probe and sample heating is often evident if you look at the lock level and see that it decreases during the acquisition time.