5
Typical parameters Pulse repetition time Pulse fl ip angle Scans
Digital resolution Sample spinning 13C decoupling
Routine ~7 sec 45° 8
0.5 Hz On Off
Quantitative >T1 x 7 90°
S/N>100 <0.25 Hz Off On
We can see the point at which the signal intensity turns to 100% expressed as a multiple of T1. According to this, setting the pulse repetition time to 7 x T1 or longer will mean the signal is nearly completely returned to the original state. This is why this is defi ned as the condition for quantifi cation. In ordinary conditions, the main focus is on the signal to noise ratio in order to confi rm the signal as fully as possible, meaning that the acquisition conditions emphasise the integration effi ciency, so the goal is different.
The pulse repetition time is the most important parameter because it is the setting that theoretically improves the quantitativeness. The other parameters are mainly for minimising the integration error as we obtain the peak areas. There are no mandatory settings for the other parameters and there should be no problems if these are varied according to the situation.
Number of scans
For quantitative conditions, the signal to noise ratio should be 100 or more. Figure 6 shows the theoretical relationship between S/N ratio and the accuracy. According to this, if the S/N is 100 or more, the integration error can be kept to an accuracy within 1%.
In other words, to obtain an integration with a slightly better accuracy, the S/N ratio must be higher. If this cannot be obtained because of the sample amount, it is important to understand that this will be a factor contributing to the quantification error. Thus, the setting for the number of scans is not a specific number. It is a setting to obtain a target accuracy for the available S/N ratio.
Figure 3: Typical parameters for routine and quantitative measurement conditions
We will look in more detail now at the pulse repetition time and the number of scans which are two of the most important parameters.
Pulse repetition time
As shown by Figure 4, the pulse repetition time is the length of time from the irradiation of one pulse until the irradiation of the next pulse. For quantitative conditions, this should be at least 7 times longer than T1 (longitudinal relaxation time).
Figure 6: Infl uence of signal intensity (S/N) to repetitive accuracy (SD) of integration Figure 4: Pulse repetition time overview
The magnetisation behaviour on the sample side corresponds to the pulse sequence of the instrument when the magnetisation is perturbed by the application of a pulse. If we wait long enough to allow the magnetisation to recover completely before applying the next pulse, the quantitativeness of the signals can be ensured. Therefore the parameter settings must ensure suffi cient delay between pulses to ensure quantitativeness while making the measurements.
The index for setting this time is T1 – time constant that is a characteristic of the signal. The relaxation time can be determined by making an inversion recovery measurement. In order to determine how much time is required for the pulse repetition time, Figure 5 shows the theoretical relationship between the signal strength and the ratio between the repetition time, and the relaxation time. The vertical axis indicates normalised signal strength, and the horizontal axis is the ratio of the repetition time to the longitudinal relaxation time.
Analysis The analysis stage includes: data processing, calculation of purity and evaluation.
Appropriate data processing and signal selection, unlike measurement conditions settings, don’t provide a theoretical improvement. In principle, the integration range must be set to between 64 and 128 times the full width at half maximum of the signal in order to obtain the true integrated value of the signal, but this means setting an extremely wide range so this is unrealistic in most cases.
For signal selection, as NMR is not a separation analysis, it is necessary to consider whether there is any overlap of signals such as from impurities. There are various approaches for checking this. First, the integrals of the signals for all analytes must be obtained, each signal checked, and a fi nal judgment made by looking at the variance between signals.
It is recommended to establish rules and to clarify the processing and signal selection used to obtain the numerical values. If the original spectrum is good, the errors in processing can be minimised. It is not a procedural point for processing, but making efforts to obtain good spectra during the measurement is important for successful qNMR.
Summary Figure 5: Pulse repetition time analysis
qNMR analysis is becoming more and more attractive in a range of sectors thanks to its versatility. To optimise the measurement conditions, there are parameters that are universal for any case, but there are also parameters that must be considered for each sample. If challenges occur during analysis, it may be required to go back to the basics, so it is a good idea to establish a simple protocol before embarking on routine qNMR analysis.
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