We have recurring interest from industrial customers (e.g., pharma, flavour/fragrances) to acquire spectra using only small amounts of analyte in our benchtop NMR instruments – considerably less than our recommended 0.1 M concentration . Why is our recommended concentration so high? Well, it’s reflective of the consequences of a low-field instrument. That is, lower-field results in inherently lower sensitivity and accordingly a decrease in observed signal to noise ratio (S/N).
n – # nuclei in resonance
γe – gyromagnetic ratio of excited nuclei
γd – gyromagnetic ratio of detected nuclei
B0 – applied field strength (T)
t – experiment run time
To circumvent the lower SNR on benchtop we decided to investigate the possibility of using low volume NMR tubes. In this blog entry, we are going to present our findings using 5mm, 3mm and a capillary tube (1.67 mm). We used a solution of ethylbenzene to measure the SNR and in order to keep things consistent we used the same sample concentration and NMR parameters (# scans, delay, spectral window, etc.) for all the measurements. We also ‘normalized’ the peak intensities to make the analysis simpler.
Table 1.0 summarizes the results and the sample volume used in each tube. In each case the volume used was meant to ensure the liquid level reached the top of the coil, so smaller volumes can be used in order to increase the concentration even more!
Table 1.0 Results obtained using NMR tubes of different sizes
|Tube (mm)||Volume (mL)||Normalized S/N|
5mm and 3mm NMR tubes are very common and commercially available from different sources. For the capillary tubes we bought the capillary kit available from New Era Glassware (figure 1.0). High quality tubes are not required in low field, so we used regular melting point capillary tubes (very inexpensive by the way!).
In table 1.0, we observe the predictable decrease in S/N with decreasing size of tube – and therefore volume. This is consistent with a decrease in the ‘number molecules’ being analyzed, as well as with the decreasing filling factor, but we can discuss that more in another blog.
Keep in mind that these samples have the same concentration; however, the small volume offers us the unique opportunity to use the same amount of analyte but, owing to the lower sample volume, significantly concentrate the sample. Let’s do a little bit of math and see how much we can actually improve our S/N by running more concentrated samples in smaller tubes. If we assume that the average molecular weight of an organic molecule is 300 g mol-1 and that we only have 0.5 mg of analyte, we can make the following solutions: 2.78 mM, 10.4 mM and 33.3 mM for the 5mm, 3mm and 1.67mm tubes, respectively (table 2.0).
From table 1.0 we see that the S/N decreases to half if we move from the 5mm to the 3mm NMR tube. Therefore, we would need to double the concentration in the 3mm NMR tube in order to get the same S/N as in the 5mm NMR tube. The decrease in volume allows us to prepare a solution that is almost 3.7 times as concentrated, which means that at the end we would have a S/N close to 1.85 times higher than the same analyte in a 5mm NMR tube.
Likewise, if the same analyte were used to prepare a solution for the capillary tube (0.05 mL) the concentration would be close to 12 times higher. This means that the improvement in S/N would be close to 3 times higher compared to the same analyte in the 5 mm NMR tube.
Table 2.0 Concentration of a hypothetical sample (0.5 mg, 300 g mol-1)
|Tube (mm)||Volume (mL)||Concentration (mmol)||Normalized S/N|
In conclusion, despite the decrease in S/N by using small NMR tubes, there is an overall increase in S/N if a smaller volume is used to make more concentrated the solutions.
Contact us about NMR tube compatibility, sensitivity and any other benchtop NMR inquiries you may have!