Fill volume for 5 and 3 mm NMR tubes

While 5mm NMR tubes are the default size for solution NMR, several alternatives are available. For those with less sample 3mm, 1.7mm, and 1mm NMR tubes are available. The 1.7mm and 1mm NMR tubes really require a microprobe with reduced diameter coils, but 3mm tubes can be used in a 5mm probe. With our 1.7mm probe out of action I purchased a 3mm spinner and ran some spectra to compare the fill volumes of 5mm and 3mm tubes in our 5mm BBI probe.

To compare filling volumes of 5mm and 3mm NMR tubes I used the procedure I had used previously for 1.7mm NMR tubes; aliquots of methanol-d₄ were added to the NMR tube and a 1D ¹H spectrum was recorded after each addition. The position of the tube in the spinner was adjusted after each addition until the tube could not be lowered any further. The sample was locked and shimmed automatically. A single 90^o pulse was used with a 2s relaxation delay and a 2.28s acquisition time. A total of 16 scans were recorded with 4 dummy scans. Aliquots were added until there was no further change in the spectra.

The figure below shows stack plots of the methanol methyl resonance. The first spectrum in the 5mm tube was recorded with 200μL of methanol and the final spectrum had 680μL. Aliquots were 20μL. In the 3mm tube the first spectrum was recorded with 50μL and the last with 240μL, with 10μL aliquots. In both tubes the initial spectra are broad and poorly shimmed, but within a few additions the linewidths reduce and the couplings start to resolve.

The linewidths from both sets of spectra are plotted against volume in the figure below. In the 5mm tube, only the first spectrum had really bad linewidths though the linewidth did improve slightly as the volume increased. In the 3mm tube the linewidths showed more dependence on the fill volume, and never became as small as the linewidths in the 5mm tube. Using a 3mm tube in a 5mm probe leaves extra space between the sample and the coils and likely prevents the shimming from being optimal.

In the 1.7mm tube there was a distinct volume below which the linewidths were large and markedly dependent on the volume, and above which the linewidth did not change. I was expecting the same behaviour for the 5mm and 3mm tubes but did not observe it. Perhaps the ability to center the solution in the coils for the 5 and 3mm tubes, but not the 1.7mm, allows better shimming for the larger tubes and a less marked dependence on the the fill volume.

To get a measure for the required volume I measured integrals, as I had previously for the 1.7mm tube, but this showed similar behaviour to the linewidths and didn't give a definitive answer. Finally, I plotted the signal-to-noise for the methyl resonance against volume and obtained the graphs below. Both the 5mm and 3mm tubes showed a consistent increase up until a particular volume after which there is little further increase. In the 5mm NMR tube the volume at which the S/N stops increasing is 500μL, and in the 3mm tube its around 200μL. Based on the difference in diameters one would expect the 3mm tube to need 3²/5² of the volume, i.e. 180μL.

For our Bruker 5mm BBI probe the recommended fill volume for 5mm NMR tubes is 500μL, while for 3mm tubes in the 5mm probe I recommend 200μL. Note that these recommendations are for one particular probe. Other probes may require different volumes. Varian probes are built with longer coils to give increased signal, but require larger sample volumes.