Sensitivity in reduced diameter tubes

Sensitivity is the bane of NMR. Spectroscopists are always looking for ways of increasing it, particularly when sample is limited. One suggested method to increase sensitivity is to reduce the volume while keeping the amount of material constant, thereby increasing the concentration. This can be done by using a smaller diameter NMR tube, e.g a 3 mm or 1.7 mm tube. I have been skeptical of this method for a long time and finally decided to test how well it works.

Natural products researchers invariably have limited access to their compounds and these investigators have led the development of reduced volume NMR such as the 1.7 mm cryoprobe¹. Not everyone has access to such instrumentation, however, and cheaper more accessible alternatives have been investigated. One of the easiest ways to increase concentration is to reduce volume. This can be done with Shigemi tubes which fill the upper and lower regions of the NMR tube with glass matched to the magnetic susceptibility of the solvent, or by reducing the diameter of the NMR tube. Using a 1.7 mm tube in a 3 mm probe has been reported to increase sensitivity², as has using a 3 mm tube in a 5 mm probe³. Using a 1.7 mm tube in a 5 mm probe reportedly decreased sensitivity³.

Explanations for the origin of the increased sensitivity in these papers have not been convincing. The NMR signal is proportional to the number of atoms and if this is not changed then sensitivity should not change. In fact, since the sample is further from the detection coils when using a reduced diameter tube the signal should be reduced slightly. I suspect that in the reported cases other factors, such as the fill height, probe tuning and matching, or pulse calibration, were not kept constant.

To test if sensitivity can be increased by using reduced diameter tubes I prepared samples with the same amount of material in 5 mm and 3 mm NMR tubes. Starting with a 5 mM aqueous solution of L-tryptophan I added 40 μL to a 5 mm tube and to a 3 mm tube. A further 460 μl of D₂O was added to the 5 mm tube to bring the total volume to 500 μL. This gave a fill height of 36 mm. I then added D₂O to the 3 mm tube to get the same fill height, which required a total volume of 170 μL. Both tubes then contained 200 nmol of L-tryptophan at a concentration of 0.40 mM in the 5mm tube and 1.18 mM in the 3mm tube.

A 1D ¹H spectrum with excitation sculpting to suppress the residual water peak was recorded on both samples. The 5mm room temperature BBI probe was tuned and matched for each sample and the 90^o pulse width calibrated. Pulse widths were similar for the two samples but the tuning and matching was significantly different. The receiver gain for both samples was the same. The figure below shows an overlay of the two spectra with expansions below.

The spectra are remarkably similar, however, the spectrum recorded in the 5mm tube is slightly more sensitive. Calculating signal-to-noise using the aromatic singlet at 7.25 ppm gave 54.08 for the 5mm sample and 44.73 for the 3mm sample. The decrease for the 3 mm sample was expected, but I am a little surprised how small the difference is.

In the spectra above the solvent was H₂O/D₂O and the reduced solvent volume did not help increase the sensitivity. However, when using a solvent with a higher dielectric constant, such as a biological buffer, the reduced volume may be helpful as indicated by the following equation¹

R_s ∝ r⁴L(σ+ωε_oε'')ω²

Here R_s is the resistance of the sample, r the radius of the NMR tube, L the length of the sample or fill height, the term in brackets is the dielectric constant of the sample, and ω is the magnetic field strength. A higher dielectric constant leads to increased sample resistance (R_s), which results in increased dissipation of the radio frequency pulses and a loss in sensitivity. When working with samples in PBS the reduced volume of a 1.7 mm tube in a 1.7 mm probe should be better than a 5 mm tube, even when not sample limited.

References

1. Martin GE.
Small-Volume and High-Sensitivity NMR Probes.
Annual Reports on NMR Spectroscopy, vol. 56. Elsevier; 2005. pp. 1–96.

2. Martin GE, Hadden CE.
Comparison of 1.7 mm submicro and 3 mm micro gradient NMR probes for the acquisition of 1H-13C and 1H-15N heteronuclear shift correlation data.
Magn Reson Chem 1999;37:721–9

3. Krunić A, Orjala J.
Application of high‐field NMR spectroscopy for characterization and quantitation of submilligram quantities of isolated natural products.
Magn Reson Chem 2015;53:1043–50

Acknowledgements

Thanks to Julius Bogomolovas for providing the L-tryptophan solution.