High-Resolution Microcoil NMR for Analysis of Mass-Limited, Nanoliter Samples

An improved nanoliter-volume NMR probe design places the microcoil and capillary at the magic angle (54.7°) with respect to the external magnetic field. Using an NMR probe which requires a total sample volume of just 200 nL, high-resolution 300-MHz ¹H-NMR spectra (line width, 0.6 Hz) are presented of 10 mM α-bag cell peptide for an observe quantity of 45 ng (50 pmol in 5 nL). For the volume of sample inside the microcoil (the observe volume, V_obs), the 3σ limit of detection (LOD) is 9 ng (10 pmol, 2 mM) for data obtained in 15 h. To reduce the data acquisition time, a probe with a greater V_obs is developed. As an example of a rapid, mass-limited analysis, a concentration corresponding to 400 ng of menthol dissolved in V_obs = 31 nL (82.6 mM) yields a spectrum in 9 min (LOD = 6.9 ng, 44 pmol, 1.4 mM). To illustrate improvements in concentration sensitivity, a spectrum is acquired in 45 min for 400 ng of menthol dissolved in a total sample volume of 200 nL (12.8 mM). Compared to a commercial nanoprobe for the same mass of menthol, these two examples reduce data acquisition time by at least 95%. Both model compounds demonstrate substantially improved concentration LODs compared to those obtained hi previous high-resolution, microcoil NMR work. These advances illustrate the utility of enhanced sensitivity provided by NMR microcoils applied to nanoliter volumes of mass-limited samples.