Small-Volume and High-Sensitivity NMR Probes

Gary E. Martin    Pfizer Global Research and Development, Michigan Structure Elucidation Group, Kalamazoo, MI 49001-0199, USA

1 INTRODUCTION

Nuclear magnetic resonance (NMR) methods are capable of providing a wealth of atom-to-atom connectivity information that has served to make NMR the cornerstone of modern structure elucidation. Unfortunately, NMR is also an inherently insensitive technique. Considerable effort has been expended on the part of thousands of scientists to enhance NMR sensitivity. Efforts have been focused in three primary directions. Magnet technology has been one very major focus. Magnets have progressed from predominantly 60 MHz permanent magnets in use when this author's career in NMR began to the first superconducting NMR magnets at 220 and 300 MHz with voracious appetites for liquid helium, and now to huge 900 MHz magnets that are intended for biomolecular NMR studies and essentially require a gymnasium-sized room to house them. At the same time, the field strength of magnets on instruments for more routine applications have also steadily climbed to the point that most instruments intended for walk-up usage by chemists in the pharmaceutical industry are now 400 or 500 MHz instruments. A second primary area of focus has been in the development of more sophisticated NMR pulse sequences to achieve higher sensitivity. Heteronuclear shift correlation experiments progressed from the fledgling heteronucleus-detected experiments in the early 1980s to the proton- or inverse-detected heteronuclear shift correlation experiments now in widespread usage for chemical structure characterization. The third major area of focus has been on the development of higher-sensitivity NMR probes. Historically, NMR probes have typically been based around an rf coil designed to accommodate a 5 mm NMR tube. With the advent of commercial Fourier transform NMR instruments in the late 1970s, probes evolved from simple 5 mm proton observe capabilities to heteronuclear probes with the heteronuclear or X-coil inboard surrounded by a larger proton-decoupling coil. These designs were quite serviceable through the late 1970s and early 1980s, but with the development of the first proton-detected heteronuclear shift correlation experiments by Müller,1 Bodenhausen and Ruben,2 and Bax et al.3 it became obvious that conventional heteronuclear NMR probe designs would be supplanted by what were originally termed “reverse-geometry” NMR probe designs. These are probes with the proton coil closest to the sample for improved proton sensitivity since 1H is the detected nuclide. More recently, “inverse-detection” terminology has come into common usage, the terminology again based on the exchange of the rf coils in the probe design. Modification of NMR probe coil geometry afforded better sensitivity than conventional designs, but sample requirements were still relatively high for investigators working in severely sample constrained areas of investigation such as the characterization of impurities and degradants of pharmaceuticals, metabolites, etc. This brief history sets the stage for the development of small-volume high-sensitivity conventional NMR probes that occurred through much of the 1990s, which has been followed more recently by the development and commercial availability of cryogenically cooled NMR probes with still higher sensitivity.

2 SMALL-VOLUME CONVENTIONAL NMR PROBES

Small-volume conventional NMR probes encompass the development of 3 mm micro-probes, followed very closely by a 2.5 mm variant. Nearly 6 years after the introduction of 3 mm probes, a 1.7 mm submicro- or SMIDG™ probe was introduced followed, most recently, by the development of a 1.0 mm probe. Given sample handling considerations, it is unlikely that smaller conventional (those using tubes) NMR probes will be developed. This section does not include the development of either magic angle sideways spinning liquids (Nano-probes™) or what are now generally referred to as μCoil NMR probes. These types of high-sensitivity probe technology are discussed in later sections of this contribution.

2.1 The 3 mm micro-NMR probes

Historically, small-volume NMR probes date back to the 1966 Report of Odelblad, which employed a solenoidal microcoil to study human cervical mucosal secretions.4 By the early 1970s, Shoolery had begun exploring the use of 1.7 mm capillary NMR probes for the acquisition of 13C data.5 These early reports essentially set the stage for the start of the development of 3 mm or micro-NMR probes that began in 1991 through a collaboration by the author and his colleagues, then at Burroughs Wellcome, Co., in North Carolina with staff at the Nalorac Corporation. The first experimental results obtained with a 3 mm inverse-detection micro-NMR probe were reported in 1992. Using the simple indoloquinoline alkaloid cryptolepine (1) as a model compound for the study, Crouch and Martin6 were able to demonstrate the acquisition of a heteronuclear multiple-quantum correlation (HMQC) spectrum recorded on 12 μg (0.05 μmol) of the alkaloid dissolved in 145 μL of d6-dimethyl sulfoxide (d6-DMSO) in 16 h (Fig. 1). Long-range heteronuclear shift correlation data in the form of a heteronuclear multiple-bond correlation (HMBC) experiment, which is substantially less sensitive than the HMQC experiment, were recorded on a 35 μg (0.15 μmol) sample of the alkaloid in the same volume of deuterated solvent in 21 h. These preliminary results heralded a significant advance in the characterization of small samples by heteronuclear two-dimensional (2D) NMR shift correlation methods. Later in 1992, Crouch and Martin7 reported a direct comparison of 3 and 5 mm NMR probe performance at 500 MHz. Using a sample of 167 μg (1 μmol; samples were prepared by serial dilution) of the simple alkaloid harmane (2) dissolved in either 160 or 600 μL of d6-DMSO for 3 or 5 mm NMR sample tubes, respectively, the authors found that it took four times as long to achieve the same signal-to-noise ratio (s/n ratio) in the 5 mm probe as for the 3 mm probe, based on the s/n ratio of a projection through the F2 frequency domain when identically parameterized HMQC spectra were run. Unfortunately, data were not recorded for the 3 mm sample run coaxially in the 5 mm probe.

Following the initial 1992 reports describing the capabilities of 3 mm micro-NMR probe technology, a 3 mm dual probe was developed with the X-coil inboard for the acquisition of 13C spectra. This 3 mm probe variant made it possible to acquire a 13C-NMR spectrum for submilligram samples overnight at a 13C observation frequency of 100 or 125 MHz. As NMR console designs continued to evolve incorporating pulsed field gradient capabilities, 3 mm micro-probes with gradient capabilities were also developed. Interestingly, based on work done in the laboratories of the...