NMR Quantitation of Natural Products at the Nanomole Scale

Abstract

We describe a simple and accurate method for quantitation by solvent 13 C-satellites (QSCS), of very small amounts of natural products using microprobe NMR spectroscopy. The method takes advantage of integration of 13 C satellite peaks of deuterated solvents, in particular CDCl 3 , that have favorable intensities for measurements of samples in NMR microcoils and microprobe tubes in the 1-200 nanomole range.Recent developments in instrumentation have greatly increased the sensitivity of NMR measurements and have facilitated structural elucidation of natural products at the submilligram level. Microcoil probes 1 and narrow diameter tube microprobes 2 offer significant advantages for measurements of small samples of natural products. 3 Reduction of the sample volume by a factor n at a constant sample mass will increase sensitivity of NMR signals in proportion to n, at the same absolute concentration-sensitivity or signal-to-noise (S/N). Microcoils typically have fill volumes of 2.5-5 µL while 1 mm and 1.7 mm tube probes offer fill volumes of about 7 and 30 µL respectively. In practice, microcoil and capillary probe designs optimized for small fill volumes sacrifice some S/N, yet the gain in mass-sensitivity more than compensates for this loss. For example, a recently developed commercial 1.7 mm 1 H-13 C-15 N CPTCI cryoprobe operating at 600 MHz achieves a nominal S/N of 1000:1 for 1 H (ASTM standard) when compared to 9000:1 for a 5 mm TXI probe at the same field strength with sample fill volumes of 30 µL and 750 µL, respectively. Thus, the increase in mass-sensitivity of a 1.7 mm cryoprobe compared to the 5 mm cryoprobe can be estimated as the product 1/9 × 750/30, or a factor of 2.7. Compared to a conventional "room temperature" 5 mm probe at the same field strength, this represents >10-fold improvement in masssensitivity. Higher gains in S/N have been realized with recent innovations in probe design; for example the 600 MHz 1 mm high-temperature superconducting (HTS) probe. 4 Natural products structure elucidation at the sub-milligram level is most suited to microcoil and microtube NMR, however independent estimation of the amount of natural product undergoing measurement is not trivial. For example, 1 nanomole (nmol) of a compound of molecular mass 1000 is equivalent to 1 µg. Gravimetric assay of this amount on a standard analytical balance of nanomoles of material is impossible as the precision of the instrument is typically limited to 0.1-0.01 µg. Accuracy suffers when the net must be calculated by subtraction of two large numbers: the gross mass and the tare mass of the sample container (often 1500-13000 mg). Submilligram samples may be weighed on a microbalance down to *To whom correspondence should be addressed: Tel: +1 (858) Results and DiscussionThe advent of micrcoil-and microtube probes has allowed NMR measurements of extremely small amounts of compounds including natural products. During the course of investigations of sub-micromole samples of new natural products from Phorbas sp., we required...

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