Hyphenated NMR Methods in Natural Products Research, Part 2: HPLC-SPE-NMR and Other New Trends in NMR Hyphenation

Abstract

IntroductionIn the first part of this review [1], the use of direct NMR hyphenation in natural products research has been discussed. The direct HPLC-NMR methods were defined as methods in which the NMR data acquisition is performed on the HPLC column eluate [1]. This implies either a direct connection between the NMR spectrometer and the HPLC apparatus, or use of storage capillaries in which the HPLC eluate is temporarily stored prior to acquisition of NMR data.Many useful results related to natural products have been obtained using direct HPLC-NMR methods [1] [30]. However, the direct combination of state-of-the-art separation technology with the most powerful and versatile tool for structure elucidation of organic molecules in solution suffers from various practical problems. In short, these practical limitations are due to somewhat contradictory requirements of HPLC and NMR with respect to solvents and time-scale of the experiment, and thus compromises are necessary in order to hyphenate these two techniques. For example, 1 H-NMR spectra are preferably acquired in deuterated solvents, but use of deuterated solvents for HPLC is expensive and the selection is limited. Some useful HPLC eluents, such as buffers with high ionic strengths, are directly undesirable in direct HPLC-NMR hyphenation. Gradient elution of HPLC columns, required in most separations, implies constantly changing magnetic susceptibility and field homogeneity in the NMR flowprobe, not to mention variations of analyte chemical shifts with solvent composition. Hyphenated NMR Methods in Natural Products AbstractThis review describes the principles and performance of a novel and highly promising hyphenated technique, HPLC-SPE-NMR, which is based on post-column analyte trapping by solid-phase extraction. The analytes are subsequently eluted from the SPE cartridges using deuterated solvents. This indirect HPLC-NMR hyphenation offers numerous advantages compared to direct HPLC-NMR methods. Multiple trapping leads to a dramatic increase of analyte amounts available for NMR, enabling acquisition of high-quality 2D NMR data within a short time. Other new developments, including combination of solenoidal coil capillary flow-probes with microflow HPLC, are also discussed. Fast extract dereplication using these techniques enables focusing of isolation efforts on truly novel and promising natural products, based on precise structural data obtained with crude extracts or fractions.

Online coupling of enantioselective capillary gas chromatography with proton nuclear magnetic resonance spectroscopy

“…18,19 In a recent work, the successful capillary gas chromatographic separation and online identification of the stereoisomers of E,Z-2-pentene and of E,Z-2-hexene using a Chirasil-b-Dex stationary phase and a 400 MHz NMR spectrometer equipped with a custom-built, double resonant solenoidal microprobe was described. 17 Both methods benefit from newer developments in the field of NMR such as microprobes 16,17,20,21 or cryoprobes [22][23][24][25] and stronger magnets, which dramatically improved the sensitivity of the relatively insensitive spectroscopic method so that the largest sample amounts, which can be separated by capillary GC are getting closer to the detection limit of NMR spectroscopy. Thus it is to be expected that many applications using a combined method of GC and NMR (offline as well as online) will emerge in the future especially if a high purity isolation is required for an unequivocal identification.…”

Design and construction of a microcoil NMR probe for the routine analysis of 20‐μL samples

“…Hyphenated microcoil NMR techniques include popular separation methods such as HPLC (15)(16)(17)(18)(19)(20)(21)(22), MS (23), SPE (16,24,25), CE (26,27), cITP (28)(29)(30)(31)(32)(33)(34), and most recently, GC (35). Although the combination of these techniques has been straightforward, there is often a potential loss of SNR and thus information, as only a small percentage of the available sample is located and detected within the probe coil (7).…”

Nuclear Magnetic Resonance Spectroscopy for Structural Characterization of Bioactive Compounds

“…A final advantage of this LC-SPE-NMR technique is the use of lowvolume probes to maximize sample concentration and NMR data quality [67]. In this field, several reports have appeared, providing comprehensive information about novel products obtained directly from plant crude or prepurified extracts [67,74]. However, the application of this methodology to marine natural products is quite scarce.…”