On‐line and At‐line Liquid Chromatography Nuclear Magnetic Resonance and Related Micro‐Nuclear Magnetic Resonance Methods in Natural Product Analysis

Abstract

Nuclear magnetic resonance spectroscopy (NMR) is essential for the de novo structure identification of natural products. This technique can be directly or indirectly hyphenated to high performance liquid chromatography (HPLC) leading to efficient LC-NMR platforms. NMR is, however, a very insensitive detection method compared to mass spectrometry and several strategies for its efficient hyphenation with HPLC have been developed. This includes on-line LC-NMR, SPE-NMR, or other at-line approaches involving microflow probes or microtubes in combination with cryoprobes. All of these various technologies and their potentials and limitations in the field of natural product research are described. Recent applications involving other complementary detection methods such as LC-MS or biological HPLC profiling illustrate its utilization

The integration of LC‐MS and NMR for the analysis of low molecular weight trace analytes in complex matrices

“…The use of long columns, however, leads to long analysis times, and requires a longer time for column equilibration between injections. For LC‐MS‐NMR, the most commonly used columns are 15‐25 cm long . The column internal diameter determines the amount (in mass) of material that can be injected on column, thus larger diameter columns allow for the injection of more material on column.…”

“…Because different analytes in a complex sample are at different concentrations, to maximize the concentration of analytes at lower levels the column should be loaded to capacity. Unfortunately, overloading of column also leads to peak broadening and in‐turn, peak‐to‐peak contamination of closely eluting analytes …”

“…Due to the differences in sample requirements between MS and NMR, a small fraction of the flow is split off for MS analysis, with the majority directed to waste or to a sample collector for further analysis. This configuration has not been used extensively because it is difficult to correlate the MS and NMR data as the analyses occur at significantly different times in each detector (and dispersion in the large NMR flowcell degrades chromatographic resolution of the LC‐MS data) …”

“…Reduced flow rates can increase the residence time for NMR analysis but may lead to broadening of the LC peaks (loss of LC resolution). Flow rates on the order of 0.1‐0.4 mL/min have been found to be a good compromise between longer residence time in the NMR probe with acceptable broadening of LC peaks …”

“…Stopping the LC pump for NMR analysis can be triggered by either UV peak detection or by the MS detector. Delays between peak UV and peak NMR intensity need to be calibrated, and prior knowledge of the elution patterns of analytes and interferences is valuable . Triggering stop‐flow from MS has an advantage over UV in detecting analytes that do not contain a chromophore or for compounds with a low UV absorbance.…”