Automated para-Hydrogen Hyperpolarization for Mixture Analysis Using ¹H and ¹³C Benchtop NMR Detection

Benchtop NMR spectroscopy is an affordable and accessible technique for mixture analysis but suffers from lower sensitivity and increased signal overlap compared to standard high-field NMR. Signal amplification by reversible exchange (SABRE) hyperpolarization can effectively address the sensitivity limitation, with automation of the hyperpolarization step enabling the multistep NMR experiments required to resolve signal overlap for complex mixture analysis. The automated workflow presented herein delivers highly repeatable hyperpolarization (with a 2.5% relative standard deviation in signal enhancement across 100 experiments) and can be integrated into any NMR pulse sequence with typical repolarization times of ∼13 s. Using this approach, a fully resolved ¹³C{¹H} benchtop NMR spectrum with signal-to-noise ratios of up to 40 for a mixture of pyridine, 4-methylpyridine, and 3,5-dimethylpyridine (3 mM each) at natural isotopic abundance is obtained with only 18 min of signal averaging. Overlapped peaks in the ¹H NMR spectrum of this mixture are resolved using a SABRE-enhanced 2D ¹³C-¹H HETCOR experiment (8 scans, 2.4 h). Analysis of a lower concentration mixture (750 μM per analyte) is exemplified with a SABRE-enhanced 2D ¹H-¹H COSY spectrum acquired in a single scan in 37 min.