A microcoil NMR probe for coupling microscale HPLC with on-line NMR spectroscopy

An HPLC NMR system is presented that integrates a commercial microbore HPLC system using a 0.5-mm column with a 500-MHz proton NMR spectrometer using a custom NMR probe with an observe volume of 1.1 microL and a coil fill factor of 68%. Careful attention to capillary connections and NMR flow cell design allows on-line NMR detection with no significant loss in separation efficiency when compared with a UV chromatogram. HPLC NMR is performed on mixtures of amino acids and small peptides with analyte injection amounts as small as 750 ng; the separations are accomplished in less than 10 min and individual NMR spectra are acquired with 12 s time resolution. Stopped-flow NMR is achieved by diversion of the chromatographic flow after observation of the beginning of the analyte band within the NMR flow cell. Isolation of the compound of interest within the NMR detection cell allows multidimensional experiments to be performed. A stopped-flow COSY spectrum of the peptide Phe-Ala is acquired in 3.5 h with an injected amount of 5 micrograms.