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 mu L and a coil fill facto r of 68%. Careful attention to capillary connections and NMR now cell desig n allows on-line NMR detection with no significant loss in separation effic iency when compared with a UV chromatogram. HPLC NMR is performed on mixtur es of amino acids and small peptides with analyte injection amounts as smal l as 750 ng; the separations are accomplished in less than 10 min and indiv idual NMR spectra are acquired with 12 s time resolution. Stopped-now NMR i s achieved by diversion of the chromatographic now after observation of the beginning of the analyte band within the NMR flow cell. Isolation of the c ompound of interest within the NMR detection cell allows multidimensional e xperiments 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 mu g.