Chemical-shift referencing and resolution stability in gradient LC-NMR (acetonitrile:water)

Wide ranges of solvent conditions are generated during solvent-gradient LC-NMR. This complicates the referencing of the chemical-shift scale of the resulting NMR data. The problems that arise when performing LC-NMR in acetonitrile:water - particularly when the mobile-phase composition can range anywhere from 0% to 100% - are examined here, and the reliability of the secondary reference signals are evaluated. It is shown that under these conditions the use of the acetonitrile signal is superior to the use of the water signal in any form (either the 1H or the 2H signal) as a secondary reference, a lock signal, and a signal for shimming. The limitations of the referencing methods and other experimental parameters, and the limitations on the solvent-gradient ramp parameters, primarily as they affect lineshapes, are all shown. These results are compared to the way some other publications have referenced the 1H chemical-shift axis (when using acetonitrile:water mixtures to perform reversed-phase chromatography LC-NMR).