Hyphenated analytical techniques (i.e., GC-MS, LC-MS) the undesired solvent signals, the four-pulse version of the have routinely proven themselves to be useful tools for many WET sequence provides superior suppression. The repetitive chemical applications (1, 2). Proton-detected high-perforpulse train in WET can be optimized to be insensitive to T 1 mance liquid-chromatography NMR (LC NMR), however, differences, B 1 -field inhomogeneities across the sample, or despite being known for over 15 years (3-10), has not yet both, by varying the tip angles of the selective RF pulses. proven to be a widely accepted technique. One of the major Our initial work focused on the B 1 -inhomogeneity optimized impediments to the wider growth of the field is the difficulty version of four selective pulses, which uses the following of observing analyte resonances in the presence of the much sequence (where G1-G4 are pulsed-field gradients): 98.2Π larger resonances from the mobile phase. While this problem (selective) -G1-80.0Π (selective) -G2-75.0Π(selecinitially appears similar to the well-known problem of water tive)-G3-152.2Π(selective)-G4-90Π(nonselective)suppression in normal liquid-phase biomolecular NMR sam-Acquire. More recently, we have found that if the T 1 -andples, it is exacerbated by three additional characteristics: (1) B 1 -optimized four-pulse WET sequence is used, it typically in typical reversed-phase HPLC mobile phases, there is more generates small but inverted solvent resonances, which can than one protonated solvent resonance (and all correspondthen be further reduced by the inclusion of a short delay ing 13 C satellites); (2) the sample in the detector coil is (0.5-2 ms) following the final field-gradient pulse ( G4). typically flowing, and this replenishment of fresh spins com-This yields the sequence 81.4Π (selective) -G1plicates many solvent suppression techniques, such as presat-101.4Π(selective) -G2 -69.3Π(selective) -G3 -161.0Π uration; and (3) the solvent resonances change frequencies (selective)-G4-dz-90Π(nonselective)-Acquire. The during the course of the solvent gradients (the chromatogradz delay is empirically optimized to minimize the amplitude phy ''method'') typically used during HPLC separations, of the small but inverted solvent signals, much like the delay thus presenting the spectroscopist with moving targets.
following the 180Π pulse in WEFT (13). While both the B 1 We present here the results of some recent developments and the T 1 -and-B 1 WET sequences work very well, we exin our laboratory which address these solvent-suppression pect that the T 1 -and-B 1 WET sequence will ultimately prove problems and greatly improve the quality of spectra generto be the method of choice due to its slightly better quality ated during both stopped-flow and on-flow LC NMR. The of solvent suppression. combination of shaped RF pulses, pulsed-field gradients Moonen and co-workers recognized that the solvent-signal (PFG), and selective 13 C decoupling allow us to acquire echos generated during repeated CHESS pulses in in vivo high-quality spectra on both flowing and nonflowing column applications could be avoided by doubling the gradient effluents, as demonstrated here using a typical CH 3 CN:HOD strength of each subsequent field-gradient pulse (14). We mobile phase. The resulting solvent suppression schemes found that the gradient echoes could be more effectively may also prove useful for conventional high-resolution NMR minimized by halving the intensity of each subsequent gradiof samples in protonated solvents (e.g., 90:10 H 2 O:D 2 O). ent pulse, yielding G2 Γ
1 2 G1, G3 Γ
1 4 G1, and G4 Γ
1 8 G1, Much of this work is based upon refinements of the WET where G1 was typically a Z one-axis gradient having an solvent-suppression technique of Ogg and co-workers (11). amplitude of 32 G/cm and a denaturation of 2 ms. This not
The WET technique uses a series of variable-tip-angle only places the strongest field-gradient pulse immediately solvent-selective RF pulses, where each selective RF pulse after the selective pulse which generates the largest amount is followed by a dephasing field-gradient pulse. While a of transverse magnetization, but also places any eddy currepetition of the simpler pulse-sequence-element CHESS rents induced by the strongest gradient pulse furthest from [90Π(selective)-gradient] (12) can also be used to suppress the acquisition. We also recognized that these techniques could be made more general by application of the shifted laminar pulse