e., 1H resonance frequency of 400 MHz selleck compound for a standard HPLC-NMR coupling. The analytical flow cell was initially constructed for continuous-flow NMR acquisition. However, the need for full structural assignment of unknown compounds, especially novel natural products, has led to the application in the stopped-flow mode. In fact, the benefits of the closed-loop separation�Cidentification circuit, together with the prospect of using all presently available 2D and 3D NMR techniques in a fully automated way, have prompted the development of stopped-flow modes, e.g., time-slice mode. A typical experimental arrangement of LC-NMR is shown in Figure 4. Figure 4 A typical LC-NMR system Generally, in LC-NMR system, the LC unit comprises autosampler, LC pump, column, and a non-NMR detector (e.g.
, UV, DAD, EC, refractive index, or radioactivity). From this detector, the flow is guided into the LC-NMR interface, which can be equipped with additional loops for the intermediate storage of selected LC peaks. The flow from the LC-NMR interface is then guided either to the flow-cell NMR probe-head or to the waste receptacle. Following passage through the probe-head, the flow is routed to a fraction collector for recovery and further investigation of the various fractions analyzed by NMR. An MS can also be attached to the system via a splitter at the output of the LC-NMR interface. In most of the LC-NMR operations, reversed-phase columns are used, employing a binary or tertiary solvent mixture with isocratic or gradient elution. The protons of the solvents of the mobile phase cause severe problems for obtaining an adequate NMR spectrum.
The receiver of the NMR spectrometer is not quite able to handle the intense solvent signals and the weak substance signals at the same time. To overcome this problem, solvent signal suppression can be achieved by one of the three major methods: presaturation, soft-pulse multiple irradiation or water suppression enhancement through T1 effects (WET) presaturation employing a z-gradient. This problem can also be minimized by considering the following guidelines: Using eluents that have as few 1H NMR resonances as possible, e.g., H2O, ACN, or MeOH. Using at least one deuterated solvent, e.g., D2O (approx $290/L), ACN-d3 (approx $1600/L), or MeOD (approx $3000/L). Using buffers that have as few 1H NMR resonances as possible, e.g.
, TFA or ammonium acetate. Using ionpair reagents that have as few 1H NMR resonances as possible, e.g., ionpairs with t-butyl groups create an additional resonance. To date, three Anacetrapib main types of data acquisition modes have been introduced: continuous-flow acquisition, stopped-flow acquisition, and time-sliced acquisition. Whatever may be the acquisition mode, an optimized HPLC separation is crucial to any LC-NMR analysis. As the sensitivity of LC-NMR is much less than other hyphenated techniques, e.g.