Modes of Operation for LC-MS-NMR

As mentioned in the section of modes of operation for LC-NMR (Section 20.3.2), with the use of shielded cryomagnets, the location of the MS instrument will follow the same rule as for the HPLC. The most common modes of operation for LC-MS-NMR are on-flow and stop-flow. With stop-flow, the MS instrument can also be used to stop the flow on the chromatographic peak of interest that is to be analyzed by NMR. These two modes are presented here with an example. In the loop collection mode, the MS of the LC-MS-NMR system may also monitor the trapping of the chromatographic peak inside the loop. 

A Varian Unity Inova 600-MHz NMR instrument (Palo Alto, CA) equipped with a H C/ N pulse field gradient triple resonance microliow NMR probe (flow cell 60pL 3mm O.D.) was used. Reversed-phase HPLC of the samples was carried out on a Varian modular HPLC system (a 9012 pump and a 9065 photodiode array UV detector). The Varian HPLC software was also equipped with the capability for programmable stop-flow experiments based on UV peak detection. An LCQ classic MS instrument, mentioned in the previous section, was connected on-line to the HPLC-UV system of the LC-NMR by contact closure. The H resonance of the D2O was used for field-frequency lock, and the spectra were centered on the ACN methyl resonance. Suppression of resonances from HOD and methyl of ACN and its two C satellites was accomplished using a train of four selective WET pulses, each followed by a Bo gradient pulse and a composite 90-degree read pulse [41]. 

TABLE 20-1. MS Data of Flavonoids in Negative Mode from the On-Flow Run in the LC-MS-NMR 

Source Reprinted from reference 40, copyright 2003, with permission from Elsevier. 

These experiments indicated that for sample mixtures, the on-flow mode of LC-MS-NMR is useful for obtaining structural information on the major components. If more detailed analysis is required, or the amount of sample is small and the compound(s) cannot be isolated because of instability or volatility, stop-flow is the mode of choice. LC-MS and LC-NMR chromatographic 

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The capability of analyzing a complex mixture in a chromatographic run by the hyphenation of several techniques, such as NMR and MS, to HPLC is becoming more popular in the pharmaceutical industry. NMR and MS data on the same analyte are crucial for structural elucidation. When different isolates such as metabolites are analyzed by NMR and MS, one cannot always be certain that the NMR and the MS data apply to the same analyte, especially when the analytes have been isolated using analytical columns and prep columns for the MS and NMR analysis, respectively. HPLC conditions are not always reproducible when analytical and prep-HPLC columns are used to isolate different amounts of the analytes of interest. To avoid this ambiguity, LC-MS and LC-NMR are combined. MS data should be obtained initially because with NMR, data collection in the stop-flow mode can take hours or days, depending on the complexity of the structure and the amount of sample. This is why it is preferable to designate this operation as LC-MS-NMR rather than LC-NMR-MS or LC-NMR/MS. 

In the last three years, there have been relatively few examples in the literature that deal with the application of LC-MS-NMR. We have been interested in evaluating this technology in our laboratory to determine the pros and cons, and to decide which cases are suitable for this application. To illustrate these modes of operation, a group of flavonoids was chosen. These compounds have simple structures with primarily aromatic protons some have low field aliphatic protons, which would not be... 

Another way to improve the S/N ratio is the recording of the spectra in stop-flow mode. Operation in the direct stop-flow mode requires that the retention times of the analytes of interest are known or that a sensitive method of detection such as LC/UV or LC/ MS is used prior to LC/NMR to trigger the detection. In practice, one of these detectors is connected online before the NMR instrument and the signal of the analytes of interest passing through this detector is used to trigger a valve that will stop the LC flow exactly when the peak reaches the NMR cell after a calibrated delay (for setup, see Figure 2). 

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