CPSA Digest 2001

Proceedings -Wednesday, October 10, 2001

WOD2

Applications of Flow NMR to the Analysis of Combinatorial Products

Background:
Mass spectrometry is an essential tool for providing information on compound molecular weights. NMR is essential for providing structural information, detecting structural changes in a metabolite or degradant and determining stereochemistry. Together as LC-NMR-MS they represent a unique and powerful combination for the analysis of complex mixtures. After many years of working through experimental considerations and constraints, such as plumbing issues, operating a MS in a magnetic field, suppression issues and working with deuterium solvents, LC-NMR-MS has gained broader acceptance and appeal. The advantages of using LC-NMR-MS include the elimination of off-line separation or isolation, better sample stability upon separation, automated operation and cleaner spectra. However, some disadvantages are present that include sensitivity (which is a general NMR problem), solvent suppression, and the use of expensive deuterated mobile phases and buffers.

Premise:
The three hardware components required for LC-NMR are an LC pump, an LC-NMR interface and an LC-NMR probe. Several methodologies for LC-NMR are referred to as "on-flow," "stop-flow" and "loop storage."

On-flow techniques obtain a quick assessment of the sample but at low sensitivity. Here, the eluent is diverted into the NMR probe and proton data is collected "on the fly."

Stop-flow techniques allow for structural confirmation using 2D experiments and provide better proton sensitivity than on-flow techniques. Here, the eluent is diverted through the NMR probe; upon peak recognition, the LC pump stops to acquire 1D and 2D spectra and then after data acquisition the LC separation resumes.

Loop storage techniques allow adequate NMR experiments to be performed on each fraction without interfering LC separation. When a peak is recognized it is diverted into storage loops and then selected peaks are analyzed. This approach does not disrupt LC separation and can be performed without the spectrometer.

Value of the Technology
LC-NMR applications include:

• Metabolite profiling
• Impurity identification
• Structural elucidation of degradation products
• Natural product problem-solving Each of the problems mentioned above is different, however, and may require alternate approaches. Sample mass is a consideration (often limited for in vitro metabolite identification and natural products) and so is the complexity of the sample components.

Future
The utility of LC-NMR and LC-NMR-MS will continue to increase as advances in consoles, magnets, cryoprobes, software, robotics and chromatography are introduced. The interdisciplinary concepts of LC, NMR and MS will allow for the sharing of ideas to promote information sharing and problem solving and are expected to lead to significant advances in the available techniques and applications.

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