Hyphenated NMR techniques

We have seen that NMR can provide detailed molecular structure information. It is possible to join together or couple an HPLC instrument with an NMR spectrometer. The HPLC performs the separation of a complex mixture and the NMR spectrometer takes a spectrum of each separated component to identify its structure. We now have a new instrument, an HPLC-NMR instmment. We call a coupled instrument like this a hyphenated instmment. The coupling of two instruments to make a new technique with more capabilities than either instmment alone provides results in a hyphenated technique or hybrid technique. HPLC-NMR is made possible with a specially designed flow probe instead of the standard static probe. For example, Bruker Instruments (www.bruker-biospin.com) has a flow probe for proton and C NMR with a cell volume of 120 p,L. Complex mixtures of unknown alkaloids extracted from plants have been separated and their stmctures completely characterized by HPLC-NMR using a variety of 2D NMR 

HPLC-NMR and another hyphenated, more powerful instrument, HPLC-NMR-MS (the MS stands for mass spectrometry) are used in pharmaceutical research and development. These hyphenated techniques identify not only the structures of unknowns, but with the addition of MS, the molecular weight of unknown compounds. The HPLC-NMR-MS instrument separates the sample on the HPLC column, takes the NMR spectra as the separated components flow through the probe and then acquires the mass spectrum of each separated component to determine the molecular weight and additional structural information from the mass spectral fragmentation pattern. The MS must be placed after the NMR, since MS is a destructive technique. MS is covered in Chapters 9 and 10. 

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As liquid chromatography plays a dominant role in chemical separations, advancements in the field of LC-NMR and the availability of commercial LC-NMR instrumentation in several formats has contributed to the widespread acceptance of hyphenated NMR techniques. The different methods for sampling and data acquisition, as well as selected applications will be discussed in this section. LC-NMR has found a wide range of applications including structure elucidation of natural products, studies of drug metabolism, transformation of environmental contaminants, structure determination of pharmaceutical impurities, and analysis of biofiuids such as urine and blood plasma. Readers interested in an in-depth treatment of this topic are referred to the recent book on this subject [25]. 

NMR metabolomics increasingly makes use of natural product structure elucidation techniques such as 2D NMR, particularly for plant-based studies. In both natural products research and metabolomics, strategies such as quantitative NMR and low-volume NMR are employed, while the use of hyphenated NMR techniques is also expanding. These techniques have been applied in numerous studies examples are given in Table 1 (see also Volume 9). 

Recent trends indicate an increased use of hyphenated NMR techniques, especially those involving NMR in combination with chromatographic methods such as HPLC, GPC, or SEC.P°l Online SEC- and GPC-NMR have found useful applications in the direct determination of molecular weight and molecular weight distribution. ... 

Sampling, sample handling, and storage and sample preparation methods are extensively covered, and modern methods such as accelerated solvent extraction, solid-phase microextraction (SPME), QuEChERS, and microwave techniques are included. Instrumentation, the analysis of liquids and solids, and applications of NMR are discussed in detail. A section on hyphenated NMR techniques is included, along with an expanded section on MRI and advanced imaging. The IR instrumentation section is focused on FTIR instrumentation. Absorption, emission, and reflectance spectroscopy are discussed, as is ETIR microscopy. ATR has been expanded. Near-IR instrumentation and applications are presented, and the topic of chemometrics is introduced. Coverage of Raman spectroscopy includes resonance Raman, surface-enhanced Raman, and Raman microscopy. 

In the case of the low abundance of some compounds, there are difficulties with signal overlap. To overcome these difficulties, there have been developments involving NMR hyphenation with techniques such as HPLC and mass spectrometry. In LC/NMR methods of analysis, NMR is used as the detector following LC separation and this technique is capable of detecting low concentrations in the nanogram range. This technique has been reported for the detection and identification of flavanoids in fruit juices and the characterization of sugars in wine [17]. 

Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools that are widely used in the pharmaceutical industry. Eor some compounds, mass spectrometry alone is insufficient for complete structural elucidation of unknown compounds nuclear magnetic resonance spectroscopy (NMR) can help elucidate the structure of these compounds (see Chapter 20). Traditionally, NMR experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less cost-effective. The... 

Two-dimensional H NMR experiments acquired in stop-flow mode represent to some extent a link between on-flow LC-NMR screening and the detailed structural elucidation of isolated compounds employing two-dimensional NMR techniques in conventional off-line probe-heads. As the LC-NMR-MS hyphenation offers the possibility of triggering the stopping of the LC pump by the MS signal, this technique is particularly well suited for the reliable detection of compounds showing only weak UV absorbances. 

Chemical structure of rubbery materials Chapters 1, 2, 3, 4, 5, 6, 9 and 11, describe applications of (multi) hyphenated TGA techniques, optical and high-resolution NMR spectroscopes for the analysis of chain microstructures and conformations, chemical composition of components, additives and volatiles in rubbery materials, vulcanisation chemistry, functional groups analysis and chemical modification of rubbery materials. 

Quality control Applications of NMR relaxation experiments, NMR imaging, NMR MOUSE and (multi) hyphenated TGA techniques for quality control are discussed in Chapters 1, 7 and 10. 

Both CE-NMR and CEC-NMR have been established as powerful hyphenated separation techniques. The NMR sensitivity should be improved further in order for CE-NMR and CEC-NMR to become more widely applied techniques. One... 

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