Hyphenated 2D NMR

NMR is one of the most important tools in structural elucidation, but since the technique cannot be hyphenated with GC like MS and FTIR and its sensitivity is poorer, it cannot always identify chemicals from high background samples. The unchallenged capability of NMR to give information on connectivity and neighboring atoms makes it a very desirable method in the analysis of chemicals related to the CWC. NMR can, for instance, normally find out the type of the carbons (methyl, ethyl, propyl, or isopropyl) directly connected to phosphorus ( H and 31P spectra) and the presence of fluorine-phosphorus fink (1H, 19F, and 31P spectra). Different 2D-NMR experiments give additional information on connected atoms. 

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). 

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... 

Four general classes of NMR experiments are routinely used to analyze metabolites (1) ID NMR experiments (2) 2D NMR experiments (3) Solvent suppression methods and (4) Hyphenated NMR experiments. The ID and 2D NMR experiments are commonly used for metabolite structure determination. The various solvent suppression techniques (Gaggelli and Valensin, 1993 Hwang and Shaka, 1995 Smallcombe and Patt, 1995) are crucial for dilute metabolite samples where the solvent peak is the most intense peak in the NMR spectrum. These solvent suppression techniques can be incorporated as needed in both ID and 2D NMR experiments. Since their introduction in the 1990s, hyphenated NMR methods have become common tools in the identification of metabolites. These methods include LC-NMR (Albert, 1995 Spraul et al., 1993, 1994), LC-NMR-MS (Mass Spectrometry) (Shockcor et al., 1996) and LC/SPE (solid phase extraction)/NMR (Alexander et al., 2006 Bieri et al., 2006 Xu et al., 2005 Wilson et al., 2006). 

The inverse-detected 2D NMR experiments that have been discussed to this point have all been discrete, single-purpose experiments, e.g. correlating protons with their directly bound heteronucHde (typically or N). There are another class of inverse-detected 2D NMR experiments that are generally referred to as hyphenated 2D experiments. These are experiments that first establish one type of correlation, followed by an additional experiment segment that then pursues a further spectroscopic task. Predecessors of the inverse-detected variants of these experiments were the HC-RELAY (proton—carbon heteronuclear relayed coherence transfer) experiments pioneered by Bolton [151—155]. Examples of these include, but are by no means hmited to HXQC-COSY and -TOCSY [156—158], -NOESY [159], -ROESY [160], and more recent gradient variants [161] etc., where X = S (single) or M (multiple) quantum variants of the experiments. 

LC/NMR in various combinations with LC/UV-DAD, LC/MS, LC/MSMS, LC/IR, and/or LC/CD has been used in many applications related to the online identification of natural products. In this field, the challenge for hyphenated techniques is important since often the characterization of completely unknown molecules is required in very complex biological matrices. In this case, LC hyphenated techniques are used for the chemical evaluation of biologically active fractions or extracts and for dereplication purposes. As full structure assignment is often needed, all online spectroscopic data are taken into consideration. Most applications are performed in the stop-flow mode and extensive 2D NMR correlation experiments are measured. For unknown online determination the need for data is often mandatory. This type of information can be deduced from HSQC and HMBC indirect measurements and very recently it has been demonstrated that even direct measurements were possible in a crude plant extract. For this application the LC peak of interest was preconcentrated by trapping on SPE and the measurement was performed on a cryogenic flow... 

G.E. Martin, B.D. Hilton, K.A. Bhnov, A.J. Williams, Unsymmetrical inchrect covariance processing of hyphenated and long-range heteronuclear 2D NMR spectra-enhanced visualization of 7/ch nd Jch correlation responses, J. Heterocychc Chem. 45 (2008) 1109. 

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