LC NMR Hyphenation

LC-NMR hyphenation consists of a liquid chromatograph (autosampler, pump, column and oven) and a classical HPLC detector. The flow of the detector is brought via an interface to the flow-cell NMR probe. Using commercial NMR flow-cells with volumes between 40 and 180 p,L, in connection with microbore columns or packed capillaries, complete spectra have been provided from 1 nmol of sample. These micro-cells allow expensive deuterated solvents to be used, and thus eliminate solvent interference without excessive cost. The HPLC eluent can be split in order to allow simultaneous MS detection. 

Table 7.70 lists some of the main features of LC-NMR hyphenation. At 11.7T (500 MHz for 1H) a 4 mm LC-NMR flow-probehead readily provides a detection limit of ca. 5 p,g for on-flow (lmLmin-1) and 150ng for stopped-flow (in 3 h) for a typical 350-Da substance. Miniaturisation and hyphenation of NMR to various capillary-based microanalytical systems (LC, CZE) was described [650]. 

Therefore, a mild and quick extraction technique is necessary to exclude the preparation of artifacts. The carotenoid stereoisomers can be quantitatively analysed, employing MSPD extraction, from plant material, as well as from serum samples, using on-line SPE without any isomerisation or oxidation of the carotenoids. The extraction step is coupled to the separation and identification steps. Here, LC-NMR hyphenation, employing C30 stationary phases, is suitable for unambiguous distinction between all of these stereoisomers. 

However, instead of inspection of the whole blood plasma, urine or other biofluids, it can be advantageous to insert a separation step before the NMR measurement, which is realized in the LC NMR hyphenation. 

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]. 

Multiple hyphenation ( hypemation ) provides comprehensive spectroscopic information from a single separation. The first doubly hyphenated HPLC-NMR-MS appeared in 1995 [661], and its value is now accepted meanwhile fully integrated on-line LC-NMR-MS and MSn systems (QMS, QTTMS) are commercially available. On-line LC-NMR-MS coupling is by no means trivial. For example, the sensitivity of NMR is limited, while MS is incompatible with non-volatile buffers. The... 

NMR/MS has also been hyphenated to LC-SPE and LC-DAD modules. Sample enrichment and exchange of the HPLC mobile phase with an NMR suitable solvent is advantageous. LC-SPH-NMR/MS gains up to a factor of four in LC-NMR S/N for a single injection. No deuterated solvents are needed for separation and trapping. Optimisation of the separation procedure is less critical than for HPLC-UV. 

LC-NMR plays a central role in the on-line identification of the constituents of crude plant extracts (Wolfender and others 2003). This technique alone, however, will not provide sufficient spectroscopic information for a complete identification of natural products, and other hyphenated methods, such as LC-UV-DAD and LC-MS/MS, are needed for providing complementary information. Added to this, LC-NMR experiments are time-consuming and have to be performed on the LC peak of interest, identified by prescreening with LC-UV-MS. NMR applied to phenolic compounds includes H NMR,13 C NMR, correlation spectroscopy (COSY), heteronuclear chemical shift correlation NMR (C-H HECTOR), nuclear Overhauser effect in the... 

The LC-NMR instrument is a very attractive analytical tool in that it has the potential to provide a great deal of data detailing many structural features in an inline mode. The principle advantage to using hyphenated NMR technology is that in most cases one can collect NMR and MS data on the same sample reducing the possibility of decomposition during the isolation and sample preparation process. 

Describe the features of a chromatographic separation that can limit the successful acquisition of NMR data in a hyphenated LC-NMR instrument. 

The high sensitivity of hyphenated techniques such as HPLC-MS has also been exploited in the identification and structural studies of flavonoid glucosides [151] and the application of other hyphenated techniques such as LC-MS-MS and LC-NMR for the analysis of plant constituents has been discussed earlier [152],... 

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]. 

The hyphenation of CE and NMR combines a powerful separation technique with an information-rich detection method. Although compared with LC-NMR, CE-NMR is still in its infancy it has the potential to impact a variety of applications in pharmaceutical, food chemistry, forensics, environmental, and natural products analysis because of the high information content and low sample requirements of this method [82-84]. In addition to standard capillary electrophoresis separations, two CE variants have become increasingly important in CE-NMR, capillary electrochromatography and capillary isotachophoresis, both of which will be described later in this section. 

In general, MS performance should not be compromised by a static magnetic field. Many factors such as the design of ion optics, selection of interface, and type of MS analyzer should be considered with regard to the construction and configuration of the double hyphenated system. The optimum design should overcome some of the mutual incompatibilities of LC-NMR and LC-MS systems. For LC-MS, ionization propensities vary considerably depending upon solvent, ionization source type, and complex matrix effects. Most NMR analyses, however, are not affected by variations... 

This new hyphenated analytical system integrates capillary LC with NMR detection. The capillary LC-NMR system is comprised of an NMR spectrometer equipped with a capillary flow probe and the capillary LC. The capillary flow probe has a flow-cell design with an active sample volume of only 1 or 1.5 pL. This volume is chosen to match the typical peak volumes of capillary LC separation. 

The cryo-LC probe offers an enhancement of np to four-fold in sensitivity over conventional LC probes. Hyphenated techniques such as LC-NMR and LC-SPE-NMR, as well as high-thronghput NMR screening methods, will benefit from this significant increase in sensitivity. 

Careful and critical use of the hyphenated techniques LC-UV-MS and LC-MS-MS can provide sufficient online information for the identification of small molecules such as flavonoids. However, in many cases, more data are required for an in-depth structural investigation and this can be supplied by the addition of an LC-NMR analytical capability (Figure 1.13). For practical purposes, LC-UV-MS and LC-UV-NMR are generally run as separate... 

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