LC NMR

In reeent years, tire use of elevated temperatures has been reeognised as a potential variable in method development. Witlr inereased temperature, aqueous-organie mobile phases separations ean improve, viseosity deereases and diffusion inereases so baek pressures are redueed. At higher temperatures (usually with superheated water > 100 °C under modest pressures) water alone ean be used as the mobile phase and eair provide unique separation opportunities. The absenee of an organie solvent enables the use in HPLC of alternative deteetors sueh as FID or on-line LC-NMR using deuterium oxide as the eluent. 

LC-MS/MS Liquid chromatography coupled with tandem mass spectrometry LC-NMR Liquid chromatography coupled with nuclear magnetic resonance TLC Thin-layer chromatography... 

The development of appropriate analytical techniques is essential for success and to proceed toward screening, such as TLC, GC, HPLC, LC-MS, and LC-NMR [11,23]. 

Schaller, F. Wolfender, J.-L. Hostettmann, K. Mavi, S. New antifungal quinone methide diterpenes from Bobgunnia madagascariensis and study of their interconversion by LC/NMR. Helv. Chim. Acta 2001, 84, 222-229. 

K. Albert 2002, On-line LC-NMR and Related Techniques, Wiley Sons, Chichester, 306 pp. Specialized, contains NMR spectroscopy but not imaging. 

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]. 

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. 

LC-NMR can be operated continuously ( on-flow ) or discontinuously ( stopped-flow ). The optimum flow-rate in continuous-flow NMR is a compromise between best resolution and sensitivity. The sensitivity in NMR measurements has been increased significantly by ... 

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]. 

In cases where 2D NMR experiments are insufficient for a complete analysis of anionic surfactant mixtures, LC-NMR may provide better information. Characterisation of fatty alcohol ethoxylate (FAE) based oligomeric surfactants by on-line 2D (GCOSY, TOCSY and Homo 2DJ) stopped-flow HPLC- H NMR has been described [655,656]. The analysis of a typical mixture comprising three components (PEG and PEOs with different end-groups) is shown in Figure 7.34. In this representation, the 111 NMR frequency domain is in the... 

Thermal degradation of Irganox 1076 in air was studied by means of HPLC-UV/VIS and by preparative HPLC-NMR. At 180 °C cinnamate and dimeric oxidation products are formed, and at 250 °C de-alkylation products are observed [660], On-line LC-NMR hardly covers a real need in polymer/additive analysis, as the off-line option is mostly perfectly adequate for that purpose. 

Table 7.71 a-Hydroxy- -aIkoxy(aryloxy) FAE structural information obtained by on-line LC-NMR ... 

Principles and Characteristics Traditional analytical approaches include off-line characterisation of isolated components, and the use of several chromatographic separations, each optimised for a specific spectroscopic detector. Neither LC-NMR nor LC-MS alone can always provide complete structure determinations. For example, MS may fail in assigning an unequivocal structure for positional isomers of substituents on an aromatic ring, whereas NMR is silent for structural moieties lacking NMR resonances. Often both techniques are needed. 

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. 

Scheme 7.12 shows some tested configurations of integrated total spectroscopy laboratories. Couplings may be parallel or in series other examples are LC-NMR-MS and GC-FUR-MS. Wilson [664] has... 

FIGURE 4.1 Extraction technique MSPD. (From Albert, K., On-Line LC-NMR and Related Techniques, John Wiley Sons Ltd., 131, 2002. With permission.)... 

In summary, NMR spectroscopy is an extremely versatile tool useful that enables researchers to understand the structure of natural products such as carotenoids. For a full structural assignment, the compound of interest has to be separated from coeluents. Thus, it is a prerequisite to employ tailored stationary phases with high shape selectivity for the separation in the closed-loop on-line LC-NMR system. For the NMR detection, microcoils prove to be advantageous for small quantities of sample. Overall, the closed-loop system of HPLC and NMR detection is very advantageous for the structural elucidation of air- and UV-sensitive carotenoids. 

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