Multiple hyphenations

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... 

Wilson et al. [662-665] have described various prototype systems for total organic analysis devices. It has proved technically feasible to obtain UV, IR, NMR and MS spectra (together with atomic composition based on accurate mass determination) following RPLC separation. The fully integrated approach offers the benefit that one chromatographic run is required, thus ensuring that all of the spectrometers observe the same separation. Such multiple hyphenations might favour the analysis of complex mixtures for both confirmation of identity and structure determination (should this represent a cost-effective approach). Table 7.72 illustrates the main features of on-flow multiple LC hyphenation. 

Wilson [664,672] has reviewed multiple hyphenation of liquid chromatography. 

Wilson ID. 2000. Multiple hyphenation of liquid chromatography with nuclear magnetic resonance spectroscopy, mass spectrometry and beyond. J Chromatogr A 892 315-327. 

The trend is toward multiple hyphenation techniques like HPLC-UV-MS and HPLC-UV-NMR. These have an enormous potential for the rapid investigation of plant extracts. Multiple hyphenation in a single system provides a better means of identification of compounds in a complex matrix. 

While the coupling of NMR spectroscopy and MS in the same system is undoubtedly the most productive of the possible multiple hyphenations, others have been performed which deserve consideration. Probably the easiest to implement is the inclusion of a UV diode-array detector (DAD) to enable UV... 

Shortly afterwards, this work was extended by the incorporation of a mass spectrometer into the system, thus enabling on-line NMR and MS data to be obtained with on-line collection of the eluent for off-line FT-IR spectroscopy [22]. The incorporation of the mass spectrometer required the addition of a small proportion of ammonium acetate, dissolved in methanol, to the deuterated chloroform used as the eluent in order to promote the ionisation of the analytes. The inclusion of methanol and ammonium acetate to the solvent obviously introduced new signals into the NMR spectra, and in addition resulted in the loss of exchangeable protons from the analytes which had been observable when chloroform alone was used as the solvent. This work demonstrated the feasibility of multiple hyphenation ( hypernation ) but the off-line nature of the FT-IR data acquisition, with the inevitable delay inherent in offline analysis, represents a slight disadvantage. In addition, volatile components may well be lost as the solvent is evaporated. This can be a problem that, together with analyte instability, is exacerbated with such interfaces when reversed-phase eluents are used since these require heating in order to ensure removal of the solvent. 

R579 I. D. Wilson, Multiple Hyphenation of Liquid Chromatography with Nuclear Magnetic Resonance Spectroscopy, Mass Spectrometry and Beyond , J. Chromatogr., A, 2000, 892(1+2), 315... 

Hyphenation refers to the online combination of a separation technique (mainly GC or LC) and a spectroscopic detection method that provides structural information for the analytes concerned. LC-MS and GC-MS are the most popular hyphenated techniques in use today, thanks to the incredible shrinking of mass spectrometers. However, MS does not provide the same information as IR or NMR. Hypernation (one higher than hyphenation) is the multiple hyphenation which may combine, for example, UV detection with PDA, NMR, FTIR, and MS as displayed in Figure 4.22, the last one is a destmctive detector. At the present time most analysts are using a single detector but it can be guessed that hypemation will be used more and more as size and cost of the instruments decrease. 

R 622 M. P. Williamson, Solving the First Globular Protein Structure BUSI IIA , Magn. Reson. Chem., 2003,41(Spec. Issue), S64 R 623 I. D. Wilson and U. A. T. Brinkman, Hyphenation and Hypernation. The Practice and Prospects of Multiple Hyphenation , J. Chromatoqr., A, 2003,1000, 325... 

Wilson ID and Brinkmann UATh (2003) Hyphenation and hypernation the practice and procedures of multiple hyphenation./o ma/ of Chromatography, 1000 325-356. 

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