LC-NMR coupling

Klaus Albert, Manfred Krucker, Karsten Putzbach, and Marc D. Grynbaum 

For structure elucidation of unknown compounds, nuclear magnetic resonance spectroscopy (NMR) is one of the most important spectroscopic techniques. Most commonly used is H NM R spectroscopy NMR measurements give additional information, but due to the low natural abundance of nuclei (1.1% the isotope 

The major benefits of H NMR spectroscopy are easy quantification and determination of steric arrangements. Disadvantages are the low sensitivity of the NMR detection and the fact that inorganic counterions and parts of the molecule without attached protons cannot be detected. Mass spectrometry, in contrast, is a very sensitive and selective detection technique steric information, however, cannot readily be obtained. Therefore, these two detection methods are mutually complementary for unambiguous structure elucidation purposes, a combined evaluation of both sets of data is highly desirable. 

To measure NMR spectra, the molecules of interest are placed in a magnetic field generated by a cryo-magnet. In this way, the polarization of the nuclei is 

HPLC Made to Measure A Practical Handbook for Optimization. Edited by Stavros Kromidas Copyright 2006 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim ISBN 3-527-31377-X 

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The theoretical basics of LC-NMR coupling have already been discussed in the previous chapter. Except for one type of experiment, the connection of the chromatographic system and the NMR detection cell via a capillary is not sufficient. Most of the experiments require a special interface with switching valves under software control for reliable and reproducible results. The level of equipment and the application field depends on the types of experiment which are being conducted and will be discussed in the following. 

From the selectivity point of view, LC-NMR coupling is especially suited to the analysis of compound classes such as nitroaromatics, phenols, aromatic amines, aromatic carboxylic acids, polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and azo- and anthraquinone dyes. Another advantage of LC-NMR coupling for the investigation of aromatic compounds in environmental samples is that the position of substituents on the aromatic ring, e.g. in unknown metabolites or degradation products, can best be determined by NMR spectroscopy. 

For aliphatic compounds with longer alkyl chains, such as surfactants, the NMR detector can contribute little to an increased selectivity of the LC-NMR coupling since, in the range of aliphatic protons, the spectra are often complex. Moreover, analyte signals around 2 ppm can be suppressed or influenced by the solvent suppression when acetonitrile is used as the organic component of the eluent. Since surfactants are present in many environmental samples, they pose problems for non-target analysis, not only because of their complex spectra but also because they can influence the separating properties of the analytical column by their surface activity [2]. 

Nuclear magnetic resonance spectroscopy (NMR) is one of the most powerful analytical methods for identification and structure elucidation of organic compounds. Since NMR spectra are recorded in solution, no phase transfer like in MS is necessary when coupled with LC techniques. Additionally, NMR is a non-destructive detection technique, allowing the analyte to be transferred for characterization using additional methods. As of today, LC—NMR coupling was used in a wide range of applications [65,66,67,68,69,70,71],... 

V. Exarchou, M. Krucker, T.A. van Beek, J. Vervoort, l.P. Gerothanassis, K. Albert, LC-NMR coupling technology recent advancements and applications in natural products analysis, Magn. Reson. Chem., 43 (2005) 681. 

From LC NMR coupling constants, the location of the hydrogen atoms can be determined. However, since this method does not give information about the absolute size, a few structural parameters are fixed. By combining the two methods, the deviations from MW spectroscopy are smaller <6USP(7)58> and this is further improved by addition of rotational spectroscopy data. 

LC-NMR coupling technology has been recently reviewed [23], and the interested reader is also referred to the detailed text edited by Albert [24]. The miniaturisation of flow probes to the capillary scale has been developed to enhance signal detection sensitivity, as discussed in Section 3.4.2, and more recently the hyphenation of gas chromatography with microflow cells (GC-NMR) has even been demonstrated [25]. 

LC—NMR coupling can be realized by four operation modes on flow, stop flow, time sliced, and loop collection. Since, in the on-flow mode, only a relatively low number of NMR pulses can be acquired for a peak or... 

Another most exciting new tool for the analysis of complex polyolefins is the direct coupling of high-temperature liquid chromatography and H-NMR. Such equipment became available only recently when a high-temperature flow-through NMR probe was introduced by Bruker. The construction and experimental setup of the LC-NMR coupling is described in detail by Hiller et al. [130]. In brief, the NMR flow probe can operate at temperatures up to 150°C. The probe has an active flow cell with a volume of 120 pL. It is a dual inverse H/ C probe with pulse field... 

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