Capillary LC-NMR

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. 

Applications of LC-NMR are still scarce but the technique will become more widely used. The main effort for efficient exploitation of LC-NMR needs to be made on the chromatographic side, where strategies involving efficient preconcentration, high loading, stop-flow, time slicing, or low flow procedures have to be developed. Microbore columns or capillary separation methods, such as capillary LC-NMR, CE-NMR, and CEC-NMR, will find increased application, one reason being that the low solvent consumption will allow the use of fully deuterated solvents. 

Although not at capillary level nor a physical separation, Johnson and He first introduced the combination of electrophoresis and NMR (E-NMR) to study electrophoretic mobilities and diffusion coefficients [27], Sweedier and coworkers described the first application of microcoil NMR probe as an on-line detector for CE [13,28], In 1998 Bayer, Albert, and co-workers reported the first CEC/NMR experiments which also included CE/NMR and capillary LC/ NMR results [29],... 

Bruker Biospin GmbH. Bruker BioSpin announces novel capillary LC-NMR system 2002. Forthcoming. 

S/N for a flow cell of only 50 nL volume. The main drawback was rather wide NMR linewidths. Behnke etal. demonstrated capillary LC/NMR in 1996 [138], using flow cells of 50 nL and 900nL and a standard saddle coil, and achieved sensitivities approximately 10 times greater than conventional LC/NMR. The approach, however, did not incorporate a UV detection cell so that stopped-flow NMR measurements had to be nuule by reference to on-flow NMR measurements. 

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]. 

The basic components of an LC-NMR system are some form of chromatographic instrument and an NMR spectrometer equipped with a flow-probe, as shown in Fig. 19.17. In terms of the chromatography of choice, there are many examples in the literature of a wide array of separation instruments employed, from SFC to capillary electrophoresis (CE) [87,88]. By far the most common method (not necessarily the best choice from a separation point of view) of achieving the desired separation is through HPLC. There are many commercial... 

What advantages are there for utilizing capillary-scale LC-NMR ... 

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. 

Both the capillary LC and NMR are controlled by the interface software, which enables the operator to use the UV-detector output for peak selection. Only peaks of interest can be subjected to NMR analysis, while minor or unimportant compounds can be directed to waste. NMR acquisition can take place in either on-flow or stop-flow mode. The combination of capillary LC and NMR is suitable for sample-limited applications (e.g., proteomics) and allows for low nanograms detection. 

The specific constraints and requirements of continuous-flow NMR will be explained in the first chapter, whereas specific applications, such as biomedical and natural product analysis, LC-NMR-MS and LC-NMR in an industrial environment, together with polymer analysis, will be discussed separately. Thus, the reader will obtain a broad overview of the application power of LC-NMR and the benefits of its use. He/She will also be introduced to the pitfalls of this technique. Special attention will be given to the exciting newer coupled techniques such as SFC-NMR and capillary HPLC-NMR. However, new emerging future developments will also be discussed thoroughly. 

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. 

At present, applications of LC-NMR in environmental analysis are still limited by the relatively low sensitivity of the NMR detector. Further progress is expected when cryoprobes and new capillary flow cells hopefully become available in the near future. 

Currently, on-line LC-NMR is just at the starting point for dramatic miniaturization and parallelization. Prototypes of NMR probes with remarkable sensitivity values are currently available in 2002, although the ease of operation of the combined separation-detection system is not guaranteed. Because the design of the current analytical LC-NMR probes is so simple, they can be used in many real-world applications. However, current research areas such as proteomics and metabolomics necessitate the utmost sensitivity, using very small amounts of sample, which can only be achieved by employing capillary probes. 

Schefer AB, Albert K. Capillary Separation Techniques. In Albert K, ed. On-line LC-NMR and Related Techniques. New York Wiley, 2002 237-246. 

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