LC-NMR system

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. 

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... 

Fig. 19.17. Schematic showing the most basic elements that should be a part of an LC-NMR system. The dotted arrows represent electronic controls and the solid lines represent the flow path of chromatographic eluent.

A different issue is one that is quite common in the Pharmaceutical industry. A relatively frequent situation that arises is the need to identify a 0.1% impurity from a reaction mixture or metabolism sample. These samples are often quite convoluted in terms of the amount of compounds present as well as the general complexity of the separation, akin to a natural products extract, as can be seen in Fig. 19.19. However, to simplify this scenario to just a two-component mixture is appropriate for this section. Under common LC-NMR systems, it is typically required to have at least 50 pg of material for a complete structure elucidation (to enable the collection of long-range heteronu-clear correlation data, HMBC). Therefore, one must be able to load 50 mg of the mixture on the column. Keep in mind, that if a ID 1H spectrum is all that is needed (in the case of a regiochemical issue in an aromatic system) this task becomes more amenable. The point trying to be made is that LC-NMR is a fantastic technique, but it must be used in... 

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. 

Whereas LC-NMR was considered to be an exotic technique in the late 1970s, today over 200 LC-NMR systems are installed world-wide. The success of LC-NMR is due to the enthusiastic work of people in both industry and academia, who have combined their skills and efforts to continuously improve the reliability of this coupled technique. Some of the early pioneers of LC-NMR are coauthors of this book and thus ensure a guarantee for competent contributions. 

The injection of the sample into the LC-NMR system can be carried out by an autosampler or a manual injection valve. The only difference which has to be considered is the fact that the amount of sample required for the NMR system is larger than that required for UV detection. Therefore, it is often necessary to inject sample volumes which exceed 100 jjlL In such cases, it is mandatory to dissolve the sample in the starting solvent phase in order to avoid additional LC gradients created by the sample solvent being injected. 

Changing solvents also has an effect on the homogeneity of the magnetic field. Again, the observed changes in LC-NMR systems are not as large as those found in normal sample changer automated devices ... 

The extension of an LC-NMR system to include mass spectrometry has been in application for several years [35,36]. By directly coupling a mass spectrometer to an LC-NMR system it is possible to obtain valuable mass spectral data. Configuring the system to have the sample reach the mass spectrometer before it reaches the NMR flow-cell enables the mass spectrometer to be employed as an experimental control device for analysing complex mixtures. Mass spectrometry is an ideal detector, provided that the molecules of interest are ionizable. It provides data rapidly and can thus yield valuable information on parent or daughter ion masses prior to initiating time-consuming NMR experiments. This synergy is not possible when the instruments are not directly coupled. 

The mass spectrometry portion of the analysis was carried out by coupling a Bruker Esquire ion-trap mass spectrometer to the LC-NMR system with a 20 1 splitter. The major portion of the flow was directed to the NMR system while the minor fraction went to the mass spectrometer. The system was plumbed such that the sample reached the mass spectrometer and the UV detector at the same time. In this configuration, it is possible to use the mass spectrometer as an intelligent detector, thus allowing stop-flow experiments to be initiated on the basis of observed molecular ions or daughter ion fragments. Data were acquired with electro-spray ionization (ESI) in the positive-ion mode. 

An alternative approach for the analysis of combinatorial libraries has been to remove the chromatographic column from the LC-NMR system to generate... 

Liquid Chromatography Nuclear Magnetic Resonance (LC/NMR) Systems... 

The Flow Control and Peak Sampling Unit for a LC/NMR System... 

The second alternative is to direct specific samples to the NMR that are of particular interest. The sample can then be trapped in the cell and data acquired from an adequate number of pulses to provide the required resolution. Subsequently, the sample can be expelled from the cell using solvent supplied directly from the chromatography pump. The third alternative is to direct the eluent from the column to a sample loop where it can be stored until the spectrometer is available to take data. If necessary, a number of solutes can be stored in different loops and they can be examined when convenient. When the data has been acquired from one sample, the solute stored in the next loop can then be displaced into the NMR cell. Samples that have been examined can either be displaced to waste or collected for further examination. A photograph of the Varian flow control device for the LC/NMR system is shown in figure 41. 

The Flow Control Device for the LC/NMR System Courtesy of Varian Inc. 

The author is thankful to Dr. Ray Bakhtiar (Drug Metabolism of MRL at Rahway) for the preparation of Fig. 1, his support, encouragement in writing this manuscript, to Dr. Byron H. Arison (Drug Metabolism of MRL at Rahway) for his interest, support, encouragement, and constructive discussions during the course of this work, and David Knapp and Uresh Parikh (Medicinal Chemistry of MRL at Rahway) for technical help connecting the MS detector on-line to the LC-NMR system. 

Unless otherwise known, proceed with a level of safety precaution at least as rigorous as for the parent species. Samples should be provided in the smallest vessel possible, preferably with a conical bottom, to reduce loss. Once the sample has been transferred to the NMR tube or syringe, inspect the tube for any residual solids. Solids decrease both the sensitivity and the resolution achievable and may produce a broad hump in the spectral baseline. Finally, before placing the sample tube into the magnet or injecting into an LC-NMR system, be sure to eliminate any air bubbles, since bubbles will make shimming more challenging and will cause adverse effects in the LC-NMR system. 

Figure 4.13. Pictures of hyphenated systems and systems for prep LC and bio-purification. Note that the magnet is not shown in the Bruker LC/NMR system.

A further sophistication is to couple the LC-NMR system with a mass spectrometer. This has two advantages. The first is that the sensitivity of the mass spectrometer is much greater than that of the NMR spectrometer, so that compounds occurring below the detection limit of NMR may be observed by mass spectrometry. The other advantage is that the combination of a ID NMR spectrum together with a mass spectrum is often enough for unambiguous... 

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

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