Introduction

Nicolau Beckmann    Biophysics Unit, Preclinical Research, Sandoz Pharma, CH-4002 Basel, Switzerland

The large number of publications and journals devoted to the technical improvements and applications of nuclear magnetic resonance (NMR) demonstrates the vitality of this technique. Since its discovery by Bloch and Purcell in 1946, NMR has been widely used by chemists and biochemists to investigate molecular structures. Novel approaches significantly broadened the aims of NMR, which has become a powerful spectroscopic and imaging tool applied in different areas, from materials science to chemistry, molecular biology, and medicine.

It is not an easy task to keep track of all recent developments, even if attention is focused on a particular nucleus, as is done for 13C in this book. The principles for these developments, which are compiled and well explained in the marvelous book by Ernst, Bodenhausen, and Wokaun (1987), have been known for a long time. However, some of the new approaches had to wait for developments in hardware and computer technology to become feasible in practice.

An example is provided by the magnetic field gradients, which are the heart of imaging and spectroscopy in vivo. The use of actively shielded gradients resulted in significant improvements in the quality of in vivo spectra and of images acquired in a subsecond scale. The recent introduction of these gradients into high-resolution NMR is helping to extend the application of NMR to molecules of increasing complexity and to reduce significantly the acquisition time (see Sections 2.4.1.5 and 6.5.3.3).

Because a technique can be better appreciated by following its progress, aspects that constitute the base of the present state of the art of 13C NMR are going to be presented from a historical point of view.

1.1 The Beginning

The first publications on 13C NMR spectroscopy were presented in 1957 by Holmes and Lauterbur1 in the same volume of the Journal of Chemical Physics. These pioneering papers contained 13C chemical shifts and J couplings to protons in some compounds. Slow-passage (or continuous-wave, cw) natural abundance 13C spectra were acquired at magnetic fields of 0.8 and 1.0 T generated by electromagnets. With sweep rates between 50 and 100 mG/s, signal-to-noise ratios ranging from 20 to 50 were reported.

During the 1960s NMR spectroscopists became aware of the proportional relationship between sensitivity and the square root of the measurement time. Field-frequency control was implemented, and signal averaging became possible. Slow-passage experiments were then substituted by averaging of rapid scans, thereby increasing the sensitivity of the measurements.

Nevertheless, the early applications of 13C NMR were hindered by the low sensitivity of the 13C signal compared to that of protons, the poor spectral resolution, and the necessity of working with highly soluble, low- molecular-weight materials. Therefore, 13C spectroscopy was initially restricted to a few groups, including those of D. M. Grant, P. C. Lauterbur, J. D. Roberts, and J. B. Stothers. The interested reader is referred to the books by Breitmaier and Voelter (1974), Levy and Nelson (1972), and Stothers (1972) for detailed accounts of the early applications of 13C NMR.

1.2 Essential Developments for 13C NMR

Two major developments contributed significantly to the establishment of 13C NMR as a routine technique: the Fourier transform (FT) approach and broadband proton decoupling. With the introduction of FT NMR as described and first implemented by Ernst and Anderson (1966), 13C spectra of much better quality could be obtained in a shorter period of time. On the other hand, the usefulness of double resonance experiments had been realized quite early, as is testified to by, for example, the fact that broadband proton decoupling was already available in 1965. The following facts were already known:

• The splittings of resonance lines caused by spin-spin coupling to a group of nuclei could be removed if a sufficiently strong transverse radiofrequency (rf) magnetic field were applied near the resonance frequency of these nuclei (Anderson and Freeman, 1962; Bloom and Shoolery, 1955). This early work revealed enhancements of 13C resonances upon proton decoupling that exceeded those expected from a simple collapse of the multiplets due to 13C-1H spin coupling. The phenomenon appeared to be similar to that discovered by Overhauser (1953) in electron spin resonance spectra and to that suggested, but not observed, by Bloom and Shoolery (1955) in their pioneering paper on heteronuclear double resonance.

• The information contained in the spin-spin couplings could be used for structural analysis. In this application, rather weak perturbations were necessary to systematically perturb the splittings but not to remove them. These so-called “spin-tickling” experiments allowed one to trace the network of coupled transitions (Freeman and Anderson, 1962).

Double resonance with an incoherent rf magnetic field, originally proposed by Ernst and Primas (1963), was primarily intended for the decoupling of strongly coupled systems. Ernst (1966) later showed that double resonance with random noise was of particular advantage if the couplings were relatively weak, even when the resonance frequencies of the nuclei causing the splittings covered a wide spectral range. Also off-resonance decoupling proved to be a very useful approach for spectral assignment in heteronuclear spin systems by scaling the spin-spin interactions (Reich et al., 1969).

1.3 13C NMR in the Solid State: Cross-Polarization and Magic-Angle Spinning

Acquisition of high-resolution spectra of low-sensitivity nuclei in solids was pioneered by the group of J. S. Waugh. By means of a method called proton-enhanced NMR conceived by Pines et al. (1973), signals of dilute nuclei were enhanced by repeatedly transferring polarization from a more abundant species to which they were coupled, for example, by Hartmann-Hahn cross-polarization (Hartmann and Hahn, 1962). High resolution was attained through decoupling of the abundant spins, usually protons.

Although with this approach spectra were dominated by chemical shifts, thereby permitting a degree of separation of different chemical groups, normally the anisotropies of the chemical shifts still led to relatively broad powder lineshapes, which tended to overlap in all but the simplest polymers. The supplementation of the proton-enhanced method with magic-angle spinning (MAS) (Andrew and Eades, 1953) removed the anisotropy broadening and resulted in a spectra of bulk polymers that resembled the highly resolved NMR spectra of liquids (Schaefer et al., 1975).

1.4 Heteronuclear Polarization Transfer

Cross-polarization as a means of enhancing the signals of low-sensitivity nuclei in solids was soon extended to measurements in the liquid state. Heteronuclear polarization transfer techniques have played a central role in 13C NMR. Basically three approaches have been explored:

1 enhancement of the initial 13C polarization

2 indirect detection of the 13C resonances by looking at proton signals

3 editing of spectra by selecting resonances belonging to specific subunits in a spin system, such as CH, CH2, and CH3 groups.

In solution, heteronuclear polarization transfer has been achieved efficiently by Overhauser polarization (Noggle and Schirmer, 1971), adiabatic J cross-polarization in the rotating frame (Chingas et al., 1980), and much more commonly by rf pulses. Sequences such as INEPT (Morris and Freeman, 1979) and DEPT (Doddrell et al., 1982) were developed based on the demonstration that coherence transfer could be attained by applying a 90° pulse to each of the two spin species coupled (Bodenhausen and Freeman, 1977; Maudsley et al., 1977).

This brief introduction should remind the reader about the unity of NMR despite the broadness of its applications. We owe this unity to the introduction of pulsed NMR, which allows the use of similar techniques for high-resolution NMR in solution, for solid-state NMR, and for in vivo NMR. The following chapters document only a small fraction of the possibilities provided by this fascinating technique.