New Vistas For Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance spectroscopy now standard in chemistry laboratories, is also fast becoming part of the technologic repertoire of biochemists, geologists, materials scientists, and even food scientists and farmers. In the early years of this analytical tool, its applications were largely confined to the physical sciences. Now, however, several major advances have fine-tuned and, at the same time, expanded the capabilities of NMR, so that today the technology is proving valuable in a wider range of scientific disciplines, from the purely academic to the highly practical. For example, NMR spectroscopy can be used to reveal structural information on such diverse substances as volcanic glass, soil, drugs proteins, polymers, chocolate, cookies, and explosives. NMR Basics The technology, developed in 1946, was introduced commercially by Varian Associates of Palo Alto, Calif., in 1953. The technique is based on the fact that several common elements, including 1H, 13C, 31P 29 and Si, have a physical property called nuclear spin. This means that these nuclei can exist in different energy levels in a strong magnetic field. If such nuclei are subjected to a magnetic field and then pulsed with radio frequency energy, the absorption and re-emission of energy as they change levels can be measured as a “resonance” frequency, or chemical shift. Depending on the chemical environment of the nuclei—whether are near a carbon, oxygen, or chlorine atom, or, alternatively, near an organic group, such as a methyl, ethyl, or phenyl—one can observe different resonance energies. The actual NMR spectrum is a series of peaks representing the different chemical environments within a molecule. One can then integrate this information to reconstruct the bonding relationships of atoms in a molecule, and thus deduce the overall three-dimensional structure. NMR spectroscopy complements other tools. For example, the classic approach to determining the three-dimensional structure of a molecule is via X-ray diffraction, which reveals the distances and angles between every atom in a molecule. However, X-ray diffraction requires the sample to be a crystalline solid. When NMR is used in conjunction with X-ray diffraction, one can obtain information on the environment of each atom in a molecule in solution, as well as in a solid. The technique is especially valuable for substances that cannot be crystallized. One area in which NMR has been helpful is in studies on nepheline, a sodium-potassium-aluminum silicate found in volcanoes. X-ray diffraction, which gives only the long-range average structure of the sample, shows a certain degree of ong-range disorder in the mineral. NMR, on the other hand, reveals complete order when one considers short-range atomic interactions. Conversely, an NMR spectrum can highlight small perturbations in order that are not apparent in the more overall picture painted by X- ray diffraction. In some cases, the fact that X-ray diffraction requires a crystalline sample may be a drawback, because the solid state’ may not accurately represent the structure of the compound in another phase, such as in solution, which is the natural environment for many biochemicals. The pancreatic hormone giucagon and the metal-binding protein metallothionein, for example, display different structures when in solution versus in the solid state. In such cases, NMR spectroscopy can be a boon to researcher. NMR spectroscopy is of particular value in biologic and medical research, because—unlike many other analytical approaches—it is nondestructive. A sunflower seed found to have a favorable oil content via NMR can actually be planted following the analysis. Evolution of NMR Spectroscopy The seemingly sudden application of NMR spectroscopy to diverse fields reflects the cumulative benefits of several major enhancements of the original technique. In 1966, Fourier transform NMR was developed. This modification allowed the simultaneous detection of all resonance frequencies and greatly sped up analysis, improving the signal-to-noise ratio. The use of superconducting magnets improved resolution of peaks and sensitivity of the technique. Perhaps the most spectacular advances were the improvement in electronics and the programming of computers to regulate radio frequency (RF) pulses, enabling more information to be extracted from a spectrum. The ability to manipulate RF pulses also led to the development, in 1975, of two-dimensional NMR. This technique, which plots two spectra on an X and Y axis, provides much more detailed bonding information. NMR spectra can also be obtained at different temperatures to track structural changes accompanying phase changes. For example, a zeolite (a ceramic with pores of a particular size, often used as a catalyst), called ZSM-5, reveals 24 different peaks for silicon when run at room temperature, but only 12 peaks at a much higher temperature, indicating that a structural change has taken place. The most rapidly growing segment of the NMR spectroscopy market is in biotechnology and pharmaceuticals, with sales expected to increase by 12% to 15% over the next few years. One area in which NMR may have a great impact is in monitoring hollow fiber bioreactors used to culture high densities of mammalian cells. These delicate cells are generally more finicky than their microbial forerunners. It is imperative to be able to monitor the chemical environment throughout the system, because “microenvironments”—pockets of stagnation— can develop, leading to localized cell death. Previously, cell number in such a bioreactor was estimated by tracking depletion of glucose or oxygen. NMR measurements can more directly assess such parameters as cell density, oxygen tension, and concentrations of enzymes and metabolites. NMR of is valuable in the field of protein engineering. Of the thousands of proteins for which we know the amino acid sequence, X-ray crystallographic structures are available for only about 400. Protein engineers are trying to discover the rules by which a protein’s all-important three-dimensional conformation arises. Once these rules are fully recognized, researchers will be better able to design proteins with specific properties. Meanwhile, two-dimensional NMR spectroscopy is adding to the list of known protein structures by revealing how far apart two hydrogens (protons) are in the protein in solution. If the primary amino acid sequence is known, then NMR data can offer clues to higher-level structure. If two protons are very close to each other as indicated in the NMR spectrum, but at opposite ends of the amino acid chain, a globular conformation is suggested. Active sites are also revealed. For example, NMR spectroscopy indicates the tight association of the three amino acids that form the active site of alpha lytic protease. NMR spectroscopy helped solve the 1984 mystery of why tiles falling off the space shuttle by identifying reaction products of the components of a silicone rubber sealant used to bold on the tiles. The usually strong polymer degraded to mush because of an incompatible waterproofing agent, and the tiles slid off. Back on earth, NMR spectroscopy has been used to identify a form of silicon dioxide called stishovite, which is found in meteorites. NMR spectroscopy detected stishovite in clay deposits from strata representing the Cretaceous-Tertiary boundary, a time when many species became extinct—perhaps owing to a meteorite impact. X-ray diffraction could not detect the telltale compound. An interesting medical application of NMR spectroscopy is in cancer detection. Apparently, abnormal glycolipid patterns and the presence of an RNA-proteolipid species in the cell membranes of cancer cells cause a proton NMR spectrum of blood serum to be altered in a characteristic manner. In yet another area, NMR can detect the adulteration, year of production, and place of origin of wines. Products In addition to Varian, major manufacturers of NMR spectrometers include Bruker (Karlsruhe, W.Ger.) and JEOL (Tokyo). Modern devices emphasize both ease of operation and versatility of applications. A laboratory technician need do little more than insert a sample and push a few buttons. Bruker’s tabletop Minispec pc 100 series is fully automated, and its software enables one to perform a range of functions. Company literature includes spectra analyzing sizes of fat droplets in cosmetics, margarine and seeds; and water detection in marzipan cold pills, and plaster A worker in a cookie factory, for example, could use the Minispec to check water content at the rate of five cookies in two minutes. The conventional route to such cookie analysis—use of a drying oven—takes hours per cookie. Purchasing an NMR spectrometer is a major investment, and price parallels megahertz of the radio frequency. For example, JEOL’s CPF 270/54, a high-resolution Fourier transformation NMR spectrometer system that offers proton NMR at 270 MHz and NMR at 67.8 MHz, goes for $170,000. The GSX-400/54, with proton NMR at 400 MHz and 13C at 100 MHz, sells for $310,000; and the top-of-the-line GSX-500/52, with proton NMR at 500 MHz and at 125 MHz, sells for $530,00. Bruker’s Minispec pc 100 series ranges in price from approximately $30,000 to $50,000; whereas the company’s more powerful MSL series averages $300,000 to $1,000,000. Varian’s product line includes the VXR Superconducting FT NMR spectrometer, offering proton frequencies of 200 to 600 MHz; the EM-360L NMR, offering 60 MHz proton, 19F, and 31P capabilities; and the Gemini FT spectrometer, with 200 to 300 MHz proton and NMR. For price information, contact the company. The next generation of NMR spectrometers promises to provide even more versatility. Varian’s UNITY NMR, unveiled last May, and Bruker’s MSL series offer in the same instrument analysis of liquids, solids, and microimaging, and the ability to easily switch between them. Ricki Lewis teaches biology at the State University of New York, Albany.

New Vistas For Nuclear Magnetic Resonance Spectroscopy

NMR Basics

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