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Routine NMR Shifts Upfield Nuclear magnetic resonance (NMR) spectroscopy for liquid samples got its start during the Second World War, and the simpler techniques are still used for structural determination in undergraduate organic chemistry laboratories. However, in the past 5-10 years, NMR has evolved to encompass some of the most powerful analytical applications available, particularly in medicine and biochemistry. In recent years, NMR has benefited from increased computer power, routine use of Fourier transform (FT) techniques, and the introduction of superconducting magnets at high magneticfieldstrength to instrument. Table 1 presents FT-NMR allow high-resolution structural determispectrometers for liquidsfromthe three nation of proteins in their native folded largest U.S. manufacturers. Otsuka Elecstate. These features have brought more tronics (formerly Chemagnetics) and Hitasophisticated methods into routine use, chi also make high-resolution spectromeparticularly in the pharmaceutical industers, and a number of companies make try, where characterization of peptides and continuous-wave lower performance inproteins plays an increasingly important struments for simpler and more routine role in drug development. experiments. More information on instruments offered by each vendor is availWe asked Gary Martin of Burroughs able byfillingout the reader service card Wellcome (Research Triangle Park, NC) for his comments on trends in NMR of liq- or by sending an e-mail message to
[email protected] with a subject line uid samples and advice on purchasing an
As demand grows for complex liquid applications, high-performance NMR becomes an industry standard
containing one of the reflector keywords listed at the bottom of the table. Experimental finesse
In addition to variations on 2D experiments, which now number in the hundreds, 3D and 4D experiments are now available through increases in computer power, the development of FT methods, spin labeling, and in particular gradient NMR, without which these higher order techniques would be prohibitively time consuming. These higher order techniques require probes with three channels (also called "triple resonance" probes). For 4D experiments, the deuterium lock channel of a triple resonance probe is time shared to provide a fourth channel in effect. Protein characterization in particular is pushing the development of experiments beyond 2D. "When you go from 20 amino acids—that is, small peptides—up to 70 amino acids, 2D methods are less useful," Martin explains. 'You have to sort the information into three or four time domains to be able to interpret it." Typical 3D ex-
Analytical Chemistry, September 1, 1995 5 5 9 A
Product
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Table 1 . Summary of representative products
I
Avance Bruker Instruments 19 Fortune Drive, Manning Park Billerica, MA 01821 508-667-9580 $190,000-$3,000,000 2 standard; up to 8 optional
Eclipse JEOL USA 11 Dearborn Rd. Peabody, MA 01960 508-535-5900 $150,000-$1,000,000 2 standard; up to 4 optional
Single, double, triple, and quadruple resonance; direct and inverse; with and without 1- and 3-axis pulsed field gradients; computer-switchable 4-nucleus probe, e.g., for Ή , ,9F, 3,P, ,3C
Multifrequency 1H standard; options: tunable for 31P to 15 N with Ή decoupling, inverse, shielded 1 H, ,9 F with Ή decoupling, low-frequency (,5N-203Rh), z-gradient, microprobe, fixed-frequency macroprobe
Tube diameter
2.5 mm, 5 mm, 8 mm, and 10 mm; 20 mm for wide bore only
Probe coil geometry
Vertical, magic angle
5 mm for standard, tunable, inverse, shielded, and 19 F/'H probes; 10 mm for tunable, low-frequency, and z-gradient; 3-mm microprobe; 20-mm fixed-frequency macroprobe Vertical
nD Both 1-axis and 3-axis + rf qradients Yes
nD 1- axis z-gradient; pulsed field optional Optional
200, 250, 300, 400, 500, 600, 750, 800 52-mm standard; 89-mm wide bore and 150-mm superwide bore optional 2-15 17-28 shims
270, 400, 500 54-mm standard or 89-mm wide bore for 270- and 400MHz; 51 mm for 500 MHz 270 MHz, 2.7; 400 MHz, 8.0; 500 MHz, 10.0 16 matrix shims for 270 and 400 MHz; 20 matrix shims for 500 MHz Silicon Graphics workstation standard with 64 MB RAM; UNIX platform with X-windows/Motif/GL; Delta FT-NMR for 1D-4D experiments and up to 8 dimensions of data standard; high-pass filter, COSY symmetry filter, MEM, BLIP, LP, and Hubert transform algorithms; high-level macro programming language
Product series Company
Price range No. of rf channels Probes Types
Experiment capabilities 3D and 4D Gradient NMR Shaped pulses Magnet Field strength (MHz) Bore diameter Drift rate (Hz/h) Shims Data handling and control
Waveform generator Non-FT methods Special features
Options
Reader Service Number
Silicon Graphics workstations standard with 32 MB RAM, 1 GB hard disk, and 2 GB DAT tape; IRIX OS with X-11 windows/Motif; XWIN-NMR with simple and advanced user interfaces and drop-down menus; automated shimming, locking, acquisition, phasing, peak selection,and plotting; optional soft ware for maximum entropy, spectral simulation, mul tidimensional assiqnment, and analysis 64 kW waveform memories standard for all rf chan nels; 256 kW waveform memories optional Linear prediction and Hubert standard; maximum entropy optional On-the-fly DSP digital filtering; digital lock for full quadrature lock detection; software controlled digital signal path router; digital quadrature detection using DSP chip; real-time acquisition parameter adjust ment; graphical pulse program display; online Hypertext manual; remote acquisition from net worked PC or workstation; gradient shimming; magic anqle qradients LC-NMR; SFC-NMR; chemically induced dynamic nuclear polarization; pulsed-gradient diffusion; rf gradients; Q-switched probes; ultrahigh- and ultralow-temperature probes; 120-, 60-, and 6-tube sample changers; solids accessories; microimaging 401 ac NMR622
NA = Not applicable ΙΝΑ = Information not available at press time
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Fixed shaped pulses optional MEM, Hubert 16-bit, 200-kHz spectral width ADC standard; modem service for applications and customer support; digital filtering; spreadsheet analysis; online help; real-time spectrometer control, queue management, and online diagnostics; spectral deconvolution; off-line processing
3-resonance broadband system; autotune probe; 3-, 16-, or 64-place sample changer; high-frequency broadband CP-MAS solids accessory; microimaging accessory
402 ac NMR623
Gemini 2000 Varian NMR Instruments 3120 Hansen Way, Mail Stop D-300 Palo Alto, CA 94304-1030 415-493-4000 $125,000-$350,000
Inova Varian NMR Instruments 3120 Hansen Way, Mail Stop D-300 Palo Alto, CA 94304-1030
415-493-4000 $230,000 and up Up to 8
3, 4, 6, 8, 10, and up to 16 mm
Single and multiresonance direct and inverse with or without pulsed-field gradients; 4-nucleus computer-switchable with or without pulsed-field gradients; high-resolution 40-μί; triple-resonance direct and inverse with or without pulsed-field gradients; 8- and 10-mm large-volume inverse 3, 4,5, 8, 10, and up to 40 mm
Sample positions for vertical magnet bore range from vertical to magic angle
Sample positions for vertical magnet bore range from vertical to horizontal including magic angle
Up to 3D 1 -axis pulsed field Yes
nD 1-axis or multi-axis pulsed-field Yes
200-400 54-89 mm
85-1000 51-400 mm
2-10 Single shim configuration
2-15 3 shim configurations for up to 40 shims
Many Sun workstation options and configura tions; UNIX with X-windows; VNMR software for UNIX standard, Silicon Graphics and IBM versions optional; customizable walk-up and advanced user interfaces with drop-down menus; macro recorder; optional software includes FRED for data processing and Felix for Windows
Many Sun workstation options and configura tions; UNIX with X-windows; VNMR for UNIX standard, Silicon Graphics and IBM versions optional; customizable walk-up and advanced user interfaces with drop-down menus; macro recorder; optional software includes FRED for data processing, Felix for Windows, EPI for fast imaging, back projection, and solids analysis
Fixed shaped pulses
Standard
Linear prediction
Linear prediction
Single and multiresonance direct and inverse with or without pulsed-field gradients; 4-nucleus computer-switchable with or without pulsed-field gradients; high-resolution 40-μΙ.
Autocalibration; fully automated locking, shimming, and data acquisition and proces sing; in-line and post-acquisition digital filter ing; linear amplifiers; on-the-fly parameter adjustment; online Hypertext-linked manuals; remote acquisition from networked PC or workstation
Fully automated locking, shimming, and data acquisition and processing; in-line and postacquisition digital filtering; DSP with digital quad rature detection; gradient shimming; linear ampli fiers; exchangeable rf and gradient modules; onthe-fly parameter adjustment; online Hypertextlinked manuals; remote acquisition from net worked PC or workstation Solids accessories; LC-NMR; microimaging; H/C or broadband configuration; 9-, 50-, pulsed field gradients; variety of rf configurations; and100-place autosamplers; pulsed-field gradients; heated sample rack; high-resolution 9-, 50-, and 100-place autosamplers; highresolution MAS probes; VT modules; deuterium MAS probes; variable-temperature modules decoupling homonuclear decoupling (standard for broadband) 404 403 ac NMR624 ac NMR625
periments include homonuclear NMR techniques, which don't require any spin labeling, and heteronuclear 3D NMR, which allows up to three different labels (13C, lr'N, and 2H) on the protein. Proteins up to 250 amino acids long or 25-30 kDa have been analyzed by 3D methods at high resolution using ~ 1 μηιοί of sample. Gradient NMR is a fairly recent adapta tion of the methods underlying magneticresonance imaging. Usually, says Martin, the aim is to make the magnetic field as homogeneous as possible, but this tech nique, which requires a gradient probe, purposely disrupts the homogeneity of the field throughout the sample volume. Gra dient spectroscopy can be used to select coherence pathways in 2D experiments or to provide solvent suppression, says Martin. Gradient NMR works by eradicating i, noise and reducing the number of tran sients needed to get acceptable S/N. A spectrum that takes 32 or 60 transients in long-range heteronuclear experiments un der a homogeneous field may only re quire 2 transients in gradient NMR. How ever, Martin notes, "As sample size de creases, the advantage of using gradients is lost, because you get more signal back without the gradient. If you have to do more than 32 transients, you don't see the advantage." Gradient NMR is a "nice op tion" for small-molecule work, he adds, but for proteins and other large molecules that generate a lot of data, it is a necessity because it cuts the spectral acquisition time significantly. Gradient probes can be used for nongradient techniques as well, offering sensitivity equivalent to that of a conventional probe when the gradient fea ture is not in use. Probes The probe, which fits inside the magnet bore and holds the sample tube, contains the rf coil and electronics, both controlled from the system computer, for perform ing NMR experiments. In the old format for NMR, homonuclear or dual probes for 5-mm-diameter tubes were standard. They were made of materials that made it necessary to restabilize the magnet, which could take from an hour to over night, whenever they were interchanged in the old iron-core magnets. Today's probes are optimized for a variety of ex periments, sample tube diameters and vol umes, and applications; the newer mag nets are stable enough and probe materi als are good enough that changing probes takes only a few minutes. Martin advises
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checking the probe performance by look ing at the linewidth at half height at the height of 13C satellites (0.55% and 0.11%) when comparing probes. The smaller the numbers, the better the probe perfor mance characteristics. Sample tube diameter is an important parameter for probe design because NMR is somewhat limited in analyte sensitivity compared with other analytical methods. In a general sense, the closer the rf coil fits around the sample, the better the sen sitivity of the instrument will be. When the sample is expensive or in short sup ply, as is often the case in the pharmaceuti cal industry, microprobes that accommo date 2.5- and 3-mm-diameter tubes can re duce the volume needed for analysis without jeopardizing sensitivity. The 3-mm microprobes may be config ured as l;iC-optimized microdual probes, as conventional microdual probes that are somewhat less sensitive for 13C but per form inverse experiments much the way 5-mm inverse probes do, or as microdual inverse probes that are optimized for inverse detection experiments. To give an example of the differences in perfor mance, Martin says that on a 500-MHz spectrometer in his lab, the 18C-optimized microprobe might give reasonably good 13 C spectra for 0.25 pmol of a sample with a mass of 200-500 Da in an overnight run but would not yield C-H correlation (HMQC) data. By contrast, the microin verse probe on the same spectrometer might allow good overnight spectral acqui sition for only 0.05 pmol of sample in an HMQC experiment. Large-diameter probes, which accom modate 8- and 10-mm-diameter sample tubes, also have their uses. Applications for these probes include experiments in which large samples are needed to com pensate for low gyromagnetic ratio nu clides such as 15N or for gradient-inverse experiments on high-MW analytes (e.g., proteins) in which dilute samples are needed to prevent aggregation. Most probes for NMR of liquids are now avail able with or without gradient NMR capa bility, says Martin. Varian has also introduced a "nanoprobe" with a 40-μί, 4-mm-diameter cell for liquid samples that resembles a solids probe and sits at the "magic angle" of 54.7° to the axis of the magnetic field. The probe is designed to remove susceptibil ity broadening effects and achieve high resolution in 'H and 13C magic angle spinning (MAS) analysis of heteroge neous materials. Applications include
NMR of analytes coated onto polymer beads in a suspension. Short DNA or pep tide synthesis products and organic monolayers formed on rigid substrates are two examples of this quasi-liquid applica tion. Many research-grade instruments also accomomdate probes for solids and microimaging as options. NMR has also been hyphenated with liquid chromatography by feeding the eluent transfer line from the column through the sample space of an adapted probe and taking NMR spectra "on the fly." LC-NMR requires careful timing be tween the column pump and the spec trometer, says Martin; he explains that the separation may have to withstand a halt in the flow for as long as overnight when spectra are being taken, but the method can be valuable if LC band broadening isn't critical. A strong attraction One of the biggest changes in NMR has been the commercial introduction of su perconducting magnets. Increases in mag-
Performance benefits don't increase linearly with field strength, especially when balanced against cost. netic field strength increase spectral dis persion, which improves the inherent res olution, as well as the Boltzmann excess (number of observable nuclei), thereby en hancing sensitivity and S/N. With a given NMR probe, a spectrometer with a 500MHz magnet may give typical S/N on one transient of 1300:1; a 600-MHz instru ment might yield S/N of 2000:1 for the same sample and experiment. Conven tional electromagnets for NMR are still used for some lower performance instru ments, says Martin, "but the instruments operating above 100 MHz now typically have superconducting magnets." Nominal field strengths for the super conducting magnets currently on the mar ket are 100,200,250, 270,300, 360, 400, 500, 600, and 750 MHz (stated as proton frequency; 100-MHz field strength is equivalent to 2.35 T). Oxford Instruments, a major manufacturer of superconduct
562 A Analytical Chemistry, September 1, 1995
ing magnets, has also been working on 900-MHz and 1-GHz magnets for the Na tional High Magnetic Field Laboratory (see the June 1,1994, issue ai Analytical Chemistry for more on this lab) and other large institutions such as Pacific North west Laboratory and the Francis Bitter Laboratory at MIT. Of course, says Martin, the higher the magnetic field strength, the higher the price of the instrument. F"or this reason, field strength is one of the factors that frequently differentiates "routine" from "research grade" systems, although the types of experiments that can be per formed on a routine instrument are in creasingly complex and the definition of "routine" use varies with the laboratory. In general, says Martin, "For walk-up rou tine usage or for monitoring synthesis of small compounds such as AZT [the AIDS drug], most of the instruments these days have 200-300-MHz magnets, al though occasionally 400-MHz instruments are also used for routine work." Because of the cost as well as the performance en hancement, he says, "Most of the instru ments at 400 MHz and up are considered research grade." However, the benefit of increased field strength doesn't increase linearly, espe cially when balanced against the cost. "The biggest jump in sensitivity for the price appears to be between 300 MHz and 400 MHz," he says. "From 400 MHz to 500 MHz and up, the price difference between successive magnets is more of a factor." Choosing the appropriate field strength for your applications depends in part on the resolution and sensitivity you need. Petrochemists may be in good shape with lower field strength magnets because their sam ples tend to have high concentrations of organic analytes. Pharmaceutical and bio technology laboratories that must perform difficult techniques such as isolating com plex natural products at low yield on a reg ular basis may well consider a 500-MHz in strument "routine" for their needs. In addition to varying in field strength, magnets may have narrow ( ~ 50 mm), wide (~ 90 mm), or superwide (>110 mm) bore diameters. The general advantage of wide-bore magnets is that they accommo date bigger probes and usually bigger samples than the standard narrow-bore magnets. However, in a wide- or superwide-bore magnet, as field strength in creases it becomes progressively more difficult to achieve very high field homoge neity throughout the active region of the magnet, even though the magnet itself is
Just Released! generally quite stable. The 500-MHz mag nets are available with superwide bores, but 600-MHz magnets are available with wide bores or narrow bores only. Above 600 MHz, only narrow-bore magnets are commercially available, although some experimental wide-bore magnets are un der construction at higher field strength. Ideally, Martin says, the best magnet is the most stable one for your applications; ideal drift rates for submicromole samples may be < 4 Hz/h; typical specifications for larger samples are generally < 10 Hz/h. Shims, which are generally used to make the magnetic field as homogeneous as possible, are another consideration. In superconducting magnets, the super conducting shims are set and fixed during installation; the room-temperature shims sit in the active region of the magnet bore. Room-temperature shims offer up to 40 different shim current options. Martin says that when working with large-diameter probes, higher order shim current control (with more than 25 options) is necessary to control lineshape and resolution. Medi um- and high-order shims must be used for large-volume aqueous protein samples to optimize the H 2 0 lineshape suffi ciently to collect good spectra. In addition, shims can be either axial (spinning) or radial (nonspinning). Axial shims for Zj-z,, are commonly used; higher order axial shims become important for large-diame ter probes. Finally, the location of the spectrome ter should be considered. High field strength magnets require careful siting to ensure that the vertical clearance for the spectrometer allows access to the top of the magnet (for replenishing cryogenic fluids in the Dewar) and that other facili ties are far enough away to be outside the 5-G line and comply with safety regula tions. Floors should be able to hold a 700800-lb magnet, and floor vibrations need to be minimized with a vibration table, es pecially for inverse-detection experi ments. Spectral acquisition and deconvolution "NMR electronics have become very sta ble," Martin says. "They enable you to per form most techniques such as the in verse-detection experiments easily." Com puter control is also advanced. Because advanced NMR techniques produce such a volume of data, Martin recommends a computer system with 1-2 GB or more of data storage for research-grade instru ments. High-density 8-mm tape drives
(the size of 8-mm videotapes) that hold 2.5-5 GB are available. Although continuous-wave spectrome ters are still widely available for simple ap plications, FT-NMR is now standard for higher-performance instruments. Auto mated phasing for routine proton and I3C spectra is now commonly offered through walk-up commands at the spectrometer controller, although Martin notes that cur rent autophasing routines don't work well for ID nuclear Overhauser effect ex periments that have both positive and neg ative phases or for many of the sophisti cated phase-sensitive 2D experiments. Shaped-pulse techniques are another recent innovation in complex experiment design, Martin says, and they often carry odd acronyms such as "IBURP" and worse. They are used to select or deselect specific nuclei for perturbation by cut ting out signals from the rest of the mole cule or to perform 2D or higher order ex periments in a single dimension. These methods, like FT-ion cyclotron resonance methods in MS, manipulate the rf pulse
Non-FT deconvolution methods are useful for increasing spectral resolution in some cases. in a complicated way to produce excitation profiles that may be, for example, per fectly square. The more complicated ones require the addition of a waveform gen erator and computer-controlled attenua tors to the computer hardware. Not all advanced NMR techniques use FT deconvolution, however. Alternate methods, available through software op tions, include Bayesian analysis, maxi mum entropy (MaxEnt), and linear pre diction. Each has its limitations and strengths, and may be useful to increase spectral resolution in certain cases. As they become better known and more widely used, these less common methods may increase in popularity along with the growing capabilities of commercial NMR instruments. In the meantime, both "routine" and "research" NMR experi ments for liquid samples are likely to keep advancing. Deborah Noble Analytical
Exploring QSAR This new two-volume set provides a thorough introduction to quantitative structure-activity relationships (QSAR). In Volume 1: Fundamentals and Applications in Chemistry and Biology the authors show how structureactivity relationships should be considered as a whole in quantitative terms, starting w i t h physical organic chemistry and including all types of biochemical and biological processes. They give a unified view w i t h extensive cross-referencing and show the interrelatedness of SAR from the ionization of acetic acid to the action of hallucinogens in humans. Throughout the book, QSAR in biological processes are related to those in physical organic chemistry. Volume 2: Hydrophobic, Electronic, and Steric Constants contains comprehen sive tables of physicochemical parameters (substituent constants and octanol-water log Ρ values) that are necessary for quantitative structureactivity relationships and qualitative SAR. These values were collected over 25 years by t w o of the most re n o w n e d researchers in the field. Volume 7 by Corwin Hansch and Albert Leo Volume 2 by Albert Leo, Corwin Hansch, and David Hoekman ACS Professional Reference Book 880 pages (1995) Clothbound ISBN 0-8412-2993-7 $99.95 ORDER F R O M American Chemical Society Distribution Office Dept 74 1155 Sixteenth Street. NW Washington, DC 20036 Or CALL TOLL FREE 1 800-227-5558 (in Washington, DC 872-4363) and use your credit card! FAX: 202 872 6067. ACS Publications Catalog now available on internet: gopher acsinfo.acs.org
ACS III PUBLICATIONS Essential Resources for the Chemical Sciences
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