product review
Liquid NMR probes: Oh so many choices Vendors offer a wide variety of probes with applications that include synthetic chemistry, protein structure determination, and metabolomics. Rajendrani Mukhopadhyay
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n an NMR instrument, the probe does all the “talking” with the samples. A superconducting magnet generates a strong, homogeneous magnetic field around the sample. A console produces the rf signal for sample interrogation and amplifies the signals generated from the sample. But it’s the probe that acts as the rf link between the sample and the instrument electronics as it sends and receives pulses to and from the sample. “The probe is arguably the most important component in the NMR spectrometer. It determines the magnitude and quality of the signals one can extract from the sample,” says Paolo Rossi of Rutgers University. “It’s also the most subject to damage, part degradation, and the most likely to break. It is perhaps the most delicate part of the spectrometer.” “A typical lifetime of a probe is 3–5 years,” agrees Arnold Schwartz, the former director of NMR R&D at Varian, Inc. He says the magnet, because it has no moving parts, typically lasts 10–20 years. The system electronics typically last 7–10 years. “But people buy new probes all the time,” says Schwartz. “Probes are the fastest-changing parts because people are always tweaking the engineering in them.” Probes are available in a variety of flavors. They can come in a range of sizes, be sensitive to different atomic nuclei, and have chilled parts to improve the S/N. “Most NMR systems are very general-purpose,” explains Schwartz. “The probe is the part of the system that is application-specific.” Bruker BioSpin; JEOL, Inc.; and Varian dominate the market of NMR probes, and Protasis Corp. supplies specialized capillary-scale NMR (CapNMR) probes (box on p 7962). Because the types of probes cover the whole gamut of size ranges, nuclei detection, and application choices, the probes listed in Tables 1, 2, and 3 only hint at the variety of probes that are available from each vendor. These tables are not meant to be comprehensive. Only probes for liquid samples are covered here, because probes for solid-state NMR are a different beast (last reviewed by Analytical Chemistry in 2002, 74, 45 A–47 A). © 2007 American Chemical Societ y
The job of a probe
The reason NMR spectroscopy can be applied to so many types of samples is that the nuclei of several atoms act as tiny magnets. When tiny magnets such as these are placed inside a larger magnet, their south poles line up with the north pole of the larger magnet and absorb some energy. They then tip over or “wobble” around their axes, emitting a particular signal. This behavior is called resonance, and the signals can be physically mapped to show which atoms are present in the molecule and where they are located relative to one another. The wobbling by the nuclei is instigated and is read by the probe. It holds the sample at the center of the magnetic field and bombards the sample with rf energy. When the nuclei wobble and emit rf signals, the probe receives those very weak signals and passes them along to the console for amplification. Experts say that the probe can be compared to a radio antenna. “View the NMR machine as a broadcast station, transmitting a radio frequency signal that’s picked up by the spins, and [the spins], in turn, have a response that’s picked up by the receiver. For all practical purposes, the probe fulfills the function of an antenna system that transmits and receives radio frequency signals,” explains Werner Maas of Bruker BioSpin. N o v e m b e r 1 , 2 0 0 7 / A n a ly t i c a l C h e m i s t r y
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to be tunable just to a single frequency; it has to be an Company Bruker BioSpin (978-667-9580, www.bruker-biospin.com) antenna that has multiple frequencies simultaneously Product 5 mm TCI CryoProbe 1.7 mm Micro-CryoProbe 5 mm BBFO Probe available to it. Cost (U.S.D.) >$120,000 >$120,000 $45,000–65,000 To make a probe ameApplications For structure determination of Suitable for quantity-limited Used for structure characnable to picking up signals biological macromolecules by samples, such as natural prod- terization of a broad range of from more than one type of using a family of double- and ucts, low-abundance proteins, organic molecules, including triple-resonance experiments and small molecules fluorine-containing compounds nucleus, several rf circuits are engineered into the part. LiqAmount of 200 µg–10 mg 100 ng–1 mg 1–50 mg sample uid probes, in general, have two rf coils. The innermost Description Provides the highest sensitivity Offers a 10× increase in mass Designed for high sensitivity of probe for 1H and 13 C frequencies, ensensitivity over conventional of nuclei in the range 31P to coil, called the observe coil, is abling access to advanced NMR probes; its extreme sensitivity 15 N, in addition to 1H and 19 F for always the most sensitive for techniques; on appropriately jump makes it an ideal tool for mid-field spectrometers; any of a given nucleus. The second, 2 configured spectrometers, H any NMR analysis with limited the nuclei can be automatically outermost coil is usually less can be decoupled, permitting sample amounts selected and optimally tuned; decoupling of up to three nuclei key features are the ability to sensitive than the inner coil in a single pulse sequence; observe 19 F with 1H decoupling and is used to decouple the 1 19 available with automatic tuning and to perform 2D H/ F specrf signals emitted from other and flow capabilities troscopy of superior quality nuclei in the sample. 1Contact the vendor for the full product line. Users have to determine whether the nucleus in their sample that requires the Table 2. Selected liquid NMR probes from JEOL, Inc.1 greatest sensitivity is highCompany JEOL, Inc. (978-536-2310, www.jeolusa.com) frequency (1H or 19F) or lowProduct Cryogenic HCN Gradient Probe Multinuclear Gradient Probe Indirect Detection Gradient frequency (15N, 13C, or 31P). System with Automatic Tuning Probe with Automatic Tuning They then have to define Cost (U.S.D.) $165,000 $25,000–35,000, depending on $ 30,000–40,000, depending on which nuclei will be observed field strength field strength and which ones will have Applications Ideal for biological NMR strucSuitable for organic, organo- Used for biological and organic their signals decoupled. ture research metallic, and general-purpose research To explain the roles of NMR observe and decouple nuclei, Amount of Micrograms to milligrams Milligrams to grams Micrograms to grams Jonathan Lee of Boston sample University says, “I grew up Description For biological and organic 5 mm multinuclear NMR probe 5 mm probe for organic and biplaying football, so I always of probe applications; has the highest for direct and indirect detec- ological NMR; autotuning prouse this analogy. When they sensitivity for 1H and indirect tion of organic samples; auvides completely hands-free detection; requires an additotuning provides completely probe tuning and matching, teach you to tackle, they say, tional cryogenic system hands-free probe tuning and which is ideal for student use ‘Watch the guy’s waistline matching, which is ideal for or remote off-site operation; because no matter what he student use or remote off-site operates in temperature range does with his hands and operation; operates in temfrom –100 °C to +150 °C perature range from −100 °C knees, his waistline is where to +150 °C his body is going.’ You lock 1Contact the vendor for the full product line. your eyes on that. Now if there’s someone physically The probe is critical for determining the quality of an attached to the guy, when he moves, then that person will experiment. “If you have a more sensitive antenna, you get move with him even if you can’t see him.” The same idea apa more sensitive signal,” says Doug Meinhart of JEOL. “If plies to a probe—a probe will lock onto a nucleus and then the antenna can’t tune to a particular frequency, you can’t do track the other nuclei attached to it. NMR at that frequency.” To come up with a piece of equipment that’s sensitive to rf But the probe is more sophisticated than a radio antenna. signals of several nuclei requires some exquisite engineering. “As you know from your radio, you tune to any radio sta“The challenge to the engineers is to build a probe that is not tion, and then you’re locked into one station at a time. You only multiply tuned but also has no susceptibility to magnetic can’t tune into multiple stations at a time. But sometimes we perturbation, has temperature control, and so on,” states Lee. want to look at proton signals, but we also want to transmit “When you’re all done, you’re talking about a piece of custo the carbon or nitrogen atoms and correlate the proton to tom-engineered mechanical equipment that has maybe a few carbon and nitrogen,” says Maas. The job of the probe isn’t hundred or thousand dollars’ worth of parts. And once you
Table 1. Selected liquid NMR probes from Bruker BioSpin.1
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for sensitivity. The closer the coils are to the sample, Company Varian, Inc. (800-356-4437, www.varianinc.com) the larger the filling fac1H{ 13 C/ 15 N} Triple Resonance Product MicroSample 1H{13 C/ 15 N} Triple AutoX 1H–19 F/ 15 N– 31P Dual tor, and the better the Resonance PFG Cold Probe Broadband PFG Probe PFG Cold Probe S/N. So, probes come in a Cost (U.S.D.) Contact vendor for quote Contact vendor for quote Contact vendor for quote variety of bore diameters, ranging from 1 to 20 mm, Applications Identification and structural Identification and structural Structural characterization of characterization of quantitycharacterization of chemical biological samples to accommodate different limited samples samples volumes. For cases in which Amount of 30–225 µL 225–600 µL 225–600 µL sample volumes are severely sample limited, Schwartz explains, Description Cryogenically cooled, tripleProbe of choice for automated Cryogenically cooled, triplesome vendors offer flowof probe resonance NMR probe optisingle- or double-resonance resonance NMR probe optithrough probes in which mized for maximum sensitivity studies of chemical samples; mized for maximum sensitivity the sample is siphoned into with small samples; designed optimized for very flexible with biological samples; dea capillary instead of a test for double- and triple-resohigh-frequency (1H–19 F) and signed for double- and triplenance 1H experiments that low-frequency (15 N– 31P) ob resonance 1H experiments tube and its nuclei are obinvolve simultaneous or single serve and decouple experithat involve simultaneous or served by a microcoil. 13 15 13 15 irradiation at C and N frements single irradiation at C and N The volume a user has quencies frequencies; can be converted into flow-cell configuration to analyze depends on the with an optional accessory sample. NMR spectroscopy 1Contact the vendor for the full product line. has two types of sensitivity limits—mass and concentraassemble the probes, it’s somewhere between $30,000 [and] tion. If a sample dissolves in only very small amounts—say, $100,000 apiece because they are all handmade, almost like a large protein that dissolves in only micromolar concentrajewelry.” tions—10 µL of the sample will contain very few molecules Probe design has undergone a dramatic transition. “When and therefore very few spins. The result will be a lousy signal. I started doing NMR spectroscopy 25 years ago, we built “It’s then advantageous to go to a larger volume, maybe 500 all of our probes ourselves, and every user knew something µL in a typical 5 mm probe, so we’ll have more spins in the about probes,” says Lee. Nowadays, the parts are sleek, comdetection circuit and get a larger signal. That is concentration mercially available black boxes. Lee adds, “Users don’t know sensitivity,” explains Maas. what’s even in the boxes or the magnet. They’re just staring Mass sensitivity comes into play when the molecules or at the computer, saying, ‘Does it tell me what I have or not?’” compounds readily dissolve in fairly high concentrations, but The probes have become automated and sophisticated the sample quantity is limited. This is a problem often seen enough that most users show up at an NMR facility, drop in natural product chemistry, where only a few micrograms their sample into a probe, click on a computer screen, and of a rare flower or deep-sea sponge extract are available. Maas have the data emailed to them. “The analytical NMR jock says, “We dissolve it, but rather than dilute it into a 500 µL part is becoming less visible to the user,” states Lee. Before, volume to fit into a big probe, we go to a dedicated NMR “users had to talk to that person and describe what they probe that has a very small coil—for instance, a 1 mm probe wanted [for their experiment]. Now users set the machine up that accommodates ~5 µL of sample.” as a wind-it-up-and-forget-about-it machine.” Lee’s analytical Users then have to make the decision about which nuclei NMR jock makes such automation possible with probes that they want to analyze. Indirect detection probes offer the are robust and applicable to a variety of samples. greatest sensitivity for high-frequency nuclei, such as 1H and 19F, whereas direct detection probes offer the greatest sensiSelection of probe tivity for low-frequency nuclei. The coil geometry differs for The decision about the type of probe to use for a particueach type of probe—for example, a low-rf coil is placed as lar experiment has to be based on sample quantity, sample close to the sample as possible in a direct detection probe to solubility, and the types of nuclei to be detected. NMR observe low-frequency nuclei. spectroscopy, despite being more information-rich than MS, Users also can opt to use indirect detection probes to get suffers from being a relatively insensitive technique. Investiga- information about low-frequency nuclei. “We sometimes want tors have to go to great lengths to boost S/N. The proper to get information about the chemical shift or position of a choice of a probe is key to maximizing the sensitivity of an carbon atom that’s attached to a hydrogen atom, but the hyexperiment. drogen atom is much easier to see,” says Lee. The hydrogen In most probes for liquids, the rf coils sit parallel, or axial, atom gets irradiated with an rf pulse and transfers its energy to the magnet. The sample sits in the bore of the innerto the carbon atom. The carbon atom wobbles for a short most coil. Experts say that the proximity of the coils to the period of time and transfers its energy back to the hydrogen sample—described as the “filling factor”—is very important atom, which then is detected by the probe.
Table 3. Selected liquid NMR probes from Varian, Inc.1
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Protasis: CapNMR flow probes Protasis manufactures CapNMR flow probes and automation systems that are based on well plates or microvials for sample introduction. The integrated systems are compatible with the NMR spectrometer platforms made by the three major vendors (Varian, Bruker BioSpin, and JEOL) at field strengths ranging from 300 to 800 MHz. A variety of detection modes are possible, including proton, carbon, and indirect nitrogen detection. CapNMR probes have the highest mass sensitivity and the lowest cost of operation available. Principal applications range from synthesis confirmation to metabolomics/metabolite identification, biofluid analysis, and investigation of natural products. A CapNMR probe consists of a capillary-scale (~1 mm) flow cell and comes in either 5 or 10 µL detection volumes. A recently introduced dual-flow cell probe incorporates two independent flow cells into a single mechanical package, providing twice the performance and sample throughput with only a single magnet and spectrometer. Samples can be recollected or discarded after the measurement is completed. Protasis’s One-Minute NMR software is web-based and independent of the operating system. Contact the company at www.protasis.com or 508-481-4163.
“We get the hydrogen sensitivity, but now the hydrogen signal also carries information about the less sensitive nuclei that it was attached to. This is used most commonly for hydrogen-to-carbon or hydrogen-to-nitrogen indirect detection,” explains Lee. “Many of the interesting nuclei, like phosphorus, carbon, and nitrogen atoms, are so insensitive that you could sit there for a week before you get a simple 1D spectrum. But in 10–20 seconds, I can get a hydrogen spectrum that carries information about the nitrogen.” A notable advance in probes has been the cryogenically cooled ones that boost the sensitivity limits of NMR spectroscopy for quantity-limited or low-solubility samples. The sample stays at room temperature, but the electronics are cooled to 20 K, cutting down the buzz and hum of the electronics’ thermal energy. But, as Lee points out, there’s a hitch. Because of the insulation that goes into the cryogenic probes, the sample can’t get as close to the rf coils as in the conventional probes. “The filling factor in a cryogenic probe is much worse than in a conventional room-temperature probe,” he says. “There’s a trade-off, and that’s the key to probe building. There are always trade-offs. Do I buy a half-a-million-dollar machine, put all my samples in a generic probe, and run them for an hour each? Or do I buy a half-amillion-dollar machine and spend another $120,000 to have this finicky cryogenic probe, but now I can run my samples in 10 minutes instead of an hour?” Other experts echo the same sentiment. Most users, they say, will buy a general-purpose probe and will make it work for their sample type. “If you have a very small amount of sample and you need to measure it just once, you would likely 7962
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do it in the 5 mm regular probe that you have and just let it run a bit longer until you get the required S/N. However, if your prevalent samples are natural products and you’re always in the microgram range then, at some point, you will consider buying a dedicated probe for that work,” says Maas. The available probes at an NMR facility may not be optimized for your particular sample, but Maas explains, “in terms of the time you spend on the instrument when you have to exchange probes, fine-tune, and optimize them, sometimes you say, ‘You know what? I’ll just let it run and come back tomorrow morning.’” Meinhart confirms this: “For the most part, customers are looking for general-purpose probes. Eighty percent of customers are going to be very satisfied with a tunable 5 mm probe that covers a large range of nuclei. That covers the largest group—your routine organic and organometallic [types] of users, with magnets in the 300–600 MHz range. All the manufacturers have a standard probe which covers that. Then you augment with specialty probes that depend on the application. Customers might want the ultimate sensitivity or to do a particular type of experiment. That’s the other 20% in terms of [sales] volume.” Most users want to work around general-purpose probes in a plug-and-play fashion because adjusting probes with samples in them before launching into the experiment is the painstaking and tedious part of NMR spectroscopy. Lee contrasts the setup of an NMR machine with that of a UV–vis spectrometer. “You bring a cuvette of sample to a UV–vis spectrometer, put the cuvette in, turn the machine on, measure the spectrum, and walk away,” he says. “When you bring a sample to a traditional NMR machine, I have to adjust the homogeneity, tune the frequencies, calibrate the pulses, and do a whole bunch of things before we can ask, ‘What does the sample look like?’ Well, you’ve left the room by that point because you’re bored.”
Probes of the future
Even if most users are satisfied with general-purpose probes, vendors and experts say the push now is to design more-sensitive probes. “Everybody’s looking for the highest possible sensitivity, and the probe is key in that equation because it’s the first step for the detection of the signal,” explains Meinhart. “Sensitivity is related to throughput. If you can make the probe more sensitive, you can collect the same data more quickly.” Schwartz says that one direction probes are being pushed in is accommodating a smaller sample size. “Because NMR is a sensitivity-limited technique, it becomes difficult to study very small sample volumes. Going to smaller probes is one mechanism for trying to do those studies. I think they’ve all had limited success so far.” Another direction, he points out, is parallel analyses of multiple samples with a single probe. If the probe can be made sensitive enough to handle small volumes, then potentially several small-volume samples can be simultaneously accommodated in the bore of the probe. A lot of the rethinking of probe design has been triggered
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by the emergence of novel applications. Maas provides an example. “Currently, one of the hot issues is metabolomics,” he says. “You need to run a fairly large number of samples in order to have some representative studies. You can then think about putting these samples in a cooled microwell plate, picking them up with a liquid handler, and flowing them through the magnet.” Pharmaceutical studies also require the analysis of hundreds of samples. Maas describes one type of automated high-throughput system in which a robot fills tubes from 96-well plates and a sample changer takes the tubes in and out of the probe. In another system, the samples are siphoned off the plates and are introduced into a flow-through probe. In either case, the system can work independently to get only NMR data, or it can be combined with an LC system to perform LC/NMR or LC/NMR/MS analyses. Maas says, “It’s getting way beyond the simple NMR probe and system. You’ll see in the coming years that NMR is starting to be more of a black box and driven by highly automated applications.” Although solid-state NMR probes aren’t discussed in this article, experts and vendors say that tremendous excitement exists in this area. Some applications include materials analysis and proteomics—where experts hope the structures of stubbornly insoluble membrane proteins can be solved.
A middle area even exists between liquid and solid-state NMR spectroscopy that affects the development of probes. High-resolution magic-angle spinning (often known as hrMAS) NMR spectroscopy can be applied to the analysis of tissues from medical biopsies. Tissues are considered to have properties of both solids and liquids. Maas explains, “It’s an area where we apply that spinning technology from solid-state probes but also the radio frequency technology from liquid probes. It’s a hybrid of the two, and the probes are different.” But, because NMR spectroscopy lends itself so well to so many types of applications, vendors are at the mercy of research funding fads—one of many challenges they must face. At any one time, some applications increase in popularity while others shrink. The companies saw a rise in sales in 2000 and 2001 when proteomics got a major boost in funding. But they saw a drop in 2005 when the proteomics funding fell away and a spate of pharmaceutical mergers occurred. However, vendors say now they see an upswing with other applications becoming more popular. With the demand for new probe designs and systems, Maas says, “it’s an extremely exciting time.” Rajendrani Mukhopadhyay is a senior associate editor of Analytical Chemistry.
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