Go out and grab a coffee when the NMR guy is refilling the liquid helium, unless you are willing to risk quick freezing of body parts or catching shrapnel from a surprise tank explosion. (image source: Dephologisticated)
Most of an organic chemist’s physical work appears to the naked eye as an interchangeable set of clear liquids and white powders (that is to say, if they are lucky enough in the lab not to produce brown sludge.) This is because atoms, even entire molecules, are too small to be seen through the lens of a microscope, so chemists must deduce their shape and structure indirectly. This is achieved with a variety of instrumentation and analytical techniques, most of which output data in the raw form of spectra, wavy lines that with a little experience can be used to paint a high-resolution image of the unseen. Because atoms and molecules, even gigantic ones such as a protein or enzyme, are smaller than a wavelength of light, they appear under even the most powerful electron microscopes as a nothing more than a fuzzy blob. Because it’s not part of our human perception, interpreting spectral data is a difficult challenge that chemists face every day starting when they are undergraduates. Operating the obscure equipment, and the hardware and software interfaces that this entails, is its own sort of challenge.
There are several types of spectroscopy, which is a broad concept that describes any kind of radiation of energy as it passes through a given material. Mass spectroscopy or Infrared spectroscopy is widely used in organic chemistry, but is mostly good for identifying mixtures. For instance, a winemaker might use one of these techniques to understand levels of eugenol in their chardonnay and therefore determine how long to toast their French oak barrels (eugenol is a compound from oak which gives the clove-like aroma and flavor to wine). Ultimately the Mass and IR techniques are too low in resolution to do what most organic chemists really need to do, which is to confirm if the thing you think you made in the lab is what it is supposed to be. Step in, NMR. Nuclear Magnetic Resonance, the work horse tool of the organic chemist, and therefore the only one I’ll get into much detail with here. It is said that if the NMR machine is shut down for some reason, then the organic chemist goes home for the day. (So in my world I guess that makes it a bit like a Starbucks.)
The ability to detect the resonance of magnetized nuclei and its implications as an imaging technique was one of the great post-war scientific breakthroughs. What is resonance? Think of how sound resonates from a crystal glass when you run a wet finger around its rim. A proton makes a similar effect when you hit it with a radiowave, and a scientist can “listen” for how it responds. Well, that is, some protons do. To be useful for NMR, an atom must have protons with a property called “spin,” which means their magnetic fields line up cleanly, sort of like a dipole magnet. These atoms (13C and 1H are the most commonly used by far) can get disrupted and relax back to their normal state without too much asymmetric rotation going on and distorting things, which makes them great for the technique of NMR.
An NMR machine is a complicated instrument. It consists generally of a large thermos bottle that contains a layer of liquid nitrogen surrounding another layer of liquid helium. This set-up acts as a large superconducting magnet. A sample of the chemical to be analyzed is inserted down into the middle of the thermos and the giant magnet causes the protons to all line up in the same direction. Then from this state, the molecule is disrupted with radio waves (or RFs, radiofrequency pulses) and the instrument records the time it takes for the protons to relax back into their normal state. This time to re-equilibriation (T1) is one of the most telling variables about a molecule’s composition and arrangement of atoms. For instance, if a proton is shielded because it is densely surrounded, it doesn’t move around much when blasted with an RF pulse. That sort of information helps chemists put a picture together of the molecule’s structure. The larger the NMR machine(typically expressed on a range of 300-600 megahtz), the greater the resolution is. And if you are a chemist working on a large molecule, say a protein with 500 hydrogen atoms in it, you need a lot of resolution to separate the signals.
An NMR machine consists of a sample, inserted via the probe, which sits within a magnetized chamber. A layer of liquid helium surrounded by a layer of liquid nitrogen turns the machine into one big superconducting magnet. Source: Rsc.org
The physical operation of the NMR process is fairly straightforward, with a few minor quibbles. Since these are big-ass magnets, one might need a ladder to insert the sample. Also, it is advised not to wear a pacemaker in the general vicinity. And, importantly, try and step out for coffee when the liquid helium guy comes to refill the machine. (Quick-freezing of body parts from escaped vapor, or catching some shrapnel from a highly pressurized tank explosion is no fun.) Most of the usability pain of an NMR is in the proper calibration and operation of the instrument via its companion software interface and the subsequent interpretation of the results – also done on a computer.
Chemistry labs are still places full of Rube Goldberg contraptions. The NMRs, particularly older ones, are no exception.
A 500 Mhz NMR spectrometer, like the one they have at Rutgers University, is large enough to require a ladder in order to insert a sample.
Try not to trip on the air hose attached to this monstrous NMR in the basement of Bagley Hall at The University of Washington.
Despite a gradually growing commitment to ease of use by the industry (primarily through automation, not necessarily design), NMR machines are a long way from “point and shoot.” Some of them can auto-lock and auto-acquire the sample. Some of them have auto-feed magazines, which can rotate in multiple samples overnight while the chemist steps out to the nearby brew pub. Some of them have web-based clients for remote analysis directly from the machine. But ultimately, there is still a lot of old-timey, goldbergian aspects to the electronics and mechanics involved. Step on the air line that is draped across the laboratory floor from the nearby air compressor, slightly choking the air flow but maybe not enough to notice, and the finicky machine will start giving odd readings with very little feedback as to what went wrong. The attached PCs run homegrown software provided by the manufacturer and there is little standardization across the industry in terms of file formats, navigational idioms, or iconography.
“They have the worst interfaces” says Dr. David Flanagan, a former Umass polymer scientist and now Editor in Chief of the journal Advanced Functional Materials. “They still had green screen, command-line interfaces when I went to grad school. And this was well after Windows 95 had been released, so the general public was used to a windows based graphic interface by then. I don’t think it’s changed much since.” According to Flanagan, the NMR software culture borrows its style cues from early silicon valley, back in the GNU era, when the place was still dominated by bearded, slightly crazed, anti-social types. NMR is more Woz than Jobs, sort of a UNIX nerd service at the heart of the larger nerd-dom of organic chemistry. “It’s all very retro, very black arts,” he told me.
In this excerpt from a user guide for Topspin (from major NMR maker Bruker), one gets a taste of the sheer number of steps and arbitrariness of how the features are named, grouped, and sequenced. If you want to adjust the number of scans, simple - just type "eda" into the command line or click on the tab called AcquPars. Got that?
The single most onerous part of getting a good reading from an NMR machine is in controlling the smaller magnetized elements that are wrapped around the sample. These are used to fine tune the way the sample is calibrated, a process called shimming. It’s the equivalent of focusing the lens of a camera in order to get a clear image – but an almost infinitely more abstract concept that can be the most difficult aspect of mastering these machines. Originally, the shimming process involved a cabinet with a bunch of knobs, cryptically labeled, that the operator tweaks while staring at the feedback coming back from an oscilloscope. Long symmetrical waveforms on the scope mean the machine is picking up the resonance clearly, sort of like tuning a guitar with a chromatic tuner. Flanagan reminisced about the university NMRs he had encountered in his student days. “The controls are not labeled clearly enough for a noob grad student. It’s a little intimidating at first. A few knobs invariably had ‘don’t touch’ signs on them.”
To shim the control magnets, an operator sits there and tweaks the knobs in trial-and-error fashion until something like a trumpet appears on the oscilloscope. If you don’t get your trumpets, you’ll get a pretty messy looking spectrum and it will be a problem if you are trying to get your results published in a top journal. The NMR guy in the lab might write down the best shims of the day nearby to help people out. The knobs may have been replaced with sliders on a computer interface in a modern spectrometer, but the challenge is more or less the same. The operator is asked to adjust controls with cryptic labels like X, Y, Z, Z1, & Z2, all of which are variables in mathematical formulas used to understand the homogeneity of magnetic fields. This requires some substantial mathematical modeling skills in order to understand, and using them is not intuitive for less experienced operators. Spectrometers with auto-lock and auto-acquire features are starting to make this process a little less onerous. The NMR technicians, or facility managers, do a lot of day-to-day coaching to help chemists figure out what they have. “Having good relationships with these guys is invaluable to a grad student’s success,” says Flanagan. “The real usability issues start to come when things go wrong. The machines don’t provide a lot of feedback or guidance in troubleshooting or recovering from mistakes.”
The spectrometer blasts a sample with (typically 16) radiofrequency RF pulses which display on the oscilloscope as "resonances."
The computer attached to the instrument then transforms the resonances into a useful spectrum using a mathematical process called a Fourier Transform.
A properly calibrated, or "shimmed," machine will give off nice little trumpet shapes.
The shimming control unit on a Bruker Avance DMX-500 spectrometer, conveniently located behind all the dangling cables. Fortunately, the shims are controlled via the computer on this model.
Hard key shim adjustments (left) and the software versions (right) of the same controls. While the machines are more automated these days, understanding how to properly shim the smaller, controlling magnets around the sample is one of the hardest things to master in NMR usage.
Assuming you can get things set up and you’ve fired your RF pulses and you’ve got nice little trumpets coming up on your oscilloscope and the NMR machine hasn’t vibrated itself off of its pedestal, the battle is still nowhere near over. It is time to analyze the results, which is another pain point in the chemist’s workflow. The traces of each proton’s activity after receiving the RF pulse show up in the spectrum itself as a series of peaks, whose areas are proportional to the number of protons they represent. The bottom axis of an NMR spectrum (usually expressed as a dimensionless unit called “ppm”) is an abstraction of the difference in chemical shift position between atoms in the molecule, which differs based on how well a proton can absorb electromagnetic radiation in its position within the compound, as well as the resolution of the machine (expressed in MHz). What is interesting is that the chemical shifts of protons in organic compounds fall into predictable ranges on the ppm range based on their type, which is what makes NMR so powerful as an analytic technique. In fact, it is possible for a chemist to interpret an NMR spectrum by a simple visual inspection once they master the basic theory. (Note: this isn’t true of IR and Mass spectroscopy)
A typical NMR spectrum. This one is from the compound strychnine. The numbered peaks from the spectrum itself (bottom part of image) can be used to draw the chemical structure (top part of image) which is used to identify the compound.
NMR spectra are an efficient way of identifying a compound and drawing a chemical structure. Since types of elements tend to appear in predictable ranges based on their chemical shift properties, it's possible to visually inspect the spectra and piece together the compound's structure. This chart shows where certain classes of elements tend to show up on an NMR spectrum, with alkanes and alcohols in the upper right region and aromatics and aldehydes in the lower left. A diagram like this helps to interpret an actual compound's spectrum like the one that appears in the preceeding figure. Source: "NMR Spectroscopy," MSU faculty website.
Upon closer inspection of a "peak," a chemist will find that they are split into particular patterns of height and spacing called "multiplets." These patterns are caused by the magnetic fields of the adjacent protons (2 adjacent protons will split a peak into three peaks with a heights ratio of 1 to 2 to 1, or a "triplet.") This phenomenon gives the chemist a great deal of information about the substance's exact chemical structure.
Interpreting multiplets of spectral peaks is an inexact science, as can be seen around 2.9 on this proton NMR spectrum. The peaks themselves are sort of a blob, but an organic chemist would infer that the short height of the lines and location of this particular cluster means that this signal is coming from a highly shielded proton (e.g. this proton is surrounded by a lot of other hydrogen protons). A chemist therefore can use several different clues to deduce the likely chemical structure of the substance.
Spectral analysis on a computer typically requires a lot of zooming. The macro display (left) needed to see the entire spectrum is too low in resolution to do proper peak analysis needed for identifying the compound. So the user mouses over an area, blows it up (left), but loses the wider context.
NMR spectra are analyzed in 1D or 2D, but typically not 3D. This creates the problem of figuring out whether certain shapes are overlapping peaks (such as those occuring around 7.5-7.8), or just strong couplings. Source: P2C2E
There is such a thing as an analytical chemist, one who works with organic chemists in the same way that a radiologist might work with surgeons – partnering with them as a diagnostic specialist and expert on the equipment. Big pharmaceutical companies have these people around. The analytical chemist is particularly adept with the equipment , and can roll up their sleeves and work with the algorithms and pulse sequence formulas that are behind the scenes of how spectroscopy works. In drug discovery, where the organic chemists are cranking out multitudes of new molecules on a daily basis chasing marketplace gold, there is considerable need for help with the identification process of novel compounds. But most organic chemists, especially in academia, have little choice but to use the machines themselves. It’s the only way a chemist can makes sense of what they’ve done in the lab. So therein we introduce the heart of the usability problem, a mainstream technology right in the middle of the experimental workflow turns out to be difficult to both learn and master. The machine needs to be operated correctly and the results need to be interpreted correctly. It turns out neither of these are very straightforward.
We NMR spectroscopists have to accept that for a majority of the scientists in todays chemistry/biology research, NMR is a black box that’s neverless – and “unfortunately”- absolutely necessary. They like to use software that seems to generate listings and plots without requiring knowledge by the operator. We still try to teach our own students about the innards of NMR-experiments. But the reality is that black-box attitude and the trend for automation are increasing all the time. – From Italian NMR Spectroscopist and Blogger, “Old Swan.”
The data itself is often transported to a chemist’s personal laptop computer for analysis, typically with a thumb drive. When I asked several academic chemists why they don’t keep the NMR PCs on the network for easier file access, most were concerned less by security (it’s not top secret what most university labs are working on) than they were by the intense desire to keep the expensive, and fickle machines operating. If the NMR machine was on the network, most felt, it would probably eventually stop working. Some spyware or virus would screw things up. And since the machines are already perceived as being finicky and expensive, this is a risk most just aren’t willing to take. But this introduces some real pain in the workflow. Files need to be transferred in physical space and then analyzed in separate desktop software. In other words, these machines are a long way from point, shoot, then see instantly what you have.
Getting spectral data into a manuscript for journal publication can require many manual steps. The mnova spectral analysis software lets you drag and drop data, such as a peak list, directly into a document from the analyzer itself. The left side of the screen shows a spectrum (with the multiplet analyzer feature toggled on), the right hand side is a printable document view where a user can drag and drop a variety of information about the spectrum as well as other text and graphical objects, such as a structure drawing from Chem Draw.
One way chemists can learn to interpret NMR is by doing exercises. The "Spectral Game" is one such example. Analyze the peaks using a java spectral analyzer, then guess which compound it is from the choices below.
Other Reading on Solid State UX:
The User Experience of Organic Chemistry Part 1 – A Chemical Language
Other Reading from Around the Web:
Topspin (NMR software) User Guide from Brown University’s Department of Chemistry
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