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Opportunities in Chemistry: Today and Tomorrow (1987)

Chapter: V. Instrumentation in Chemistry

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Suggested Citation:"V. Instrumentation in Chemistry." National Research Council. 1987. Opportunities in Chemistry: Today and Tomorrow. Washington, DC: The National Academies Press. doi: 10.17226/1884.
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Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

CHAPTER V Instrumentation in Chemistry All scientific knowledge is rooted in our abilities to observe and measure the world around us. Thus, science benefits enor- mously when more sensitive measuring techniques come on the scene. This is the situation in chemistry today. The discussions to follow will identify a number of powerful instrumental methods that are now the everyday tools of re- search chemists. We will focus on the capabilities of today's instruments and on how much they have changed over the last decade or two. ~67

Al Hi, A Laser Flashlight A laser flashlight? Sounds like something out of Buck Rogers or Star Trek! What would that be? Well, to bring this down to Earth, let's first think about what a laser is and then how to make it into a flashlight. Lasers are very special light sources. They put out pencil-sha~p beams of pure color end so intense they can be used to cut patterns in steel. Also, they can be focused so sharply they are better than a surgeon's knife in mending the retina in your eye. Finally, they can give light pulses as short as a millionth of a millionth of a second! That's what's called a picosecond. With shutter speeds that fast, chemists can now `'photograph'' the fastest chemical changes known. And how do lasers work? It all begins with a whole bunch of atoms or molecules all ready to emit light of exactly the same color. Atoms and molecules usually absorb light, not emit it, so somehow we've not to DUmD them UD in energy SO Hat ~ ,&. ~ &. ~ ~ they're more inclined to emit than absorb. This is called a "population inversion.' Once we've got a population inversion, there are some tricky things to do with mirrors to make a laser out of it but we don't have to go into everything. So how do we pump up the molecules to get that population inversion? One good way to do it is to use electrical energy, like we do In a fluorescent light. That's what you might use to light up a dark closet to look for your missing sneaker. And it works fine as long as you have a long enough extension cord. But think of looking for your jacl: ~ the back of your car when you have a flat out on a dark highway. That's where a flashlight comes In handy. In a flashlight, the energy comes Tom a chemical reaction. That's what the batteries are all about. Could we use a chemical reaction to pump a laser? If so, it would be a "chemical laser,'' our laser flashlight. But that would require a chemical reaction that produces a population inversion. Trying to find out whether there are such reactions led chemists to the discovery of the first chemical laser. O[course, it's hardly news that chemical reactions can emit light. Candles do it all the fume. And thirds about a firefly—he (or she) Carl do it without an extension cord. These emissions show that a reaction follows special pathways, and when energy Is released, these preferred pathways might be a super ~ ~ way to get a population inversion. Mar W '599~ ._ . _ 'it '~_- ~ ._ 168 I, The suspense, though, was that chemical lasers were not discovered by looking at bit flames or by copying the firefly. Chemical lasers were discovered to operate best In the infiared, where the human Or eye is blind. This spectral region is where molecular vibrations ` ~ can cause molecules to absorb or emit light. From these lasers cat . . . ` we reamed that quite a few reactions prefer reaction path- .~ Sways that put most of the available energy into vibrational `~' motions of the final products. Why this happens still isn't "I clear, but we're working on it. In the meantime, we have a whole bunch of fine chemical lasers. They can be very efficient, which has made at least one chemical laser a candidate to be the match to lift the nuclear fire of nuclear fusion. If that worked, chemical lasers would help us get '~clean" nuclear energy for the rest of tune. Chemical lasers can also be very intense, as shown with the fluonne-hydrogen flame laser. What good is that? Well? that gets us back to Buck Rogers, Star Trek. and Star Wars. If you want a laser out in space, you'll either need a chemical laser or a mighty long extension cord. So wraths next? We're still after that firefly.

V-A. INSTRUMENTATION FOR STUDY OF CHEMICAL REACTIONS V-A. Instrumentation for Study of Chemical Reactions Section IV-A indicated how the chemist's use of the most modern instrumenta- tion is making it possible to investigate even the fastest chemical processes in intimate detail. We are witnessing a quantum jump ahead in our understanding of the factors that control the rates of chemical reactions. Among the tools respon- sible for this rapid advance are lasers, computers, molecular beams, synchrotrons, and, on the horizon, free-electron lasers. We will consider each in turn. LASERS Chemical lasers have been discussed in "A Laser Flashlight" on page 168. For a laser to work, it needs a "population inversion" in which there are more molecules that have enough energy to emit light than there are molecules ready to absorb light. To maintain such an inversion, energy must be injected somehow. Some energy-releasing reactions do this (resulting in chemical lasers), but the energy can be injected in other ways. The simplest way is through irradiation with a conventional light source. However, electrical energy input is probably the most convenient way to establish a population inversion. The ap- paratus need not be much dif- ferent from a fluorescent light fixture. Whatever the manner of en- ergy input (the "pumping" method), the special qualities of laser light arise from "stimu- lated emission," which can be regarded as the inverse of light absorption. A photon of light of the exact energy needed to excite a molecule from one energy level to another, higher level can stimulate emission of a second photon from a molecule already in the higher level. The second photon that is thus produced turns out to be perfectly in-phase ("coherent") with the electromagnetic wave of the first photon that started it all. This coherence gives lasers their distinctive character. It accounts, for example, for the pencil- sharpness that permitted us to reflect a laser `'searchlight" beam offa mirror placed on the Moon by the Apollo astronauts. The remaining feature of a laser is a set of accurately focussed mirrors that cause any stimulated emission to go back and forth many times through the population inversion. These mirrors are called an optical cavity; they permit and cause the buildup of the special qualities of laser light. Lasers bring to mind a brilliant beam of light cutting through a sheet of steed or shining deep into space. But to a scientist, the beauty of the laser lies in its ability to deliver light of extremely high intensity, extremely high power, extremely high spectral purity, and/or extremely short duration. For a given experiment, laser design is dictated by the one of these features of greatest value to the experiment at hand, and usually at some sacrifice in the others. Some of this trade-off is PUMP I NO LIGHT hv~ coo o Font ax' 0 0 0 o E2 LIGHT ABSORPTION CAN ESTABLISH A POPULATION INVERSION 169

170 MIRROR F LUORESCENT M I RROR Dl SCHARGE Z=~=~ ~ .. ~ AM I TG.NT ~ _ _ HIGH —VO LTAGE ~t ~ ~ A LASER NEF.D NOT BE; COMPLICATED INSTRUMENTATION IN CHEMISTRY imposed by the Uncertainty Principle. This fundamental premise of quantum mechanics states that the duration of a light pulse is related to and limits the spectral purity. Thus, the Uncertainty Princi- ple tells us that if a pulse is as short as one picosecond (10-~2 seconds), there will be an uncertainty in the frequency (the color) at least as large as 5 cm-. With this much frequency spread, most information is lost about molecular rotations of gaseous molecules. On the other hand, if a line width of 0.005 cm~~ is needed to detect individual rotational states, then the molecule of interest must be examined by a light pulse at least as long as one nanosecond (10-9 seconds). This limitation depaves us of time information about species or events with shorter lifetimes than the nanosecond probe. PULSE DURATION ~ > SPECTRAL PURITY ,- ONE MICROSECOND = .000 OO1 SECOND ONE NANOSECOND S .000 OOO OO 1 SECOND ONE PICOSECOND = .000 OOO OOO OO 1 SECOND ONE FEMTOSECOND - .000 OOO OOO OOO OO 1 SECOND - .ooooo5 cm l .005 cm l < - 5 cm 5OOO cm l PULSE DURATION LIMITS FREQUEN CY ACCURACY AND V ICE VERSA Developments in the Last Decade There were three crucially important developments in laser technology that took place during the 1970s and they are having a great impact on chemistry. First, several types of tunable lasers were developed and became commercially available. A "tunable laser" is one whose color (wavelength) can be selected according to need. The wider the range of the spectrum that lasers can work over, the more valuable they are as a research tool. The most important of these was the dye laser, which gave continuous color tuning throughout the visible region of the spectrum and a bit beyond into the near-infrared and near-ultraviolet. Dyes are chemical compounds whose intense color causes them to absorb light efficiently so that they can then emit coherent laser light. Second, the invention of efficient ultraviolet lasers gave scientists access to the photochem~cally important ultraviolet region at wavelengths shorter than 300 mm. These include "excimer lasers'' that are based upon light emitted from molecules formed from electronically excited reactants. An example is the krypton fluonde laser. Krypton is an inert gas that does not form bonds in its ground state. After one of its valence electrons is excited, however, the

V-A. INSTRUMENTATIO1V FOR STUDY OF CHEMICAL REACTIONS resulting krypton atom has the chemistry of rubidium. Thus, the molecule formed between Kr and F has the bond strength and stability of RbF. This is a desirable factor in building up concentration to reach a popu- lation inversion, so that it can emit laser light. The third de- velopment was the discovery of methods of laser operation that gave short-duration light puIses~ne picosecond or Kr electron Krypton ' coll~S'°~ (ground state) 5s O 4p Is 3d ~ ~ 3) ~ fin -17~1 Kr. Krypton (excited state) Hi, ~0 }fir (gas) + F2 (gas) ~ (KrF)~(gas) + F(gas) Pb~gas) ~ F2(gas) - > RbF(gas) + F(gas3 Rb Rubidium (ground state) 0 AFTER EXCITATION KRYPTON REACTS LIKE RUBlI)IUM THAT MAKES EXCIMER LASERS POSSIBLE less. In 1970, the tunable dye laser did not exist except as a laboratory curiosity. In the early 1980s, almost every chemistry research laboratory had more than one tunable laser source. Tunable lasers can now be conveniently operated over the wavelength range from 4 microns in the infrared (40,000 A) to 1,600 A in the ultraviolet beyond the wavelength at which air becomes opaque (i.e., into the range called the "vacuum ultraviolets. Already in the state-of-the-art stage are lasers that extend the wavelength range to beyond 20 microns (200,000 A) in the infrared and to less than 1,000 A in the vacuum ultraviolet. Chemical Applications Table V-A-l lists many chemical applications of lasers. It is important to note that most of the more powerful lasers are not continuously tunable; they have only particular output wavelengths. They are most useful in the study of solid matenals, which will usually absorb a wide range of wavelengths of light. For most chemical applications, tunable sources are critically important, and these lasers are often TABLE V-A-1 Some Research Areas Utilizing the Laser Area Research Application Solar energy research, photosynthesis Uranium, plutonium isotope purification Trace element analysis, environmental monitoring Probing flames, explosions Monitoring industrial processes Cell discrimination and separation Photochemistry within biological cells Gas-phase deactivations, Chemical reactions Laser Used - Excimer, dye Excimer, dye, TEA CO2 Continuous ion, color center Photochemistry Isotope separation Atomic absorption, fluorescence Combustion diagnostics Atmospheric gas analysis Biological cell sorting Cell bleaching Microsecond kinetics (1-100) x 10-6 see Nanosecond kinetics 10-6 to 10-9 see Picosecond kinetics 10-9 to 10-'2 see Subpicosecond kinetics <10-'2 see Excited state lifetimes, very fast reactions Fast electronic state deactivation, coherence decay in liquids Vibrational deactivation in solids and liquids Solid-state, dye Semiconductor diode Ion laser Dye laser Flashlamp dye, TEA CO2, chemical Solid-state, excimer Ion, solid-state Ion, solid-state

172 INSTRUMENTATION IN CHEMISTRY excited with another powerful, single-frequency laser. Having the best suited laser system is essential for work at many of today's most exciting chemical research frontiers. COMPUTERS The use of computers by chemists has paralleled the tremendous computer development of the last three decades. The size of this growth is reflected in the number of industrial installations of the largest IBM computers over this same time penod. In the mid-1950s, there were 20 or 30 such machines (IBM 701s). By the mid-1960s, the much more powerful 7094 and 360 systems numbered about 350. Today, there are perhaps 1,700 indus- tnal installations of IBM 3033s. This numerical growth has been accompanied by a phe- nomenal increase in computer power. The extent to which chemis- try has benefited from this growth can be seen by compar- ing two landmark calculations. For polyatomic molecules, the first theoretical calculations based upon the Schroedinger Wave Equation without any simplifying assumptions (an ab initio calculation) appeared in the 1960s. Of special impor- tance was the study of rotation around the carbon-carbon bond of ethane, C2H6. As the hydrogen atoms at one end rotate past the hydrogen atom at the other end, the energy rises to a maximum. To learn the height of this internal rotation barrier, theoretical cal- TABLE V-A-2 Relative Computing Speeds of Computer Levels 2000 ,_ 1 500 <5: I: cat z ~ Too lo: - o ~ 500 :~ MA I NFRAME COMPUTERS _ _ t ~ 1 1 1 1 1 1 1 1 t I I I 1 960 1 970 1 980 YEAR INDUSTRIAL USE OF LARGE COMPUTERS Computing Superminicomputers Mainframes Supercomputers Example DEC VAX 11/780 IBM 3033 CRAY IS (1) 10-15 80-120 culations ("self-consistent field" method) were based upon a ba- Relative SiS set of 16 functions. Speed This can be contrasted with a recent and similar study of deca- methy! ferrocene, ECs(CH31512Fe. This calculation used a basis set of 501 functions. Since such stud- ies require computing effort proportional to the fourth power of the number of basis functions, the decamethy! ferrocene computation involves (501/1614 or one million times more computation than the ethane problem! \

V-A. INSTRUMENTATION FOR STUDY OF CHEMICAL REACTIONS Superminicomputers This level of computer has become a workhorse in chemistry. Instruments like the DEC VAX 11/780 are comparable to the worId's largest mainframe computers available in the late 1960s. They have revolutionized computing in chemistry because of their substantial capacity, high speed, and lowered cost, which is now in the range $300,000 to $600,000. The last 20 years have also seen three important develop- ment phases for the use of com- puters in chemical expenments. In the first, computerization phase, advances in both hard- ware and software greatly im- proved our ability to accumulate measurements (data acquisi- tion). Then an automation phase increased the possibilities for experiment control through continuous morutonug of cntical parameters. F~nady, a "knowledge engineer- ing" phase ushered in an era in which computers perform high-level tasks to interpret collected information. An excellent example is the Fourier Transform aIgonthm which permits us to record spectral data over a long time period, thereby to achieve high speck resolution. Because this allows detection of quite weak signals, this aIgonthm is now routinely used to record ~3C NMR sign~s and to transform infrared inte~erograms. Because of the success of these instruments, the Fourier Transform aigonthm is now being incorporated into ad sorts of equipment: electrochemical, microwave, ion cyclotron resonance, dielectnc, and solid-state NMR instrumentation. \ ASH C_H me, ETHANE 1 6 FUNCTIONS CH3` C ~ _CH~ - ~ -C CH3 C - C C—CH3 HA ~ , HI ~ CH3—Cx —C ACHE ARC . '-Can CH , CH3 3 (50 1)4 _ 1 0 DECAMETHYL FERROCENE 50 I FUNCTIONS A MILLION-FOLD ADVANCE IN TWO DECADES Mainframe and Supercomputers Some needs for computation in chemistry can be met only with the greater capacity and capability of the largest scientific computers (Cray/M and X-MP or CYBER 205), coupled with specialized resources such as software libranes and graphics systems. This is most notably true for electronic structure studies for many atom molecules beginning with the complete Schroedinger Equation and without approximations tab initio calculations). Another area that will benefit from supercomputers is computational biochem- istry. Most dynamical simulation procedures applicable to biological molecules require calculation of the simultaneous motions of many atoms. A conventional LOO-picosecond molecular dynamics simulation of a small protein in water would require about 100 hours on a DEC VAX Il/780 or 10 hours on an IBM 3033. Calculations of the rate constant for a simple activated process require a sequence of dynamical simulations to determine the free energy barner, and additional simulations to determine the nonequilibrium contributions; the times can now 173

174 INSTRUMENTATION IN CHEMISTRY reach 1,000 hours on a DEC VAX 11/780. More complicated processes or longer simulations become impossible without the much higher speeds of supercomputers. MOLECULAR BEAMS The advances of vacuum technology over the last three decades have made it possible to reduce the pressure in an experimental apparatus to a point at which molecular collisions become quite improbable (e.g., at pressures below 10-9 torr). Under these conditions, molecules that enter the vacuum chamber stream to the opposite chamber wall without deflection. Such a situation is called a "molecular beam." This provides a special opportunity to study chemical reactions. The most obvious application is to cause two such molecular beams to intersect. When a molecular collision does occur, it is almost always in this intersection zone. If the collision causes a chemical reaction, the product fragments leave the reaction zone with energies and directions that provide information about the reactive collision. By measuring the spatial distribution and fragment energies, we can learn intimate details about single-collision chemistry. Capabilities A typical, crossed molecular beam apparatus can contain as many as eight differentially pumped regions provided by various high-speed and ultrahigh-vacuum pumping equipment. It may be necessary to maintain a pressure differential from one atmosphere of pressure behind the nozzle of the molecular beam source to 10-i ' torr at the innermost ionization chamber of the detector. What is glibly ceded the "detector" is likely to be an extremely sensitive mass spectrometer with which to measure the velocity and angular distributions of products. By replacing one of the beams by a high-power laser, molecular beam systems are now giving new kinds of information on the dynamics and mechanism of primary photochem~cal processes. In the past 5 years, molecular beam experiments have played a crucial role in advancing our fundamental understandings of elementary chemical reactions at the microscopic level. These advances provide deeper insights with which to build our explanations of macroscopic chemical phenomena from the information gathered in microscopic experiments. The pervasive importance of these deeper insights was recognized in the award of the 1986 Nobel Prize in Chemistry to those responsible for bringing molecular beams into chemistry. SYNCHROTRON LIGHT SOURCES Characteristics of Synchrotron Sources The most intense, currently available source of tunable radiation in the extreme ultraviolet and X-ray region is synchrotron radiation, which is produced when energetic electrons are deflected in a magnetic field. That happens, of course, all the time in a synchrotron, which is an instrument that accelerates electrons to very high energies for particle physics studies. To reach these high energies, the electrons must be "recycled" through the accelerating zone many, many times. "Recycling" requires bending their trajectories through four successive 90-degree turns. At each

V-A. INSTRUMENTATION FOR STUDY OF CHEMICAL REACTIONS of these turns, the acceleration needed to change direction causes intensive radiation over the entire spectral range from the far-infrared to the X-rav range. This has, in the past, been looked on as an imtating energy loss. Now, however, synchrotrons are running out of things to do in high-energy physics. Hence, attention has turned from synchrotrons as accelerators (with radiation seen as undesired energy loss) toward snychrotrons as sources of light. Devices are placed inside the accelerator that increase the number of sharp bends in the electron trajectories to increase these radiative properties. These devices are descriptively called "wigglers" or "undulators." They show potential for intensity increases by several powers of 10 over the already bright radiation emitted by an ordinary synchrotron. Principal current use of tunable synchrotron radiation fails in the X-ray energy range, 1-100 keV. — ~.~ ~ —^'D— ' Applications of Synchrotron Sources in Chemistry Extended X-ray Absorption Fine Structure (EXAFS) has been one of the more fruitful applications of synchrotron radiation to solid substances. When one of an atom's inner-sheD electrons is excited by an X-ray photon, the atom emits light that is then diffracted by neighboring atoms. The result is a diffraction pattern that contains information about the interatom- ic spacings of these neighbors. ~ ° ~ Much attention has been di- ~ BW5 FREE ELECTRON . C 10 . "' i; LASERS . ~ r~£ 1 o1 o 1 0 LO Con 15 10 ol 4 <i 103 Cal rected toward crystal structures of inorganic solids, some of it seeking information on ox~da- tion state when other methods are not definitive. Since heavy atoms are most readily detected, EXAFS has been usefully em- ployed to learn the immediate chemical environment of transi- tion metal atoms as they occur in biologically important mole- cules, including maganese in chlorophyll. FREE-ELECTRON LASERS When a beam of electrons 1-2 GeV SYNCHROTRON (.~%BW) . , ,,, ~ [ASERSt x,.................... (.ooo4~ BW) me_ I I I I , . I 10 100 1000 10,000 PHOTON ENERGY (eV) with velocities near the speed DESIGN GOALS ARE AMBITIOUS - AND PROMISING of light moves through a peri- odically alternating magnetic field (a wiggler), light is emitted in the direction of the electron beam. The wavelength of the light is determined by the period of the wiggler field and the energy of the electrons. This causes it to behave like a population inversion; i.e., if it is placed between the mirrors of a conventional laser, 175

176 INSTRUMENTATION IN CHEMISTRY stimulated emission can occur to produce laser light. Such a device is called a Free-Electron Laser (FEL). Potential Capabilities Experience to date indicates that high-efficiency wavelength tunability and high average and peak power will all be forthcoming over a wavelength range extending from microwave frequencies through the infrared and visible to the vacuum ultraviolet spectral ranges. Average bnghtnesses several powers of 10 greater than those provided by conventional tunable lasers or synchrotron sources might be possible, particularly in the ultraviolet. An FEL has been operated at Los Alamos National Laboratory, based on a linear accelerator 2 or 3 meters long. Once a second, the device provides a train of pulses of tunable infrared radiation currently, in the 9- to 11-micron wavelength range, with 30-picosecond pulses, peak power of 5 megawatts, and 50-nanosecond spacing between pulses. Such perform- ance extended over the mid-infrared spectral region (4 to 50 microns) would open the way to many novel applications in chemistry. Examples are vibrational relaxation, multiphoton excitation, nonlinear processes in the infrared region, fast chemical kinetics, infrared study of adsorbed molecules, and light-catalyzed chemical reactions. As the wavelength is moved through the visible and toward the ultraviolet, a variety of novel chemical applications could be explored in photo- chemistry and fast chemical kinetics, as well as multiple photon and other nonlinear processes. SUPPLEMENTARY READING Chemical & Engineering News "Laser Vaporization of Graphite Gives Sta- ble 60-Carbon Molecules" by R.M. Baum (C.&E.N. stab, vol. 63, pp. 20-22, Dec. 23, 1985. "Imaging Method Provides Mass Transport' (C.&E.N. staff, vol. 63, p. 29, Sept. 23, 1985. '~Computers Gaining Fiery Hold in Chemical Labs" by P. Zurer (C.& E.N. staff), vol. 63, pp. 21-31, Aug. 19, 1985. "Supercomputers Helping Scientists Crack Massive Problems Faster" by R. Dagani, , . vol. 63, pp. 7-14, Aug. 12, 1985. "Spectroscopic Methods Useful in Inorganic Labs" (C.&E.N. staff), vol. 63, pp. 33-39, Jan. 14, 1985. "Technique Allows High Resolution Spec- troscopy of Molecular Ions" by R.M. Baum (C.&E.N. staff), vol. 62, pp. 34-35, Feb. 20, 1984. "Extreme Vacuum Ultraviolet Light Source Developed" by R.M. Baum (C.&E.N. stab, vol. 61, pp. 28-29, Feb. 7, 1983. "Synchrotron Radiation" by K. O. Hodgson and S. Doniach, vol. 56, pp. 26-27, Aug. 21, 1978.

The Ant That Doesn't Like Licorice While touring through the Costa Rican jungle recently I stumbled on a terribly wide path completely devoid of plant life. The path must have been 6 feet wide, and as I strolled along it I tried to keep out of the way of the native ants who were bustling past me. Each of the ones going the other way was carrying a big piece of leaf overhead; the whole bunch looked like a fleet of Chinese junks sailing along. Suddenly, I was oversalted by this very attractive native ant. `'Hi there!" I introduced myself, '`My name is Red Ant. What's your name?" Blushing, she answered,"My last name is Formicidae, but they call me L`eafeutter.'' "Say, that's a pretty name. Why do they call you that?" Giggling, she said, `'Everyone knows why it's because that's my business." She gracefully pointed an antenna at a pitifill-looking tree up the path. "See that?" she asked, "My sisters and I did that. We cut every one of the leaves off that tree in only 5 ~~_ days. Enough to feed the whole family for 2 months.'' ~ She turned to leave. "Don't go!" I exclaimed, "I'll Go' ~ , _ get you a leaf from this tree right here." Reaching ~ ~~' toward a lush tree that all the other ants were passing by, pulled oiT a leaf and presented it to Leafcutter. "Pew!" she ~~''~'~' Am' said, holding her nose, "Take it away—~ hate licorice." Sure enough, the leaf ~ held smelled just like liconce. I wondered what was wrong with licorice Leafcutter explained `'I'm not sure why, but Mama doesn't like it when we bung leaves that smell like that into the anthill." I was still poled so I asked her if she'd show me her home. Leafeutter lived in this gorgeous anthill along with her 5 Anion sisters, 500 brothers, her Mama, and, believe it or not, a Angus! Her lazy brothers never lifted a feeler to bring in even one leaf—all they seemed to do was amuse ~ Mama. And guess what? The ants didn't even eat all ~7: ~~' those leaves they brought home at Soothe fungus did! Apparently, the ants don't have the right enzymes to metabolize carbohydrates. But the fungus thrives off those leaves, and in gratitude to the ants for supplying them, it coIlveIts their carbohydrates into delicious sugars that the ant family lives on. '`Mama says we're symbiotic," Lealcutter exploded. Scientists have also taken an interest in Lealcutter and her family. They have concentrated on the leaves that Leaicutter doesn't like, trying to find out what protects these leaves over the others. Using liquid chromatography, they've extracted 10 to 15 milligrams of about 50 different compounds from great piles of the rejected leaves. Then they've worked to purify and identify these compounds. NMR studies have shown that every one of those trees that L~eaicutter dislikes contain compounds with molecular structures like that of carophyllene oxide, the compound that gives Sconce its flavor. They've adso got this notion that it's the fungus that gets sick on those leaves. And when the fungus gets sick there's no sugar for the ant family. So it looks as though the liconce-flavored trees have learned to synthesize their own fungicide to protect themselves from the Leatcutters The next step for those scientists will be to try to synthesize some similar compounds to combat harmful fungi elsewhere. The next step for me is Into a cozy little anthill with Leaicutter we're getting hitched in the spring. 177

178 INSTRUMENTATION IN CHEMISTRY V-B. Instrumentation Dealing with Molecular Complexity The chemical identification and synthesis of complex molecules ultimately depends upon the chemist's ability to bring about a chemical change and then ascertain the composition and three-dimensional structures of the products. The fact that chemists are now active in the biological arena shows the capability that now exists. It permits us to expect to understand the chemistry of life processes at the molecular level. All this is within reach because of diagnostic tools invented by physicists and sharpened by chemists to meet the analytical and structural challenges presented by extremely complex molecules. Foremost among these tools are nuclear magnetic resonance, X-ray diffraction, and mass spectrometry. NUCLEAR MAGNETIC RESONANCE (NMR) The nucleus of an atom carries electrical charge, and its behavior in a magnetic field shows that it acts like a tiny magnet. To help explain the existence of this magnetic property, we attribute to the nucleus a rotational movement, or spin. If the electrical charge of the nucleus is distributed over the volume of the nucleus, then nuclear spin implies that some of this charge would move in a circle around the axis of rotation. Such a charge movement would generate a magnetic field, so the spin concept "explains'' why the nucleus acts like a tiny magnet. When placed between the poles of a large magnet, the nuclear magnet will, like a compass, try to align itself parallel to the field. It will then require an input of energy to flip the magnet to an orientation opposing the field. Through fine spectroscopic measurements, scientists have found that both the nuclear spin and the energy of interaction between the nuclear magnet (the "magnetic moment") and an external field are "quantized," as are all atomic properties. In contrast to the behavior of macroscopic magnets, only particular values of nuclear spin are found in nature, and these values determine sharp "energy levels." These discrete energy levels provide the basis for a nuclear spectroscopy called nuclear magnetic resonance, or NMR. Spectroscopic studies guide us in assigning quantum numbers to the spin of a particular nucleus. Thus, electrons and protons are found only with spin quantum numbers of +~/2 or - I/2. A deuteron (a nucleus containing a proton and a neutron) has a spin of ~ . The nuclei of i2C and ~60 each have spins of zero (O) (i.e., they have no nuclear magnetic moment). In contrast, the isotopic nuclei ]3C, ON, '5N, and |70 have nuclear spins, respectively, of3/2, 1, 1/2, and 1/2. These nuclear spins determine the number of energy levels that will be seen if the nucleus is placed between the poles of a large magnet. A nuclear spin of zero (O) means that there will be no interaction with the field (i.e., i2C and ~60 will be invisible). A spin of I/2 implies two energy levels which correspond to the nuclear magnet oriented either parallel to the field (+1/2) or opposing the field (-1/2~. A spin off implies three energy levels that correspond to the nuclear magnet parallel to (+ 1), opposing (-1), or perpendicular (O) to the field. In general, if the spin is 5, there are (25 + 1) energy levels. As usual in spectroscopy, we can sense and measure these energy levels through the absorption of light.

V-B. INSTRUMENTATION DEALING WITH MOl FCUL OR COMPI F:XlTY The spacing of the energy levels depends, first, upon the magnitude of the applied mag- netic field. It also depends upon the magnitude of the nu- clear magnetic moment, which is not fixed by the spin quan- tum number but is dependent upon the nuclear structure. For a given nuclear-moment, the energy level spacing can be in- creased by increasing the ap- plied field. That makes the en- ergy spacing easier to measure and improves the resolution. Hence, progression in the field of NMR spectroscopy has been connected with, and lim- 7 ~ C:) EXTERNAl FT ELD ~ .112 . . I.. ~' \; _ 1/2 +1 EXTERNAL FI ELD . . . · . — EN ERGY LEY ELS 0 ~ N U CLEA R MA ON ETS IN AN EXTERNAL MAGNETIC FIELD ited by, our ability to produce very high and very uniform magnetic fields. Present-day NMR performance takes advantage of the compactness of supercon- ducting magnets to produce magnetic fields of tens of thousands of gauss (Io-~5 tesla). But in the 1950s, long before superconducting magnets, physicists began measuring the magnetic properties of the nucleus to learn about its structure. Their precision was sufficiently great that the physicists discovered, a bit to their dismay, that the measured nuclear resonance frequency depended not only on the magnetic properties of the nucleus but also upon the chemical environment the nucleus found nearby. Chemists were elated, however, since they saw the method as a new probe of molecular structure to supplement the rapidly developing infrared spectroscopic methods. Instrument developers quickly responded to the many opportunities seen for applications in chemistry. The outcome surpassed the most extravagant dreams. Today, NMR is surely one of the most important diagnostic tools used by chemists. It has had momentous impact in such diverse areas as synthetic chemistry, polymer chemistry, mechanistic chemistry, biochemistry, medicinal chemistry, and even clinical diagnosis. For example, we are now able to distinguish the chemical neighborhoods of the hydrogen atoms in molecules as complex as segments of DNA. Solution NMR Thus far, most of the chemical applications of NMR have involved liquid solution samples. This is because differences in chemical environments are sharply revealed because of averaging effects of the random motions in the liquid state. Performance has been limited by the uniformity of the high magnetic fields required, which also limits sample size and sensitivity. Through the 1960s and 1970s, technological developments (including superconducting magnets) permitted steady increases in magnetic filid intensity and uniformity. Now a barrage of new developments in 179

180 INSTRUMENTATION IN CHEMISTRY other factors, including Fourier Transform methods, high-resolution solid-state techniques, and a variety of pulsed measurements, is opening new dimensions for NMR. Fourier Transform NMR (FT NMR) Modern computers make it possible to record data continuously for some time period and then to transform the accumulated information into a frequency spectrum (See Section V-A, Computers). This Fourier Transform method was first applied to NMR in 1966; because of the better perfor- AROMATIC malice it brings, virtually all commercial research instru- ments now use FT. For exam- ple, it permits detection of the ~3C isotopically labeled mole- cules in an organic compound based on the '3C present in nature (1 in 100 carbon atoms iS TIC). At the same time, im- provements in superconduct- ing magnet technology raised the magnetic field intensity al- most threefold (from 5 tesla in 1966 to 12-14 tesla in 19791. Together, those two improve- ments provided 100-fold in- creases in sensitivity and 10- fold increases in resolution. Chemists can now ascertain the proton positions in the molecular structure of the anti-Parkinson's disease drug ~-dopa with as little as 5-10 milligrams of sample. NMR spectra of such complex molecules as insulin and abnormal hemoglobin (e.g., sickle cell) can be studied. Such instruments are now essential for research on all new pharmaceuticals, novel anticancer drugs, hormones, and products of recombinant DNA technology CARBONYL ALIPHATIC HEME ..<I..I klkl,i:,~i~'~l~, ok ~ ~ ~ - l 3C NMR OF CYTO CHROME c ~ 5 00 Mhz circa 1 984 ( NOT POSSI BLE I N 1 969 ) Solid-State NMR In the late 1960s, a variety of pulsed NMR experiments was introduced which reawakened interest in obtaining high-resolution NMR spectra of solids, despite the fact that molecules in solids are fixed in position, so that the averaging effects of molecular motions in liquids are lost. Initially, abundant and sensitive nuclei ASH, OF) were studied with resolution near one part per million. Then, in the penod of 1972-1975, methods were developed in which the tube containing the sample is rapidly spun around an axis tilted relative to the magnetic field. Then, the spectrometer sees a "blur," an average, of the NMR spectra of all of the angles through which the spinning sample moves. The blurring effect is quantitatively calculated with an "averaging function," (1-3 cos2 D), where ~ is the angle of tilt. If this tilt angle is fixed to be 54.7 degrees, the averaging function tI-3 (cos 54 7~21 is

V-B. INSTRUMENTATION DEALING WITH MOLECULAR COMP' FXITY equal to zero. This angle is ADAMANTANE called '`the magic angle." NMR spectra of solid samples spun at this magic angle pro- vide band sharpening ap- proaching those available for liquids. Today, both organic and inorganic solids can be studied at 0.01 parts per million resolution. Novel applications that have been made to inor- ganic samples include observa- tions of quartz formed at me- teor impact in which silicon atoms are found in unusual, six-coordinate crystal positions. Structures in rubbers, plastics, papers, coal, wood, semiconductors, and high-tech ceramics can be examined over wide temperature ranges, from 4K to 500K. 1972 _~,~, ~ it_ 1 I 984 WHERE IS IT? CROSS-POLARIZATION CROSS-POLARIZATION AND MAGIC ANGLE SPINNING IN NMR, THINGS ARE GETTING BETTER! Two-Dimensional NMR By means of cleverly timed pulsed radiofrequency excitation techniques, it is now possible to observe multiple-quantum transitions and to record NMR spectra in "two dimensions." Such 2D spectra appear as contour maps in which different types of interaction spread out resonances along two axes. In addition to the characteristic frequency shifts (e.g., by which we can distin- gllish between CH2 groups and SHALE 01L ~ ~ D NOR CH3 groups), the new dimen- sion reveals more distant inter- actions. Thus, information about molecular shapes can be determined for complex mole- cules even when single crystals cannot be obtained (so X-ray techniques cannot be used). z This is quite crucial for biolog- ical molecules because it gives ! access to conformational infor- mation under conditions close to the living conditions in ! which biological molecules ac- tually function. Imaging In 1973 the first spatial reso- lution by NMR was reported by chemists. Today, there ex- 1 caused bv atoms in the immediate neighborhood CH — 2 j ~ Q :0. ~ .. .. 1 ,A, ,\ 1 . .... 2D 3C NMR GIVES MORE INFORMATION ABOUT COMPLEX COMPOUNDS 181 to 10 - loo

182 INSTRUMENTATION IN CHEMISTRY ist instruments capable of "mapping" in three dimensions the NMR chemical shifts and nuclear concentrations for objects as large as a human patient. Such NMR scanners, comparable in some respects to X-ray CT scanners, appear to have considerable potential for diagnosis of diseases, possibly including multiple scle- rosis, muscular dystrophy, and malignant tumors. Most important, this diagnostic method does not require surgery or other invasive techniques. Further increases in field strength should permit real-time imaging of, for example, a beating heart. In a closely related, but invasive, medical application, NMR measurement coils have been surgically placed around intact and functioning animal organs. These have been used to study metabolism by measuring high- resolution phosphorus, carbon, and sodium NMR spectra in the organ while it is operating. These remarkable uses of NMR place before us the possibility of studying the chemistry of a living system truly in viva. NMR Performance, Availability, and Costs Resolution and sensitivity of an NMR instrument depend upon the interplay among the magnetic field intensity, sample volume, and field uniformity over that sample volume. As chemists are working with more and more complex molecules, better resolution advances research capabilities as soon as it becomes technolog- ically feasible. This can be seen in the steady rise in the magnetic fields available in commercial NMR instruments (as expressed in the proton NMR frequency, usually given in megahertz, MHz). Over the last 25 years, the highest field available has increased by a factor of about I.5 every 5 years or so. Unfortunately, the resultant higher performance, coupled with other improvements, has exponentially in- creased the cost, hence, the availability, of the highest performance machines. Thus, the price of commercial NMR instruments has risen from about $35,000 in 1955 to $850,000 in 1985, a few percent per year faster than inflation. The critical importance of state-of-the-art NMR instrumentation is reflected in the annual sales of NMR instruments, which, in 1984, totaled about $100 million. The most advanced NMR spectrometers were 500-MHz instruments, and about 70 such instruments had been produced worldwide. Many of these are in U.S. industrial laboratories; a number of them are in Europe, Japan, and the Soviet Union; and about 17 of them are placed in U.S. academic institutions. Magnet technology will presently permit commercial production of 600-MHz instruments at a cost of about $850,000, and on the horizon are 750-MHz instruments with an expected cost near $1.5 million. Applications of NMR by chemists have revolutionized much of chemistry; and they are having profound influences on adjacent research fields in biochemistry, materials research, geochemistry, botany, physiology, and the medical sciences. Thus, while the costs of modern NMR instrumentation are high, the potential rewards are so great that we cannot afford to lose them. MASS SPECTROMETRY (MS) In a mass spectrometer, a molecule of interest is converted to a gaseous ion, and the ion is accelerated to a known kinetic energy with an electrical field. Then its

V-B. INSTRUMENTATION DEALING WITH MOLECULAR COMPLEXITY mass can be measured, by tracking either its curved tra- jectory through a known mag- netic field or its time of flight through a fixed distance to the detector. The first step, the production of molecular ions, causes some of the molecules to fragment and give a collec- tion of ions whose masses are determined by the structural units in the original molecule. Thus, experience leads us to . ~ . . . MAGNETIC DEFLECTION t4S Applled O Ev / : ' ~ ml' If ~ ;—kJ _~\ m'm2 Faster ions Deflect More TIME OF FLIGHT MS Magnetic o En Detector ma ~ Faster ions Get There First For fused accelerating voltage Ev d~flerent masses have different veloc~t~es- ~v = 2 move = 2 m v2 ~o v~='~ ~2 ml expect that the mass spectrum of CF3-CH3 will include a mass peak at 84 due to the "parent" ions (CF3-CH31+ but also a prominent peak at mass 69 due to (CF31+ and another at 15 due to (Calm+. Thus, the mass spectrum gives far more information than just the molecular weight of the parent molecule. Furthermore, the mass spectrometer can be coupled to other techniques, such as infrared spectroscopy or gas chromatographic fractionation, to add greatly to the significance of the mass spectrum. These coupling schemes are discussed in Section {V-C as a part of Analytical Chemistry. Applicability Some scientists feel that gas chromatographic fractionation (see Section V-C) followed by mass spectrometric analysis provides the best general purpose analytical instrument for handling complex mixtures drawn from chemical, biolog- ical, geochemical, environmental, and crime lab applications. Until recently, however, such analytical use was limited to compounds that would vaporize at a temperature within their range of thermal stability. Now, over the last decade, applications of mass spectrometry are rapidly widening because of a recently developed series of related techniques using ion, neutral, and photon bombardment to desorb ions from solid samples (see Table V-B-11. These techniques dramatically increase the molecular weight range of mass spectrometry. Plasma Resorption under bombardment by the fission fragments from the radioactive Californium isotope 252Cf has given molecular ions of molecular weight 23,000 from the polypeptide trypsin, while Fast Atom Bombardment (FAB) has provided extensive structure information on a glycoprotein of molecular weight about 15,000. Laser and field Resorption have produced molecular ion mass spectra displaying the oligomer distribution of sections of DNA. Now molecular weights of 20,000 can be measured, and mass resolution of one part in 150,000 is available in commercial instruments. Perhaps 5- to lO-fold higher resolution can be achieved with Fourier Transform techniques for relatively low-mass ions. Extremely high resolution can be quite useful to distinguish between the masses of one deuterium and two hydrogen atoms (seven parts per ten thousand) or between one i3C atom and a t2C plus a hydrogen atom (three parts per ten thousand). This becomes extremely important as we decipher the mass spectrum of a large molecule because both ~3

184 I~ISTRUMEN'TATION IN CHEMISTRY TABLE V-B-1 Desorption Ionization Techniques for Analysis of High-Molecular- Weight Substances Field Desolation (FD3. Samples placed on a fine, carbon-coated wire are subjected to heat and high electric fields. Commercially available, somewhat erratic, but has been productively employed. Plasma Desolation (PD). Samples placed on thin foil are bombarded with high energy fission fragments from radioactive californium (252Cf) or ions from an accelerator. Not commercially available. Secondary lore Mass Spectrometry (SIMS). Solid samples are bombarded with kilovolt electrons. Low electron fluxes are used for molecular SIMS; high fluxes for inorganic analysis and depth profiling. Commercially available. Electrokydrodynamic Ionization (EHMS). Samples are dissolved in a glycerol-electrolyte solvent. Desorption from solution occurs under high electric fields and without heating. Almost no molecular fragmentation! Not commercially available. Laser Desorption (LD). Both reflection and transmission experiments and various sample preparations can be used. Tendency toward thermal degradation. Commercially available with time-of-flight mass analysis. Thermal Desorption (TD). Sample is placed on probe tip which is heated to desorb ions (no ionization filament is used). Useful for inorganic analysis; recently applied to organic salts. Fast Atom Bombardment (FAB). Supples in solution (usually glycerol) are bombarded with k~lovolt- energy atoms. Fluxes higher than in SIMS. Wide applicability to biological samples, including pharmaceuticals. Commencally available. deuter~um and ~3C are present in nature. Consider, for example, that a molecular weight near 900 implies 60 or more carbon atoms. For such a molecule, the i3C present in nature hi. ~ percent) is sufficient that about half of the molecules will have at least one ~3C atom. The breadth of use of mass spectrometry is shown by the fact that about $200 million worth of instruments are purchased each year. Several thousand people in the United States are engaged fulI-time in using them, more than double the number so employed 15 years ago. The chemical, nuclear, metallurgical, and pharmaceu- tical industries all make extensive use of mass spectrometry. Environmental regulations (particularly those covering organic compounds in water supplies) are written around mass spectrometry. Established and emerging methods of geologic dating and paleobiology are based on this technique. Research applications in chemistry are innumerable, ranging from routine analysis in synthetic chemistry to beam detection in a molecular beam apparatus. Sensitivity and Selectivity An unknown sample can be i`4entipe~ using MS with as little as 10-~° grams (100 picograms), while a specific compound with a known fragmentation pattern can be detected with as little as 10-~3 grams (100 femtograms). As a striking example, a O.l-milligTam dose per kilogram of body weight of A9-tetrahydrocannabino] (an active drug from marijuana) can be tracked in blood plasma for over a week down to the 10-~ grams per milliliter level using combined gas chromatography and tandem mass spectrometry. As an example of specificity, in a simple MS exami- nation of a coal sample containing a small amount of trichIorodibenzodioxin, interference by the great variety of similar compounds in the sample ("chemical noise") can completely hide the offending molecule. However, the parent mass of the desired compound (288) can be extracted from this background in a tandem MS/MS apparatus, in which two mass spectrometers are used in series. This extra

V-B. INSTRUMENTATION DEALING WITH MOLECULAR COMPl;F:XITY step of separation produces a mass spectrum essentially identical to that of the pure compound. Costs Just as for NMR, costs of mass spectrometers have in- creased exponentially over the last few decades but, again, these increasing costs carry with them enormous increases in capability. For example, in 1950, for about $40,000, the best instrument available had a resolution of about one part in 300, and it could be used for molecular weights up to 150. Assuming an average inflation of 6 percent over the 30-year penod, this same instrument would cost $230,000 in 1980 dollars. But in 1980, the best instrument available cost about $400,000, less than double this amount, but resolution had been raised 500-fold (to 150,000) and, at the same time, the mass limit had been raised over 10-fold (to 2,000~. Along with these performance im- provements, scanning speeds have been greatly increased, and data processing is done by built-in computers. Again, as for NMR, no first-rate research laboratory (academic or indus- tnal) can operate without mod- e~n instrumentation of this type. X-RAY DIFFRACTION S500 K 5400 S300 K S200 K (COST IN THOUSANDS OF DOLLARS) RESOLUTION MOLEC. AT. LIMIT _ Sloop _ AS 217 182 1~161~ 247 200 LASS 250 22s 1 MS/MS 1 288 97 ~ . 300 253 1 111 l ~ l 200 MASS 250 TRICHLORODIBENZODIOXIN IN COAL MS CAN'T FIND IT MS/ MS CAN MASS SPECTROMETRY HICH /4''''"'M^SS 1; 00,000 ~ A/ - ^CHETS , 2 5,000 J 74 LASER, ~ ~18~ FD,F^s, / Molec z'2 cl 150.000] ~~~ FOUR I ER 2.000 by TRAnSFORI4 /~ LClMS / Microanalysis /. ~ COMPUTER COnTROL . ~ 2 5,()00 _/ CHEM ICAL, Fl ELD ~ ooo r l ON ~ Zip l I /4 - GCl"5 /Sod Sampllng / Elemental Composition 300] /4 ~ DOUBLE FOCUS MS ~ l~otccular Structures gas Analysis I ~ ~ I t I 1 I . t 950 1 960 1 970 1 980 ~ 990 YEAR FAB . FA" ATOM - ~BARDMERT LC ~ LIQUID CHROM^T - ALPHA 352 C! - CALIFORn1~M 252 CC ~ GAS CHROMES - R^PHT FD ~ FIELD DESORPTION MS ~ MASS SPtCTROM"R7 INCREASING CAPABILITY INCREASING IMPORTANCE INCREASING COST 300 Molecular structure is concerned with the bond lengths, bond angles, and spatial placements of the atoms in a substance. Knowledge of such arrangements cianfies the physical and chemical properties of materials, it points to reaction mechanisms, ADS

186 INSTRUMENTATION IN CHEMISTRY and it identifies new compounds. At present, X-ray diffraction techniques offer the most powerful route to {earning molecular structures for any substance that can be obtained in crystalline form. When light shines on a mirror with regularly spaced straight lines scratched on it (a grating), the mirror still reflects somewhat. However, something special happens if the wavelength of the light, A, is about the same as the spacing, a', between the lines on the mirror. Then the reflec- tion pattern includes bright re- gions at special angles that are determined by the ratio of A to d. This pattern is called a ~if- fraction pattern. it arises from constructive and destructive interference between light waves, similar to that which occurs when two water waves merge. If the line spacing, 4, is known, then the wavelength of the light, A, can be determined by measuring the angles at which the bright regions of the pattern are found. X-rays are light rays just like visible light, except that the C2RYST`~ human eye cannot see them and their wavelengths are only a few A (green light has a wavelength of 5,500 A X-rays are around 2 A). No machine shop can scratch a mirror with fines only a few A apart to make a grating for X-rays. However, Nature provides us with excellent X-ray gratings in the form of natural crystals. The regular spacings of the atoms serve as regularly spaced scattering centers and so X-rays are diffracted by a crystal. In this case, we know the wavelength of the X-rays, so we use the angles at which bright regions appear (e.g., on a photographic plate) to determine the atomic spacings. A crystal made up of single atoms (as in a pure metal) gives quite a simple diffraction pattern. The atomic spacings are regular, too, in a molecular crystal, like solid naphthalene, C,oHa. But now, there are several types of spacings that will contribute to the diffraction pattern. First, there is the spacing between the centers of adjacent C,oH~ molecules. In addition, there are the spacings determined by the fixed carbon-carbon and carbon-hydrogen bond lengths and the molecular bond angles. Now, the diffraction pattern becomes much more complex. Nevertheless, with precise instrumentation and modern computers, the complete molecular structure can be deduced from this pattern. Given a perfect crystal of the pure `_ I N(: I DENT r3 i FFRACTED LIGHT LIGHT . .. . .. ~ d_. GRATING I NC I DENT X-RATS D I FFRACTED 1- RAYS ~ d_ (:ItYST~ I ~ I. 1~; I; (;RA TIN(;S. n l ~~ PA CT 1 1GI1T

V-B. INSTRUMENTATION DEALING WITH MorEcuL4R COMPl;FXITY substance, this type of analysis can be used whether the crystal is an inorganic, organometallic, or organic substance, or a metal, a mineral, or a macromolecule of biological origin. The X-ray diffraction pattern reveals which atoms are bonded to which, the bond lengths and bond angles, and the molecular geometry; and it even indicates how the atoms are moving and how charges are distributed among them! It is as close as we can come to "seeing" the atoms in a molecule. Applications The X-ray diffraction technique has become an integral part of inorganic, metal-organic, and organic synthesis. Whenever an unknown substance can be crystallized, an X-ray structure determination is liable to reveal the identity, molecular structure, and conformation of the molecule. With present computer- automated data interpretation, molecular complex- ity is not a great obstacle. In fact, the requirement that the substance must be available in single- crystal form emerges as one of the major limitations to the range of applicability of this powerful tech- nique. When single crystals can be obtained, even the most complex biological molecules can be ex- amined. For example, X-ray structure analysis has be- come a vital too! for understanding the specific mechanisms for drug action. Such studies of molec- ular substrates, inhibitors, and antibiotics give in- formation on the special geometry of the receptor site, a first step toward drug design. An example is the recent determination of the manner in which the beneficial drug tr~ostin A attaches itself to a piece of DNA. When a natural product has been shown to have useful biological properties, the molecular formula must be known before progress can be made toward chemical synthesis. If the active substance can be crystallized, X-rays can furnish that crucial information. Examples already mentioned in Section IlI-A extend from insect pheromones for pest control in agnculture and forestry to growth hormones to increase food, forage, and biomass production. In a similar way, the structures of toxins from poisonous tropical frogs, poisonous sea life, and poisonous mushrooms have advanced studies of nerve transmission ion transport, and antitumor agents. Recently the seeds of Sesbania cirummondii, a perennial shrub growing in wet fields along the Florida to Texas coastal plain, were found to yield a possible antitumor compound. The most active compound found in the seeds is present at only 1/2 a part per million, so 1,000 pounds of seed provided only milligram quantities. The structure of this molecule )~ tric`stin-A ~2 fragment X-RAYS SHOW HOW A OR UG Bl NDS TO DNA 187 His; AH Own' O SESBANIMIDE ANTI-TUMOR DRUG? X-RAY ANALYSIS WITH ONLY TEN MICROGRAMS!

188 __— INSTRUMENTATION lN CHEMISTRY called sesbanimide, was determined by X-ray diffraction of a crystal weighing only 10 micrograms. This analysis displayed a novel tncyclic structure previously unknown either in nature or among synthetic organic compounds. With this knowledge, organic chemists have begun devising synthetic approaches to make sesbanimide and related compounds. Molecular Graphics For some time, computer-driven graphics modeling and fitting structures to X-ray-derived OF Em_ COMPUTE R G RAPH I CS SH OW MOLECULAR STRUCTURES IN 3D come more widely available, they are sure to analytical too! for connecting molecular structure to molecular function, partic- ularly for biological molecules. programs have been used for electron-density maps of mole- cules. In the last few years, however, new developments have appeared that greatly in- crease the ability to picture complex molecular arrange- meets. Computer-automated graphics units have recently become available that present the molecular structure in three dimensions, together with the capacity to rotate the molecule slowly and to high- light with color those molecu- tar components of particular interest. Even an untrained eye can perceive three-dimen- sional spatial relationships that might go unnoticed with- out these instrumental fea- tures. As such capabilities be- be regarded as an essential NEUTRON DETRACTION Complementary to X-ray diffraction and of considerable use to structural chemistry is neutron diffraction. Neutrons with room temperature velocities have wavelengths that are comparable to the atomic spacings in crystal lattices, so when they are scattered from crystalline matenals, they give rise to diffraction patterns. To be practical, high-intensity neutron beams are needed, and these can be obtained only from nuclear reactors. If available, however, there are two unique advantages of neutrons over X-rays. First, their scattering from protons is of comparable intensity to that from heavier nuclei, so that neutron diffraction gives more precise information on positions and bonding of hydrogen atoms. Second, the neutron has a magnetic moment, so that neutron diffraction can be used to study magnetic structures.

V-B. lNSTRUMENTATlON DEALING WITH MO~FCUL~AR COMPLEXITY Applications Among the accomplishments of neutron scattering research in the past decade are the determination of structures of magnetic superconductors, determination of the spatial organization of macromolecular assemblies such as ribosomes, and the location of hydrogen atoms in the hydrogen bonds that determine protein structures. ELECTRON SPIN RESONANCE Most molecules contain an even number of electrons that occur in pairs with opposite spins. However, a reaction in which an electron is transferred can generate species with an unpaired electron (e.g., free radicals and radical ions). The unpaired electron gives the molecule magnetic properties that allow detection and charactenza- tion by the technique of electron spin resonance (ESR). The ESR instrument consists of a strong magnet, microwave equipment (ong~naDy based on radar technology), sensitive electronic apparatus, and, frequently, a dedicated computer. Applications Even though molecules with unpaired electrons tend to be reactive, they are important in many chemical and biological processes, usually as transient interme- diates. For example, samples of photosynthetic materials give rise to ESR signals when they are irradiated. These signals arise from primary electron-transfer events initiated by the absorption of light by the photosynthetic pigments, and their study has been important in understanding the mechanism of photosynthesis. Organic radicals and radical ions produce a unique ESR spectrum that allows their identification. In addition, the pattern in the spectrum provides information about the electron-density distribution in the molecule. SUPPLEMENTARY READING Chemical & Engineering News "Founer-Transform Mass Spec Joins Analyt- ical Repertoire" by S.C. Stinson (C.& E.N. staf0, vol. 63, pp. 18-19, Mar. 18 1985. "Modern NMR Spectroscopy" by L.W. Je- linsky, vol. 62, pp. 2~40, Nov. 5, 1984. "Field Flow Fractionation Used to Separate DNA" (C.& E.N. staff), vol. 62, pp. 23-25, Apr. 30, 1984. "Potentiometric Electrode Aims to Measure Antibody Levels" by R.L. Rawls (C.& E.N. staff), vol. 62, pp. 32-33, Apr. 2, 1984. "ZermField NMR Advances Molecular Struc- ture Determinations" by R.M. Baum (C.& E.N. staff), vol. 61, pp. 23-24, Dec. 12, 1983. "Multiple Quantum Technique Extends NMR" by R.M. Baum (C.& E.N. stay, vol. 61, pp. 30-31, Jan. 3, 1983. "Mass Spectrometry/Mass Spectrometry" by R.G. Cooks and G.L. Glish, vol. 59, pp. 40-52, Nov. 30, 1981. Science "The Use of NMR Spectroscopy for the Understanding of Disease" by G. Radda, vol. 233, pp. 640-645, Aug. 8, 1986. "Multiple Quantum NMR Spectroscopy" by M. Murowitz and A. Pines, vol. 233, pp. 525-531, Aug. 1, 1986. `'Two Dimensional NMR Spectroscopy" by A. Box and L. Lerner, vol. 232, pp. 960- 967, May 23, 1986. "The 1985 Nobel Prize in Chemistry" (for x-ray crystallography) by H.A. Hauptman and J. Karle, vol. 231, pp. 309432, Jan. 24, 1986. `' High Resolution NMR of Inorganic Solids" by E. Oldfield and R.J. Kilpatrick, vol. 288, pp. 1537-1543, Mar. 29, 1985. 189

H—N—~ N - Pt C! H' CQ Cisplatin— The Strong, Silent Type cis H`\ -Pt — N bans Cancer's a tricky dude to fight. Somehow it takes over a cell that's been functioning redly for years. Then, it fiddles with the control center of the cell the nucleus- and causes it to reproduce more cancerous cells at an alarming and unhealthy rate. This enemy seems unstoppable. Luckily, scientists have found an ally in the war on cancer a shy and unobtrusive molecule called cisplatin. Cisplatin is merely one fob of a platinum compound called diamminedichloroplati- r~um. In spite of its long name, it Is a surprisingly simple molecule consisting of two ammonia groups (NH3) and two chlorine atoms bound to a platinum atom. This compound comes in two shapes (cis and trans), with two very different personalities. Cis-DDP, or cisplatin, is the form most effective in fighting cancer, even though trans-DDP seems to work in a similar manner. It was perplexing at first why one form of DDP would be so much more effective than the other when their mode of operation seems so similar. Cisplatin is very good at infiItrahng enemy lines without detection; then, once inside the cell nucleus, Cisplatin works undercover to block cell reproduc- tion. Trans does basically the same thing but always manages to blow his cover before cell reproduction. He is recognized and removed from the nucleus before completing . . . . 11S ~SSIOI1. How is this possible? WeD, research has shed some light on the story. Apparently, both DDP molecules are easily taken into the cell and bind readily with DNA, fanning what ace caned DNA "adducts." The key seems to lie in the fact that cis-DDP binds consistently between two adjacent guan~ne rings in the DNA, while trans-DOP produces a variety of cross-links between bases with one or more nucleotides in between. All cells have a mechanism by which they notice and repair *egulanties when possible. In the case of trans-DDP, it has made the mistake of being too obvious the bans adducts are awkward and are recognized and removed within a few hours of having formed. But sneaky Cisplatin is not noticed so easily and manages to stay it. 190 in place, interfenng with the DNA's attempts to replicate. We now believe this Is precisely how Cisplatin fights cancer growth. Even though cis-DDP is more toxic to cancer cells than it is to normal cells, it, like many other forms of chemotherapy, cames with it serious side effects for the patient. But hopefully, inquiries into the workings of this simple drug will point the way toward similar antitumor agents that are just as effective but without the bummer side effects. We'I1 get that tricky dude yet!

V-C. INSTRUMENTATION AND THE NATIONAL WELL-BEING V-C. Instrumentation and the National Well-Being As discussed in earlier chapters, sophisticated instrumentation has figured prominently in our discussions of environmental monitoring and economic appli- cations of chemistry. The techniques of the surface sciences are of dominant importance to the advances being made in catalysis, upon which so many industries depend. Chromatography joins mass spectrometry and laser spectroscopy as an everyday too! in analytical chemistry. Infrared spectroscopy is typical of the several spectroscopic methods that are finding effective use in environmental monitoring as well as in research applications. SURFACE SCIENCE INSTRUMENTATION Surface science is a rapidly growing area. The development of powerful instruments that can reveal the atomic structure and chemical composition of surfaces has been largely responsible. The field has been stimulated, as well, by a wide range of important applications. For example, the electrical properties of surfaces and films are important in the miniaturization of semiconductor devices. Consequently, surfaces and thin films have attracted the interest of both physicists and chemists. They are investigating surface etching to permit removal of a layer a few atoms thick in an accurate pattern (a circuit). Another problem of current research interest is the growth of a semiconductor film (for example, a silicon fiIm) when vapor is condensed on a cold surface. It is found that when silicon atoms are condensed on a crystalline surface, the electrical properties of the film can be fixed by the underlying crystal structure (epitaxial growth). And the prospect of understanding catalysis on a fundamental level is one of the most exciting and significant frontiers opened by these new instruments. Instruments for the Study of Surfaces The various techniques of surface science probe the surface with particles or with light (photons). Among the particles that have proven useful are electrons, ions, neutral atoms, neutrons, and electronically excited atoms. Photon probes extend from the X-ray region to the infrared. When particles are used, ultrahigh vacuum environments are essential (10-9 to 10~~° torr). In contrast, photon probes can be effective when the surface is in contact with a gas at high pressure or with a liquid, the conditions under which surface catalysis actually occurs. A key question about chemistry as it takes place on a surface is the molecular structure of the molecules that have become attached to the surface. If each molecule is essentially intact (physisorbed) with its structure and bonding little changed, the surface is serving only as a site for reaction, immobilizing the reactant as it awaits its fate. But if the molecule reacts with the surface (chemisorbed), it acquires a new molecular identity with changed chemical behavior. Table V-C-1 lists six types of surface science measurements that are the most informative about the structure of the adsorbed molecules. There are several other instruments that tell about the surface structure, composition, and bonding in the 191

192 INSTRUMENTATION IN CHEMISTRY first few layers. Two or more complementary methods used together can greatly enhance the significance of any single measurement used alone. TABLE V-C-1 Instrumentation Relevant to Chemistry on Surfaces . . Method Electron energy loss EELS spectroscopy Bombard or Irradiate Acronym with: Electrons, 1-10 eV Physical Basis Information Obtained Molecular structure, surface bonding of adsorbed molecules Molecular structure of adsorbed molecules Energy of surface binding Vibrational excitation of surface molecules Infrared spectroscopy IRS Thermal desorption Auger spectroscopy Low-energy electron diffraction Secondary ion mass spectroscopy TDS Auger LEED SIMS Heat Infrared light Vibrational excitation of surface molecules Thermally induced desorpiion of adsorbates Electron emission from surface atoms Back-scattenng, diffraction Ejection of surface Electrons, 2-3 keV Electrons, 10-300 eV Ions, 1-20 keV atoms as ions Surface composition Atomic surface structure Surface composition Electrons are useful surface probes because their energies, and hence, their wavelengths, can be accurately controlled with their accelerating voltage. At low energy, near 25 electron volts, the wavelength of an electron is close to the atomic spacings in a metal, so a beam of such electrons reflected from the surface will show diffraction ejects. Thus, low-energy electron diffraction (LEED) can play the same role in determining bond distances and bond angles in surface chemistry as X-ray diffraction plays in the structural chemistry of solids. LEED reveals the atomic structure of clean surfaces, as well as any regulanty in the packing of atoms and molecules adsorbed on the surface. In the Auger (pronounced "Ohjay'') effect, high-energy electrons (2,000-3,000 eV) striking an atom cause the atom to eject a secondary electron from an inner shell. The energy of the ejected electron is determined by the energy levels of the atom it came from, so measurement of the electron energy identifies the atom. Since the bombarding electrons do not penetrate deeply, these secondary electrons reveal, with high sensitivity, the composition of the first few surface layers. This information can be important because surface impurities and i'Te~ulanties can . , , ~ ,~ ~ _ _ ~ _ dominate surface chemistry. Hence, the combination of Auger and LEED is used routinely to verify the cleanliness and perfection of the surface under study. Electron energy loss spectroscopy (EELS) is of particular value because it detects the resonant vibrational frequencies of atoms and molecules bound to the surface. Chemists routinely use such vibrational frequencies for gaseous molecules to decide which atoms are hooked to which, how strong the bonds are, and their molecular geometry (see Infrared Spectroscopy later in this section). In EELS, an electron beam of known energy is bounced off the metallic surface into an energy analyzer. If the electrons hit an area where a molecule is adsorbed, the molecule can be left vibrating in one of its characteristic motions. The energy needed to do this, determined by the frequency of the motion, is taken away from the kinetic

V<. INSTRUMENTATION AND THE NATIONAL WELL-BEING energy of the electron. The measurement of these electron energy losses of the reflected beam gives a vibrational spec- trum of the adsorbed mole- cules. Ion scattering from surfaces has been used for surface com- positior~ analysis with great sensitivity, 109 atoms/cm2. In secondary ion mass spectros- copy (SIMS), neutral and ion- ized atoms and molecular frag- ments are ejected by bombard- ment with high-energy (1-20 keV) inert gas ions. Ton scat- tenng spectroscopy determines the surface composition by the energy change of inert gas ions upon surface scattering. Ton etching removes atoms from surfaces layer by layer. The combined use of ion etching and electron spectroscopy yields a depth profile analysis of the chemical composition in the near-surface region. This combination of instrumental methods is called "dynamic SIMS." The availability of high-intensity laser sources is now awakening the develop- ment of a new set of surface-sensitive techniques. Surface infrared spectroscopy, laser Raman spectroscopy, and second harmonic generation surface spectroscopy all provide information about the surface chemical bonds of adsorbed atoms and molecules. All of these emerging surface science techniques will permit us to watch chemical reactions as they occur on well-charactenzed and clean surfaces. This is an important development in chemistry because surfaces provide the two-dimen- sional reaction domain that accounts for heterogeneous catalysis. reflected electrons E2-E1-1200 \ incident Of electrons p' / ;~ ~,~ ASH \ He C / \ ~ ~ ~ ~ M E, / EELS Vibrations of Surface Molecules The Energy Loss }dentifies the Vibrational Mode SURFACE ANALYSIS Any sensitive measurement technique can be used as an analytical tool. This is the case in the surface sciences. Every one of the capabilities listed in Table V-C-1 can be put to analytical use in the pursuit of questions that may be only remotely connected to the surface sciences. As an example, a state-of-the-art laser micro- probe device designed to desorb (remove) molecules from a solid surface can be used to detect the presence of a pesticide on the leaf of a plant. Such a capability was quite impossible only 10 years ago; today it permits us to contemplate tracking the amount, stability, weathering, and chemistry of a pesticide in field use. Of course, the analytical technique may just as well be concerned with monitonug or clarifying chemical changes that take place on a surface or with a surface. Many of these analytical studies relate to catalysis. In Section IV-C, examples were given of the use of EELS to determine the molecular structures that exist on a catalyst surface as it functions. Such applications have given rise to surface analysis, a new subdivision of analytical chemistry. 193

194 INSTRUMENTATION IN CHEMISTRY The elective sampling depth is a most important feature of any surface analytical technique. Sampling depth is important, because the measuring technique must be appropriate to the phenomenon under study. For example, bonding to the surface, wettability, and catalysis in- OOO LATERS valve only a few atomic layers, whereas surface hardening treatments involve 10 to 1,000 atomic layers. Typical sam- pling depths for the primary surface analytical techniques are one or two atomic layers for low-energy ion scattering, 5 A depth for SIMS, 20 A for the Auger technique, and 100 ~ for ion etching coupled with SIMS. Laser mass spectrome- try, the Raman microprobe, and scanning electron micros- copy (SEM) reach from 1,000 to 10,000 A (i.e.' to one micron). l he shallower the sampling depth of the tech- nique, the more finely it is able to define the surface composi- tion of a sample. A major challenge in the de- velopment of surface analytical instrumentation is the reinforcement of its quantitative dimension. Most of the examples given have been concerned with what is there. We must also be able to determine how much. Another important problem is the development of micro- probes which can provide both chemical and positional information about surface species. Currently, Auger and ion microorobes are useful in this respect for _ it, ,,, ,,,, ~ ,,, ~ ~ CATA LTSI S ~ //// WETTAB I L I TT _~ URFACE BOND I NG cn ::~ E EECTR I CA ~ CONDUCT I V I TTl l ~/~< //////././//, . <~//,//~/.~,///////////////////////~/////~ PASSIVATION, SURFACE TREATMENTS I ~7~s~//////~ _OPT I CA L AB SORPT ~ ON //D ~,/,~,~i'~'~6,Y~/,~,~Y,~,~,.,^~/,~,,Y/~/i~//~//////~/y~y~///~/~ CORRO S I ON ~_~ ~~ ~~__~ - Low ILM STRUCTURES ~ ~'~///~////~///~/~///////~/////////////////////////////~/////~///////////////~/~ VISUAL EFFECTS, COLOR ISS SIMS ESCA SIMS LASER SEM (STATIC ) AUGER (DYNAMIC ) MS RAMAN HOW DEEP IS THE SURFACE? IT DEPENDS ON WHAT YOU CARE ABOUT At. . .. .. mapping elemental composition, as in revealing both the presence and location of the trace contaminants phosphorus and lead in silicon chips. However, they are not yet able to detect and map large organic molecules such as carcinogens or therapeutic drugs. Characterization of small particles is another important chal- lenge for surface analysis; this is particularly important in environmental monitor- ing where the analysis of carcinogenic hydrocarbons on atmospheric dust and other particulates is a current problem. CHROMATOGRAPHY Chromatography separates molecules or ions by dividing species between a moving phase and a stationary phase. A liquid or a gas flowing continuously through a tube (called a "column") provides the moving phase. The stationary phase can be either small solid particles packed in the tube or, for a small-diameter

V-C. INSTRUMENTATION AND THE NATIONAL WELL-BEING tube (a capilIary), the walls of the tube itself. If a pulse or a squirt of soluble substance enters the tube at one end, that substance will have some tendency to stick to the stationary surface, becoming adsorbed. However, the continuing flow of fresh solvent keeps acting to redissolve this adsorbed matenal, moving it forward in the tube. How fast the process of adsorbing and desorbing takes place depends sensitively on the composition and structure of the substance. Conse- quently, different substances that entered the tube together in the same pulse will move at different speeds through the tube, so they will exit at different times. This separation technique takes advantage of small differences in properties such as solubility, absorbability, volatility, stereochemistry, and ion exchange, so that understanding the fundamental chemistry of these interactions is basic to progress in the field. Liquid chromatography has shown an impressive growth since 1970. The current $400 million annual sales are mainly by U.S. manufacturers. This growth has come through innovations such as high pressure and moving phases of changing composition ("gradient moving phases") to give greater speed and resolution. "Bonded-molecule" stationary phases are chemically designed to increase selectivity and to extend the useful lifetime of a column. Detection also has improved with electrochemical, fluorometric, and mass spectrometric detectors, reaching sensitivities as low as 1o-~2 grams. Although gas chromatography is a more mature field by perhaps a decade, important advances continue to appear. High-speed separations can now be accomplished in a few tenths of a second; portable instruments the size of a matchbox are in use outside of the laboratory. A complex mixture can be separated into literally thousands of components, using fused-silica capillary columns that are a direct spin-off from optical fiber technology for communications. It is even possible to separate compounds that differ only in isotopic composition. , . _ High-Performance Liquid Chromatography (HPLC) Dunng the 1970s, theoretical understandings of the complex flow and mass transfer phenomena involved in chromatographic separation helped perfect column design. During this same period, small-diameter (3-10-micron) silica particles with controlled porosity were introduced. Synthetic advances in silica chemistry led to the tailoring of particle diameter, pore diameter, and pore size distribution. Today, 15-cm columns with efficiencies exceeding 10,000 distillation steps ("theoretical plates") are routine. Still another major advance of the 1970s was the introduction of chemically bonded phases in which surfaces of porous silica are covalently coated with organic molecules containing silicon (organosilanes). Especially important is the use of hydrocarbon attachments (such as n-octy} and n-octadecyI) to make the surface look like an organic solvent. Then, the mobile liquid phase is typically an organic-aqueous mixture. This is called reversed phase chromatography (RPEC), and it currently provides well over 50 percent of all HPEC separations. It is especially well suited to substances that are at least partially soluble in water (drugs, biochemicals, aromatics, etc.~. Finally, the microprocessor/computer is playing an increasing role. "Smart" HPEC instruments are under development to program the performance. New 195

196 INSTRUMENTATION lN CHEMISTRY detectors of greater sensitivity and selectivity are on the horizon. In particular, laser spectroscopy promises to yield highly sensitive devices for subpicogram detection (less than 1o-~2 grams). Because of performance improvements, HPEC is having a major impact on diverse fields of biochemistry, biomedicine, pharmaceutical development, environ- mental monitonng, and forensic science. Today, peptide analysis and isolation requires HPEC because of its separating power and speed. Analysis of amino acids in protein/peptide sequencing is conventionally accomplished by RPEC. In clinical analysis, therapeutic drug monitoring can be accomplished by HPEC. The analysis of catecholamines (important as "neurotransmitters") is typically accomplished by RPEC with electrochemical detection. Isoenzyme analysis, which is important, for example, in assessment of heart damage after an attack, can be rapidly accom- plished by HPEC. The analysis of polar and high-molecular-weight organic species in waste streams in sewage or factory treatment can be performed by HPEC, while the separation and analysis of phenols in water supplies by RPEC is recommended. Analysis of narcotics, inks, paints, and blood represent only a few of the forensic applications in police laboratories. Capillary Chromatography This version of chromatography uses an open capillary tube with a thin liquid layer on its inner wall. It began with capillary gas chromatography (GC), but the fragility of glass as an inert matenal for GC capillary columns discouraged many potential users. Now we have flexible, fused-silica capilIanes with a polymer overcoat, a spin-off of fiber optics technology. These advances in capillary column technology led to intensive commercialization dunng the 1970s. Today's capillary columns exhibit efficiencies between ]05 and 106 distillation steps ("theoretical plates") and are capable of separating literally hundreds of components within a narrow boiling point range. Direct introduction of samples at the nanogram (IO-9 grams) levels has been developed, and much effort has been directed at perfection of gas-phase ionization detectors. Combined advances in the column and detector areas now make trace analytical determinations below 10-~2-gram levels practical by capillary gas chromatography. Of particular note is the combination of capillary GC with powerful identification methods such as mass spectrometry and Fourier Transform infrared spectroscopy, as mentioned in Section IV-C. The combined techniques are now routinely capable of identifying numerous compounds of interest that are present in complex mixtures in only nanogram quantities. They have been used in identification of new biologically important molecules, as well as in drug metabolism studies, forensic applications, and identifications of trace environmental pollutants. Every fluid has a characteristic temperature and pressure above which its gas and liquid phases become indistinguishable. Above these critical conditions, the "supercritical" fluid displays exceptionally low viscosity, and it can become a much better solvent. Consequently, the use of supercntical fluids in capillary chromatography has recently emerged as a promising approach to the analysis of complex nonvolatile mixtures. As the solute diffusion coefficients and viscosities of supercritical fluids are more favorable than those of normal liquids, chromato-

V-C. INSTRUMENTATION AND THE NATIONAL WELL-BEING graphic performance is substantially enhanced. Furthermore, the optical transpar- ency of supercritical fluids makes them attractive for certain optical detection techniques. Field-Flow Fractionation (FFF) Chromatography becomes more difficult to apply as molecular size grows, and it becomes ineffective in separating macromolecules and colloidal particles in the size range 0.01 to 1 micron in diameter. A recent innovation, field-flow fractionation, may fill this need. In FFF, a liquid sample flows through a thin (0.~-0.3 mm), nbbon-like flow channel. A temperature difference or electric field is maintained across the ribbon. Each constituent in the sample distributes itself in a way that is determined by its diffusional properties and its response to the applied thermal or electncal field. Since flow through the channel is fastest near the middle of the ribbon, substances that are pulled close to the ribbon wall move more slowly than substances that reside near the middle of the flow channel. Separations are thus achieved. A useful aspect of this technique is that the strength of the applied field can be varied in a deliberate and programmed way by a computer during the course of the separation. Such thermal gradients are effective in separating most synthetic polymers. The mass range of molecules and particles to which FFF has been applied extends from molecular weights of 1,000 up to 10~8, that is, up to particle sizes of about 100-micron diameters. FFF appears to be applicable to nearly any complex molecular or particulate material within that vast range. Applications have so far included macromolecules and particles of biological and biomedical relevance (proteins, viruses, subcellular particles, liposomes, artificial blood, and whole celIs), of industrial importance (both nonpolar and water-soluble polymers, coal liquid residues, emulsions, and colloidal silica), and of environ- mental significance (waterborne colloids and the tiny particles called fly ash in smoke plumes). INFRARED SPECTROSCOPY A molecule can be pictorially, but accurately, viewed as a collection of wooden balls held together in a fixed geometry by springs. The masses of the balls are proportional to the atomic masses, and the strengths of the springs are proportional to the strengths of the chemical bonds. Such a "ball-and-spring" mode! will have resonant vibrational frequencies in which the wooden balls move back and forth ire regular patterns. These frequencies are determined by the masses, the spnug constants, and the geometry. A molecule is exactly the same. If measured, the resonant fre- quencies give direct informa- tion about the molecular archi- tecture. Consider, for example, the water molecule. This bent, Resonant Vibrational Motions of H2O it'd \~ /~/ All or ~ '0 'A %~` it\\ 3587 cm~ 1 35(30 cm~ 1 1600 cm~ Vibrational Frequencies Reveal Bond Strengths and Bond Angles 197

198 At A: o a: INSTRUMENTATION IN CHEMISTRY tnatomic molecule has three resonant vibrations. In one of these, the two bonds stretch back and forth in phase, and in another, the two bonds both stretch, but this time out of phase. In the third characteristic vibration, the bond angle alternately opens and closes. Such molecular vibrations do not break bonds, so they require little energy. Absorption of light is one way to excite these vibrations, but photons of appropriate energy are in the infrared spectral region, far beyond the visual sensitivity of the human eye. A typical molecular vibration, such as the bending motion of the water molecule, has a frequency of 4.S x 10~3 vibrations per second. This unwieldy number is usually brought into reasonable magnitude by dividing it by the speed of light, which changes the dimensions to I/cm or cm- ("reciprocal centimeters". 4.8 X 10~3 vibrations/sec _ = 3 x 10~° cm/see 1,600 cm-~. Infrared vibrational frequencies are always expressed in reciprocal centimeters (cm-~) (sometimes called "wave numbers". The measurement of these molec- ular frequencies is called vibrational spectroscopy or infrared spectroscopy. These vibrational frequencies are so characteristic that they furnish a distinctive and easily measured "fingerprint" for each molecule. This spectral fingerprint, once measured for a particular molecule, can be used to determine whether that molecule is present in a sample and, if so, how much. The vibrational frequencies also reveal the molecular structure and bond strengths in the molecule, so they can be used to learn about the molecular architecture. When an unknown compound is under study, the infrared spectrum provides one of the easiest ways to decide what the compound is likely to be. Because infrared spectroscopy is so informa- tive, it has become one of the routine diagnostic tools of chemistry. A large, research-oriented chemistry department might operate 5 to 10 infra- red spectrometers with capabilities ranging from rugged, low-resolution instrument for instruction in an advanced first-year chemistry course, to high-resolution Fourier Transform Infrared Spec- trometers (FTIR), suited to molecular structure determination and specialized research use. DIFLUOROPROPENE 1R SOLID KRYPTON, ~ 2 Mel cis and gauche em_ JE - ~£ PBOlOLTSIS (hi me_ ~1 ~ gaucaet l ~_J ~ By- --or- ~ cis1 . I I I' I t 1400 1200 1000 V(cm l) _~ ·"E. (hi)—BE=RE (hi) ~""""'B~"~\~"'~"'~ FTIR DIFFERENCE SPECTROSCOPY SHOWS ROTAMER INTERCONVERSION Computer-Aided Spectrometers Modern research infrared spectrometers incor- porate computers to permit programmed opera- tion, data collection, and data manipulation. The major impact of computers, however, has been their influence on the performance of Fourier Transform interferometers. The perfection of the

V<. INSTRUMENTATION AND THE NATIONAL WELL-BEING Fourier Transform algorithm (program), plus the reduction in accompanying computer costs, brought the interferometer from a trouble-plagued, research- only instrument to a routine, high-performance workhorse. A notable capability brought by the computer is the ease and accuracy with which one spectrum can be subtracted from another to emphasize small changes. This is called a difference spectrum. One important application relates to infrared spectra of biological samples in which evidence of a chemical change associated with a certain specific biological function can be completely covered up by the heavy infrared spectrum of the inactive substrate in which the sample is located. The digitized data permit precise spectral subtraction so that the back- ground spectrum can be virtually eliminated to reveal the spectral changes of interest. Another vivid display of the value of the difference capability is provided by photolysis of molecules suspended in a cryogenic solid ('`matrix isolations. If the digitized spectrum before photolysis is subtracted from the spectrum after photolysis, only the features that change are seen. Any molecule that is being consumed presents its spectral features downward, while spectral features of the growing product extend upward. This has been used, for example, to distinguish the two forms (cis- and gauche-) of the 2,3,-difluoropropene in the cluttered spectrum of a complex mixture. A laser tuned to an absorption frequency of one of the "rotamers," say, the cis- form, is used to irradiate the cold sample. Absorption of this light adds enough energy to the absorbing cis- molecule to permit it to convert to the gauche- form. Then in the difference spectrum, the cis- molecule spectrum appears as a negative spectrum and the gauche- molecule spectrum as a positive one. Absorptions due to other molecules do not change, so they simply do not appear at all. Applications The coupling of FTIR with gas chromatographic separations in a variety of analytical uses has been dis- cussed. Also, as noted earlier, infrared spectroscopy is a spe- cially effective method for monitoring and studying at- mospheric chemistry. This is because gaseous molecules of Laser magnetic low molecular weight are im- portar~t, including formaide- hyde, nitric acid, sulfur diox- ide, acetaldehyde, ozone, ox- ides of chlorine and nitrogen, nitrous oxide, carbon dioxide, and the Freons. These sub- stances are influential partici- pants in photochemical smog production, acid rain, strato- TABLE V-C-2 Additional Instrumental Techniques in Modern Chemistry Instrument Information Obtained Reaction rates of gaseous molecular ions Precise molecular structures, gaseous free radicals Vibrational spectrum Lifetimes9 electronically excited molecules Stereo confol~llations Laser-activated cell sorter Automated analysis of protein sequence Automated synthesis of designed DNA segments Molecular structure, gases Tracking radiotracers Ion cyclotron resonance resonance Laser Raman Fluorimeter Circular dichroism Flow cytometer Protein sequencer Oligonucleotide synthesizer Electron diffraction Scintillation counter 199

200 INSTRUMENTATION IN CHEMISTRY spheric disturbance of the ozone layer, and the "greenhouse effect.'' Infrared spectroscopy shows where they are and how much is there. OTHER INSTRUMENTATION In Sections V-A, V-B, and V-C, there has been detailed discussion of over a dozen different classes of instrumentation which are important in defining and advancing the current frontiers of chemistry. By no means, however, is the list all-inclusive. Table V-C-2 lists additional types of equipment and what kinds of chemical information each one provides. The length of this table is only one more signal of the crucial importance of instrumentation in modern chemistry. SUPPLEMENTARY READING Chemical & Engineering News "Instrumentation '86~0ptical Spectroscopy" (C.& E.N. staff), vol. 64, pp. 3442, Mar. 24, 1986. "Instrumentation 'chromatography" (C. & E.N. staff), vol. 64, pp. 52-68, Mar. 24, 1986. "Instrumentation '8~Mass Spectrometry" (C.& E.N. staff), vol. 64, pp. 70-72, Mar. 24, 1986. "Low Cost FTIR Microscopy Units Gain Wider Use in Microanalysis" (C.& E.N. stab, vol. 63, pp. 15-16, Dec. 9, 1985. "pity Chromatography" by Parikh and P. Cuatrecasas, vol. 63, pp. 17-31, Aug. 26, 1985. "GC Detector Uses Gold Catalyst for Oxi- dation Reactions" by W. Worthy (C.& Em. staff), vol. 63, pp. 42-44, June 24, 1985. '~X-Ray Technique May Provide New Way to Study Surfaces, Films" by W. Worthy (C.& E.N. stab, vol. 63, pp. 28-30, April 8, 1985. "Centrifugal Force Speeds Up Countercur- rent Chromatography" by S.C. Stinson (C.& E.N. staid, vol. 62, pp. 35-37, Nov. 26, 1984. Science "A New Dimension in Gas Chromatogra- phy" by T.H. Maugh II (Science stair), vol. 227, pp. 1570-1571, Mar. 29, 1985. "Ion Beams for Compositional Analysis" (SIMS), by A.L. Robinson (Science stab), vol. 227, pp. 1571-1572, Mar. 29, 1985.

CHAPTER VI The Risk/Benefit Equation in Chemistry

-I ~- ~o',~ ~ f — ~ ~ —~<~ ~~ OF <~lnvestigat~ng Smog Soup ~c ~ . I,=, ~0 ~ o'N`o ~ ~ Air pollution Is a visible reminder of the price we sometimes pay for progress. Emissions from thousands of sources pour into the atmosphere a myriad of molecules that react and re-react to forth a "smog soup." We are already aware of some of the potential dangers of leaving these processes unstudied and unchecked: respiratory ailments, acid rain, and the greenhouse eject. Sur~ns~ngly, you and ~ are the principal cuipnts in generating much of this unpleasant brew~veryt~me we start our cars or switch on our air conditioning or central heating! Transportation, heating, cooling, and lighting account for about two-thirds of U.S. energy use, almost all derived from combustion of petroleum and coal. Pinpointing cause and effect relationships begins, inevitably, with the identification and measurement of what is up there, tiny molecules at parts-per-billion concentrations in the mixing bowl of the sky. Finding out what substances are there, how they are reacting, where they came from, and what can be done about them are all matters of chemistry. The first two questions require accurate analysis of trace pollutants. Physical and analytic chemists have successfully applied to such detective work their most sensitive techniques. An example is the Fourier Transform Infrared Spectrome- ter. This sophisticated device can look through a mile or so of city air and identify all the chemical substances present and tell us their concentrations down to the parts-per- billion level. Recognizing a substance at such a low concentration is comparable to asking a machine to recognize you in a crowd at a rock concert attended by the entire U.S. population. ~ . . . How does this superb device work? "Infrared" means light just beyond the red end of the rainbow visible to the human eye. Hence infrared light is invisible, though we can tell it is there by the warmth felt under an infrared lamp. But molecules can '~see" inhered light. Every polyatomic molecule absorbs infrared "colors" that are uniquely characteristic of its molecular structure. Thus each molecular substance has an infrared absorption "fingerpr~nt"~i~erent from any other substance. By examining these finge~pnnts, chemists cart identify the molecules that are present. Boa An example of what can be done is the measurement of formaldehyde and nitric acid as trace constituents in Los Angeles smog. Unequivocal detection, ~ using almost a m~le-long path through the polluted air, revealed the growth _ ~ \ dunug the day of these two bad actors and tied their production to photochemical processes initiated by sunlight. Continuing experiments led ~'J \ to detailed characterization of the simultaneous and interacting concentra- @_: ~ tions of ozone, peroxyacetyl nitrate (PAN), formic acid, formaldehyde, and nitric acid ir1 the atmosphere. These detections removed an obstacle to the complete understanding of how unburned gasoline and oxides of iitrogeI1 leaving our exhaust pipe end up as eye and lung irritants in the atmosphere. This advance doesn't eliminate smog =~ soup, but it is a bin steD toward that desirable end ~ n' ~ ~ _ ~ i1~ =~w 202

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Experts agree that the nation would benefit if more young people "turned on" to the sciences. This book is designed as a tool to do just that. It is based on Opportunities in Chemistry, a National Research Council publication that incorporated the contributions of 350 researchers working at the frontiers of the field. Chemistry educators Janice A. Coonrod and the late George C. Pimentel revised the material to capture the interest of today's student.

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The book concludes with a discussion of chemistry's role in society's risk-benefit decisions and a review of career and educational opportunities.

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