Serendipity, TECHNOLOGY, and
Challenges
in Chemical Instrumentation Edward S. Yeung Ames Laboratory, U.S. Department of Energy and Department of Chemistry Iowa State University Ames, Iowa 50011
One of the things that is not taught in graduate school is how to formulate ideas for research. During our research, we often stumble upon those ideas that eventually lead to success. After a while, we seem to get the feel of it, and ideas begin to flow more readily. Looking back at some of the major developments in our laboratory in the past few years, I can identify specific instances when at least one of three factors—serendipity, technology, and challenges— played a key role in our approach. It is well known that the development of science is often influenced by serendipity. Examples in analytical chemistry have been cited in an earlier REPORT (1). I will not describe accidental laboratory events that led to discoveries. Rather, I will describe here some unexpected situations that brought us to the right place at the right time in order to generate research ideas. Capacities of chemical instrumentation depend on the availability of appropriate technologies. Sometimes the driving force is in the reverse direction. Big lasers and fancy gadgets cry out for incorporation into new instrumentation. We have certainly benefited from being in close contact with technological developments in other fields. Finally, the presence of challenges is important to good progress in any endeavor. Potential satisfaction derived 0003-2700/88/0360-441 A/$01.50/0 © 1988 American Chemical Society
REPORT from improving the capabilities of chemical instrumentation often keeps us from quitting too soon. Collecting enough data for a conference presentation is another strong incentive for moving things along. Case in point #1 About 10 years ago, then-budding graduate student J. C. Kuo stopped me in the hallway and indicated an interest in working on a project dealing with the detection of lipids (possibly because his wife was a student in the Food and Nutrition Department). I pointed out that others had already done that with laser light scattering (2). I was just about to leave for an appointment when it occurred to me that sterols are optically active. I wondered if perhaps there could be some merit to developing an optical activity detector for liquid chromatography (LC). After searching the literature for an afternoon, I concluded that the easy route— using a commercial polarimeter—does not work because of poor detectability. We subsequently turned our attention to the laser. It seemed that the key technology would involve the extinction ratio, which is the imperfection in polarizers allowing the transmission of light of the wrong polarization. Commercial polarizers are specified at one part in 106. Our approach was to use two polarizers and two analyzers and to hope for one in 1012. It became obvious that things were not that simple (otherwise, that ap-
proach probably would have been attempted previously). The idea was totally wrong, and the proposal would not have been funded. We had not considered that imperfections in the second half of the Glan-Thompson polarizer and in the first half of the analyzer cause depolarization and allow transmission. We then lowered our expectations and decided just to put something together and hope for the best. At that time, Larry Steenhoek was helping out in the evaluation of polarizers because he was our expert in photon counting. I walked by his desk one day and noticed a loose sheet of paper with photon counts written all over it. What stood out was that some numbers were 3 orders of magnitude larger than others. It occurred to me that if the worst of these were true to the manufacturer's specifications, the best were substantially better. We promptly confirmed that specific faces of polarizer-analyzer combinations exhibited much better extinction ratios than expected. The small size of the laser beam further allowed us to search for special locations on each polarizer. Eventually, we achieved extinction at one part in 1010. The initial trials were tedious and resembled a "shotgun" approach. Worse still, we had no idea what "best" results would be. The challenge was there to achieve a level of performance that would hold up over time. It is amazing that one can look This R E P O R T is based on the award address given by Edward Yeung when he received the 1987 ACS Division of Analytical Chemistry Award in Chemical Instrumentation at the ACS fall national meeting in New Orleans (September 1987).
ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988 · 441 A
directly into an argon ion laser at 1W through crossed polarizers and see only a faint spot of light. The rest was hard work. We had to put a flow cell in the cavity without degrading the good extinction. Draw ing on our experience with polarizers, we tried just about every type of win dow material without success. Eventu ally we came across thin windows that were none other than microscope cover slips. They worked! In effect, we low ered the probability of having imper fections in the window material by us ing a short path length. We were so confident then that we submitted an abstract for the ACS national meeting, which would be held in six months. We had not anticipated problems such as mounting the windows, putting togeth er the proper electronic modulation system, and dealing with flow turbu lence. The deadline for the conference presentation really made us bear down. We produced the first chromatogram one week before the meeting. Over the years, more improvements have been introduced, so that the de tection of 0.1 ppm of fructose in a l-/iL flow cell has become feasible (3, 4). This detectability is about 1000 times better than that in commercial instru ments. It took us a long time to address the determination of sterols, because for a while we concentrated on refining the system. The experiments were triv ial once we had the instrument, but the results shown in Figure 1 (5) attracted a lot of attention, mainly because of the key word—cholesterol.
ing the second chromatogram, provid ed that the two solvents have different RIs, leading to different responses for the unknowns. Figure 2 schematically depicts these responses and demon strates how one can determine Cx with out analyte identification. On the trip back from Phillips, I was very anxious. My new ideas sounded so simple that I felt there had to be a flaw somewhere. We teach undergraduates that one must identify the analyte be fore quantitation. Was conventional wisdom justified in this case? After finding no precedents in a brief litera ture search, I persuaded Rob Synovec to give the idea a try. Armed with sim ple simultaneous equations based on the linear dependence of the measured signal on the difference in RI between the solute and the solvent, we took some measurements on an Abbe refractometer. The results were terrible. The predicted concentrations for the simu lated "unknowns" were far from the actual concentrations. I then took it as a personal challenge to show that the idea was sound. An afternoon spent flipping through phys ics books provided some answers. The concentrations are related to the RIs quadratically and not linearly. With new equations, we were able to use the old data from the refractometer to pre dict the concentrations with good accu racy (6). The linear equations quoted in undergraduate textbooks are ap-
Case in point #2 At about the same time that we were addressing the sterol issue, Stan Spurlin of our group received a fellowship from the Phillips Petroleum Company. He and I were invited to visit their ana lytical laboratories and to give a pre sentation. I learned there that the stan dard practice in the petroleum field is to collect fractions using size exclusion chromatography (SEC), to vaporize the solvent, and then to use the weight or volume of the residue in each frac tion to characterize the crude oil. Mil lions of dollars each day depend on this tedious and unreliable method. I was convinced that there must be an alter native method. It then occurred to me that the re fractive index (RI) detector sitting right next to the SEC column has some special properties. Its signal depends on the solvent as well as the solute. In any chromatogram, the concentration (Cx) and the RI of the solute are the two unknowns. If one can somehow change the response to obtain a second chro matogram, then the two unknowns can be determined from the set of simulta neous equations. Obviously, changing the solvent is one approach to obtain
Figure 1. Optical activity chromatogram of cholesterol (A) and its various fatty acid esters (B-E) derived from 50 μ ι of human blood. Reversed-phase chromatography was used with 76:24 THF:water as the eluent. (Adapted with per mission from Reference 5.)
442 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 7, APRIL 1, 1988
Cx in solvent 1
s1
Cx in solvent 2
s2
C2 in solvent 1
s3
C1 in solvent 2
s4
Figure 2. Scheme for quantitation with out identification in LC. The analyte χ gives different responses, S\ and S2, when eluted by solvents 1 and 2, respectively. The solvents are then "calibrated" by using the responses S3 and S4, produced when they are eluted by each other. For the case C = C1 = C2, the unknown concentration is simply given by Cx = C(S,/S3 + S2/S4).
proximations that are applicable to high analyte concentrations. At low concentrations, the curvature is pro nounced. Later, we found that at the low concentrations typical of LC, the response is linear once again, but it is associated with a different slope. Drawing on our experience of nonlin ear detector response, we expanded our efforts to understand the response in the common LC detectors to see if these also fit into the "quantitation without standards" scheme. We had high hopes for the thermal conductiv ity detector in gas chromatography (GC), but we were unable to unravel the complicated response functions. Kristy Skogerboe was finally successful in using the ultrasound detector in GC (7), extending the scheme to picogram levels of analyte. Don Bobbitt extend ed the scheme to the polarimeter (8), which still stands as the best detector in this mode for LC. This is because a mechanical adjustment can be made to suppress the high intensity that occurs when an optically active eluent is used. Good detectability thus is possible in either eluent. Another important ad vantage of polarimetry is that chro matographic retention does not change when different enantiomers are used as the eluents, except in the rare case of chiral interactions. It is interesting that technology did not play much of a part in the develop ment of this concept. All instruments except the polarimeter were standard commercial devices. We simply found a new use for old instruments. For the
polarimeter, we did need the high performance offered by the laser to achieve useful levels of detection. We also benefited from the emergence of microcolumn separation technology to reduce solvent cost when chiral eluents are used. In fact, this becomes a justification for moving toward small columns, which allow the use of exotic solvents to provide unique information. Eventually we returned to the problem that started all of this—crude oil characterization (9). We spent more time getting the crude oil samples than doing the experiments. We did, however, begin to appreciate the other unknown in the equations—RI. It gave a clear picture of saturated versus unsaturated versus aromatic hydrocarbon contents. As scientists, we often discount simple experiments because they are not challenging, and consequently we miss opportunities for new insights. Bobbitt's results included the detection of optically inactive species using the polarimeter. This indirect detection mode led to a flurry of other activities in our group. When instruments do not respond to a particular analyte, it is still possible to use them for detection in chromatography. The eluent, if properly chosen, can create a background signal at the detector. This is represented by the open circles in Figure 3. When an analyte elutes, as shown by the solid circles in Figure 3, there will be less of the eluent species at the detector because of displacement, and a decrease in signal (negative peak) will occur. This greatly extends the usefulness of instruments in general and provides universal detection with uniform response. Previous articles have dealt with this idea, notably with regard to indirect photometry (10) and vacancy chromatography (11). The polarimeter is unique in having
Figure 3. General scheme for indirect detection. The detector sees a large constant background produced by species (open circles) in the eluent (left). When the analytes (solid circles) elute, they displace some of the eluent species (right) to cause a decrease in the detector response, that is, a negative peak.
a high dynamic reserve, which is the ability to record a small change on top of a large background signal. This is easily seen from Figure 4. For this reason, simple volume displacement is sufficient, as opposed to the requirement of charge displacement in ion chromatography. The stability of the laser intensity rather than its raw power becomes the critical issue. If that can be controlled, indirect detection in all optical schemes will gain in power. Case in point #3 Another serendipitous event happened during that period. Dick Keller of Los Alamos National Laboratory invited me to a conference in San Diego. Although that was outside my usual circuit of conferences, I went only because we were to hold a mini Gordon conference there to assess the field. A heated discussion between the physicists about shot-noise-limited detection took place. Jargon aside, the main point was that absorbance at the level of 1 ppm could be detected. That represented a small change on top of the large laser intensity. The trick was to suppress flicker noise, which depends inversely on frequency, by using highfrequency modulation. The physicists are helped by the fact that atomic lines are narrow and that wavelength modu-
Figure 4. The importance of dynamic reserve (DR) in indirect detection. The background signal is necessarily large. The stability in the background signal determines how small a change on top of that is detectable.
lation at high frequencies—in order to approach the shot-noise limit—is relatively easy. How could we adapt it to measurements in liquids, where wavelength modulation could not be used? Mike Morris of the University of Michigan and I continued the discussion at poolside. It was a challenge that neither of us had a good answer for at that time. I was convinced that the technology was out there but that we had to find it. For years, people have inquired about our new "CD" detector. When
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they do, I respond that our success was in polarimetry and not in circular dichroism. Although these two phenome na are easily confused, the instrumen tation is very different. Polarimetry re quires working at low light levels, whereas CD involves measuring the small difference between two large sig nals. It became clear to us that CD can be improved by high-frequency modu lation. A subtle aspect is that polariza tion is modulated between the left and right circular components, but every thing else is identical in the optical path. It is, in fact, a double-beam mea surement in a single-beam arrange ment. It took some time to find the correct laser and the correct driving electronics to do the experiment. Rob Synovec was able to suppress noise to one-half of a micro-absorbance unit (12). This provided CD measurements in LC with a detectability about 110 times better than that of commercial instruments. We attempted to extend high-frequency modulation to stan dard absorption measurements, but because we needed to use alternating beams in separate optical paths, true compensation was not possible. At the microabsorbance level, even a dust par ticle passing through one beam and not the other causes noise. The factor-oftwo improvement over conventional
in indirect fluorescence. Performance is therefore degraded. However, 104 is still quite an improvement over the 102 level inherent in the laser intensities. At that stage, detectability in indi rect fluorometry for anions was compa rable to, but not superior to, detectabil ity resulting from indirect photometry or conductivity. We knew that more work lay ahead of us. The next step had to involve chromatography. For a given dynamic reserve, detectability is pro portional to the concentration of the fluorophore in the eluent. In ion chro matography, this is the fluorescing eluting ion. Standard columns simply have too large a capacity to allow the use of eluent concentrations much be low 1 mM. We accepted the challenge and began to dynamically modify our own columns to achieve low capacities. Toyohide Takeuchi eventually found the right combinations. Eluent concen trations as low as 1 μΜ could be used. By using open tubular capillary col umns, the noise levels were equivalent to 1 pg of CI" or N0 3 ~ injected (14). Actually, we were able to improve the concentration detectability by only 1 order of magnitude. The smaller vol umes and our weak (8 mW) UV laser beam further degraded the dynamic re serve. Furthermore, the relatively large i.d. of our capillary column did not fa-
spectrometers does not justify all the complications introduced into the in strument. Still, we were able to partially over come the intensity fluctuations in laser beams in general. This immediately made some improvements in the polar imeter (4). It also brought new life to our pursuit of indirect detection meth ods. The challenge was to develop indi rect fluorescence. Perhaps one could transfer the advantages of fluorometry, such as low detectability and small vol umes, to nonfluorescing species as well. Sun-il Mho subsequently developed a double-beam fluorometer suitable for LC {13). Whereas in normal fluorome try we try to reduce the background as much as possible, indirect fluorometry depends on having a large background. The double-beam arrangement is es sential for good detection. The dynam ic reserve in fluorometry has only ap proached 104, whereas we were able to achieve 105 in absorption, 106 in CD, and 108 in polarimetry. This demon strates the subtle features of modula tion. The reference beam provides compensation only if the reference cell and the sample cell are exactly matched. Vibrations, heat-induced turbulence, photobleaching, light scat tering, and other phenomena are not identical in the two capillary flow cells
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vor producing very sharp analyte peaks. Ironically, the results were limited by the laser and the column technology in our laboratory. With smaller columns and higher laser powers, it should have been possible to apply indirect fluorometric detection to the profiling of the fluid inside a single cell. Our optimism for indirect fluorescence, however, goes beyond further improvements in mass detectability. We note that the analogous absorption techniques cannot be miniaturized or scaled down in concentration because of decreasing sensitivity. Indirect fluorescence also allows one to choose the fluorophore and thus the laser source for excitation, greatly reducing the complexity of the instrument. In addition, it is not restricted to ion chromatography. Competition for adsorption on the stationary phase produces a similar displacement in reversed-phase chromatography (15, 16). The idea is also applicable to thin-layer chromatography and to electrophoresis. Spots can be visualized without derivatization or staining, resulting in increased reliability and added convenience. Our work in chemical instrumentation has been influenced by serendipity, technology, and challenges. A casual conversation in the hallway, an unexpected visit to an analytical laboratory, and attendance at an odd conference were the seeds of our accomplishments. Ideas came to fruition because of the availability of the appropriate technology such as lasers, polarizers, fast modulation electronics, and microcolumn separation. The rest was hard work, facing the challenges posed by ourselves and by others. We had a lot of fun.
(13) Mho, S. I.; Yeung, E. S. Anal. Chem. 1985,57,2253-56. (14) Pfeffer, W. D.; Takeuchi, T.; Yeung, E. S. Chromatographia, in press. (15) Stranahan, J. J.; Deming, S. N. Anal. Chem. 1982,54,1540-46. (16) Takeuchi, T.; Yeung, E. S. J. Chromatogr. 1986,366,145-52. Edward S. Yeung is a professor in the department of chemistry and program director of environmental sciences at Ames Laboratory, Iowa State University. He received his A.B. degree from Cornell in 1968 and his Ph.D. degree from the University of California, Berkeley, in 1972. He is an Associate Editor
of ANALYTICAL
CHEMISTRY.
His research interests include spectroscopy, chromatography, photochemistry.
laser and
Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract no. W-7405-Eng-82. The work described here was supported by tfie director for energy research (Office of Basic Energy Sciences), by the Research Corporation through a Cottrell grant, and by the Dow Chemical Company. References (1) Lott, P. F. Anal. Chem. 1983, 55, 245 A-248 A. (2) Jorgenson, J. W.; Smith, S. L.; Novotny, M. J. Chromatogr. 1977,142, 23340. (3) Bobbitt, D. R.; Yeung, E. S. Anal. Chem. 1985,57, 271-74. (4) Bobbitt, D. R.; Yeung, E. S. Appl. Spectrosc. 1986, 40, 407-10. (5) Kuo, J. C; Yeung, E. S. J. Chromatogr. 1982,229,293-300. (6) Synovec, R. E.; Yeung, E. S. Anal. Chem. 1983,55,1599-1603. (7) Skogerboe, K. J.; Yeung, E. S. Anal. Chem. 1984,56, 2684-686. (8) Bobbitt, D. R.; Yeung, E. S. Anal. Chem. 1984; 56,1577-81. (9) Synovec, R. E.; Yeung, E. S. J. Chromatogr. Sci. 1986,23, 214-20. (10) Small, H.; Miller, T. E. Anal. Chem. 1982,54,462-69. (11) Scott, R. F. W.; Scott, C. G.; Kucera, P. Anal. Chem. 1972, 44,100-104. (12) Synovec, R. E.; Yeung, E. S. Anal. Chem. 1985, 57, 2606-10.
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