J. E. Katm G. E. Pacey J. F. O’Keefe Molecular Microspectroscopy Labwatory Department of Chemistry Miami University Oxford, Ohio 45056
The rapid pace of technology in recent years has placed great demands on analytical chemistry to develop methods and techniques for the identification and quantitation of ever smaller amounts of various materials. Although this challenge, and the instrumental methods developed to meet it, has received much attention with respect to elemental composition of samples, it has only recently been addressed in a general way with re,spect to molecular species. General identification of compounds a t the picogram level can now be accomplished through the techniques of molecular microspectroscopy,i.e., molecular spectroscopy through a microscope. Although there are many different molecular spectroscopies, the most versatile ones for identification are infrared (IR) and Raman spectroscopies. Those molecular spectroscopies involving electronic transitions in molecules (UV-VIS absorption, fluorescence, and phosphorescence), although considerably.better for quantitation, are not sufficiently selective to he widely applicable. Other spectroscopies, for example, microwave and nuclear magnetic resonance, are limited by their experimental requirements. IR spectroscopy has long been a mainstay of the analytical chemist’s arsenal for identification of molecular systems because of its high selectivity and applicability to essentially all molecules in all physical phases. Raman spectroscopy has been less widely used because of its high instrumental 0003-2700/86/0358-465A$I S O / O
@ 1986 American Chemical Society
Figure 1. Chronology of major contributors to vibrational molecular microspectrosCOPY
costs but is recognized by most vibrational spectroscopists as being useful because it provides, in many cases, data complementary to the IR data. Both techniques are nearly universal and very selective. Both may be applied to mixtures hut often give better results if coupled with some preliminary separation method. Until the advent of molecular microspectroscopy,IR and Raman were generally considered applicable to microgram quantities of materials with use of good sampling technique and special accessories. If a material were volatile, GC/FT-IR could he used to identify it a t the tens-of-nanograms level or even lower. Molecular identifi. cation of material in quantities less than the nanogram level was not practical. The development of microprobe Raman and microscopic IR makes it possible to identify picogram quanti-
ties and to detect femtogram levels of many substances. When new techniques and methods come upon the scientific scene it is often because of recently discovered principles, advances in theory, or the development of a new instrumental concept. Molecular microspectroscopy did not develop in any of these ways. Rather, it is a method that developed naturally because of a series of progressive improvements in other techniques. Finally, each of these techniques reached a state of sophistication such that practical molecular microspectroscopybecame a reality. A sort of family genealogy of IR and Raman microsDectroscoov ._is .oresented in Figure 1. In the late 1800s Michelson developed his incerferorneter for studying the speed of light. Some 70 years earlier, in France, Fourier had developed
ANALYTICAL CHEMISTRY, VOL. 58. NO. 3, MARCH 1986
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hnald D. Bills, Editor I S . Department of Agriculture anthia J. Mussinan, Editor nternational Flavors and Fragrances
Figure 2. Cutaway schematic of a Cassegrainian lens
ixamines the sensory, chemical, and ihysical methods for characterizing ind measuring flavor compounds. iighli hts state-of-the-art instrumena1 techques and their application 0 studies of flavor. Also covers ienso methods as well as new nethoTs for extracting, derivatizing, md manipulating flavor c m m n d s .
the mathematical technique that sewed to convert the output of the Michelson interferometer, an interferogram, into a spectrum. In the 192Os, W. W. Coblentz, working a t the National Bureau of Standards, showed that IR spectra of compounds were characteristic physical properties of these compounds and could he used for their identification. At about the same time, C. V. Raman, in India, showed that Raman spectra were similarly useful. The Raman effect, because of its inherent weakness, did not become a generally effective method of studying substances until after the development of the laser, pioneered by C. H. Townes. Both IR and Raman spectroscopy received a final impetus and advance through their coupling with the modern digital computer, fathered by von Neumann. Only after all these major developments (and a host of minor advances) had occurred were IR and Raman ready to be coupled with the optical microscope, an instrument whose uses had been developed in the seventeenth century by van Leeuwenhoek. The final result is two complementary methods for identification of molecular species a t the picogram level with promise of extension in the near future to the femtogram level. To study these techniques, their complementarity and application, the
:ONTENTS jensary Evaluation of Food Flavors * Sub ltances That Modify the Perception 01 Sweeti e s * Sensory Responses to Oral Chemical ieat * Analysis of Chiral Aroma Components n Trace Amounts * New Analytical Method or Volatile Aldehydes The Use of High'erhrmance Liquid Chromatography in :law Studies * Capillary Gas Chromate iraphic Analysis Of Volatile Flavor Comwunds High-RemlutionGab Chromatogra. )hy-Fourier Transform IR Spectroscopy in 'lawn Analpis * Tandem Mass Spedrometr) Wplied to the Charaderimion 01 Flavor :ompounds Automated Analysis of Volatile 4avor Compounds * Supercritical Fluid ixtraction in Flavor Applications
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) e v W f m m a symposium sponswed by he Flevw Subdivism of the Oivisim of Igricunural and Food Chemistry of fhe Imerican ChemicalSociety
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Molecular Microspectn DPYLaboratory (MML) at Miami University was designed and developed to perform research and method development in this new area. This laboratory became operational in early 1985. Although there are papers scattered in the literature that are concerned with specific applications of IR or Raman microspectroscopy, there appear to be few general reports concerning their capabilities. Several samples of the work performed in the MML, covering a broad range of interests, follow. It is hoped that they will serve to give the reader a suitable introduction to the possibilities of the techniques and a feeling for the developments to be expected in the near future.
lnshumentatlon A few general principles with respect to the two methods are worth noting. Raman spectrometry is normally carried out in the visible region of the spectrum, and thus normal microscopes, with the usual glass optics, are quite satisfactory for coupling to the spectrometer. Because the source for Raman spectrometry must be monochromatic. an argon ion laser is typically used. It should be noted that the power requirements of the laser are not very high. Although the systems typically use beam splitters, which transfer a relatively small
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
amount of the input energy to the sample, the focusing property of the microscope greatly increases the flux (power/area) at the sample; The laser beam in a typical system enters the microscope with an input power of 31 mW and a diameter of 2 mm. The internal beam splitter might transfer only 20% of this power to the microscope objective and this, along with reflection losses, results in a power of 4 mW at a 2-pm-diameter sample (minimum sample area is 1pm). For comparison, the input beam has a flux of about mW/pm2, whereas the output beam has a flux of about 1mW/pm2. On the other hand, glass is not transparent in the IR region. Therefore, all IR microscopes must use reflecting optics and Cassegrainian objectives, or mirrors of the proper configuration, for focusing and collecting the incident and transmitted beams, respectively. A cutaway diagram of a Cassegrainian lens showing ray paths is given in Figure 2. The many reflecting surfaces further decrease an already weak beam. Such low-energy situations are, of course, best handled by FT-IR systems because they use available energy much more efficiently than do dispersive systems. Furthermore, if one is to obtain the best performance, a liquid-nitrogen-cooled MCT (mercury-cadmium-telluride) detector is necessary. Most commercial units use a dedicated MCT detector within the system whose size is matched to the size of the image of the illuminated sample. However, one system uses the detector in the instrument to which it is fitted. This results in a substantial cost reduction, but in return performance is somewhat degraded. Simplified block diagrams of Raman and IR microspectrometers are given in Figures 3 and 4.
Experimental factors Materials to be investigated are customarily mounted on standard glass microscope slides or small salt windows for Raman or IR study, respectively. Minimal sample handling is necessary and is usually performed with the aid of a stereomicroscope. If the particle of interest is one of several particles or is particularly small, the area around the particle may be marked with a felt tip marking pen. Or, if the slide or crystal is to be reused, a larger particle or fiber may be placed beside the particle as a marker. With Raman microspectrometers the marked area of the sample slide is centered above the substage condenser, and the condenser diaphragm is closed down to form a narrow cone of white light. The slide is moved with the mechanical stage controls until the particle is positioned directly over the con-
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Figure 3. Block diagram of microscope coupled to a Raman spectrometer denser, which is indicated by a change in intensity of the transmitted light. The condenser diaphragm is then opened wide and the stage moved vertically to bring the sample into the focal plane of the microscope objective. If the object of interest is a small portion of a substance, such as an inclusion, successively smaller sections of the sample can be viewed by using several parfocal objectives of varying magnification on a rotating nosepiece. Location of an inclusion or surface feature in an opaque sample requires incident illumination. The white light can then be directed along essentially the same path traversed by the laser beam inside the microscope. Frequently the use of the monochromatic light provided by the laser gives a clearer visual image of a surface than does the incident white light. Some manufacturers’ objectives have antireflection coatings that lead to spectral artifacts. The additional spectral features can be avoided by placing an aperture behind the microscope to eliminate all but the centermost rays of scattered radiation. This will, of course, reduce the signal. With IR microspectrometers sample location is seldom troublesome. The small salt windows confine the sample to an area only several millimeters in diameter. Because the detectable size limit is larger with IR than with visible radiation, lower magnification microscope objectives are used. However, 470A
Figure 4. Block diagram of typical IR microscope accessoly coupled to an FT-IR spectrometer Cassegrainian lenses: a. b. and c
a single, clear, very small crystal sometimes may still be difficult to locate on a salt window. Probably the most active area of research in microspectroscopy is that of method development aimed a t finding ways of decreasing the troublesome sampling problems currently experienced. Fluorescence is by far the greatest problem encountered hy the Raman spectroscopist. Although methods to decrease fluorescence when working with macro systems are known, there seems to have been no effort as yet to adapt these to micro systems. The one notable exception is the use of alternative exciting frequencies. The most frequently discussed approach is to use a laser wavelength in the red. The fluorescence excitation band for many organic compounds is in the blue. Therefore, one can often remove fluorescence by exciting with a red line. In principle, ultraviolet (UV)excitation would he even better, as the dependence of scattering intensity on frequency to the fourth power could be effectively used. To date, the necessary equipmentlaser, optics, etcis expensive. Furthermore, there is a much greater chance of photoreactions of organic compounds in the UV region. However, the specific nature of fluorescence can often he profitably used to simply shift excitation frequency and obtain a Satisfactory spectrum. This is illustrated in Figure 5.
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This figure reproduces the Raman spectrum of a single fiber of acrylic excited by the 514.5-nm argon ion line in one case and by the 476.5-nm argon ion blue line in the second. Note that the blue line is not the more common 488.0-nm line. The fluorescenceis due to an impurity, and the band is quite narrow for an organic compound. A second, very common approach to reducing fluorescence in macro work is “baking” with the laser. The sample fluorescence often decreases with time as the sample is allowed to remain in the laser beam. This approach can also be used in Raman microspectroscopy (and in fact demands less time), but care must be used to avoid sample decomposition. The most common experimental problem in IR microspectroscopy is an excessive sample pathlength. This frequently occurs with small particles and can represent a very serious difficulty in obtaining a satisfactory spectrum. The best approach for such small samples is the use of the diamond cell to thin the sample. Two such cells, one for high pressure and one for moderate pressure only, are available from High Presaure Diamond Optics (Tucson, Ariz.). The usefulness of one of these cells is illustrated in Figure 6. The upper portion of a refrigerator door gasket that exhibited a problem had a thickness of 0.83 mm. This represents a sample pathlength that is almost two orders of magnitude
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Flguro 5. Raman spectrum of 18-pm-diameter acrylic fiber excited with the 514.5- and 476.5-nm lines of an argon im l a w
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Flgure 6. IR spectrum of refrigerator door gasket material obtained in moderatepressure diamond cell accessory
Figure 7. IR spectra of three different crystals obtained by evaporation of solvent
from a “simple” solution
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too great for obtaining a satisfactory IR spectrum. Figure 6 shows an IR spectrum of a portion of this sample recorded in the moderate-pressure diamond cell. Total sample preparation time was perhaps %3 min. I t is clear that the IR spectrum is quite satisfactory for identification of the material. The diamond has an absorption in the 2200-1900 em-’ region. This can he adequately subtracted from the spectrum in the case of the moderate-pressure cell, hut the diamond thickness is too great in the high-pressure cell for this procedure to he used. The cell is also a convenient holder for very small samples. One can also record Raman spectra on samples contained in the cells, as diamond has only one very
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strong, sharp Raman band at 1332 cm-’. A long working-distance objective is required, however. The sampling problems discussed so far, although real and sometimes formidable, can be approached in general terms. Of equal, or perhaps greater, importance are the very specific sampling problems that must be faced and solved by the individual analyst. A moment’s thought will reveal the fact that sample homogeneity is orders of magnitude more important for data interpretation in microspectroscopy than in the more conventional methods. Impurities may play a much more critical role in solving problems. An example is provided hy a project carried out recently in the MML in
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
which the identification of the active ingredient in a “simple” aqueous solution was sought. Evaporation of a few drops of solution yielded crystals whose IR spectra could he easily obtained. There were, however, three different types of crystals. The spectra are reproduced in Figure 7. Without further information there is no easy way of choosing which spectrum is that of the active ingredient and which spectra are due to impurities. The middle spectrum is that of cellulose, a common impurity on the microscopic level. Such occurrences are common when solvents are evaporated for analysis of solutes. Often several different materials are found, and their relative quantities can only he estimated by counting crystals. The method is so sensitive that the presence of sample inhomogeneitiestends to range between being greatly inconvenient and nearly intolerable. Of course, if the degree of homogeneity of a sample is sought, the method is extremely valuable. Because of the nature of the sample, other problems may arise. As a part of the same project at the MML, a second sample was analyzed. In this case the solute was dissolved in alcohol and was present in higher concentration. Evaporation of the solvent gave a rather large amount of residue. The IR spectrum of a “macro” sample of this residue is presented in Figure 8; that of a very small particle recorded with the microspectrophotometer is reproduced in Figure 9. The spectra are clearly very different. In this case an impurity was not the culprit, however. I t was found on further investigation that the solute reacted with the moisture in the air to form a coating that
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Figure E. IR spectrum of macro sample of residue obtained by evaporation of solvent from a solution prevented further reaction. The surfacebulk ratios were very different in the two samples, and this was the cause of the spectral differences. The reaction would have gone undetected if only macro methods had been used. Applications The early chemical applications of micruspertroscopy principally involved two rypea of cummerrial niaterials-semiconductors and plastirs. In both cases the applications involved the identification 01'very small amounts ufcontaminants that degraded the electrical properties of the semiconductors and the optical properties of the plastics. The physical neture of these materials provided the impetus for one of the two comple-
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Figure 9. IR spectrum of micro sample of residue obtained b evaporation of solvent from solution of Figure 8
mentary spectroscopic techniques. A large number of commercial plastics are fluorescent, usually due to unknown impurities. Because fluorescence causes a severe background vrohlem in Raman svectroscovv. IR apectroscopy is the method ofchoice. Samvle thickness uroblems exist for IR riansmitranre studies. but the method works well for plastic films. With semiconductors, however, fluorescence prohlems are few. Thin films are not easily made, and entraneous materials are often inorganir in nature. All of these facts make Raman the method of choice in the majority of applications in this field. In addition. spatial resolution is an order of magnitude better in Raman as the radiation used is in the visible region.
-FI-IR (absorbance units) Raman (counts)
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Wavenumbe Figure 10. IR and Raman spectra of a 17-j~mdiameterpolyester fiber 4 7 4 A * ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
However, as both methods developed, it became clear that it is very convenient to have both methods available because of their complementarity. As a general rule, Raman is a better method for identification of inorganic substances because their Ramanspectra are usually strong and because most, i f not all. of their vibrational transitions ocrur at low frequency. IR is generally more suited to identification of organic compounds becaust their Raman spectra may be weak and herause fluoresrence is not a problem for IR analysis. Sample preparation methods become necessary in many IR studies. This is seldom needed for Haman microprobe spectroscopy, and, rhus, the undisturbed crystalline phase can he determined from the spectrum. The sample history can frequently he inferred from the crystalline phase. An example of the complementary nature of the two methods is provided in Figure 10, which shows a Raman spectrum (lower) and IR spectrum (upper) of a 17-pm-diameter fiber. In both cases, the spectra were recorded from a circular portion of the fiber about 15 pm in diameter. Thus the fiber need not be long. Note that the Raman spectrum extends to about 125 em-' whereas the IR spectrum stops at about 700 cm-'. This is typical for the two methods using commercially available instrumentation. The two spectra are quite different, and each allows structural features to be identified. The very strong carbonVI hand in the IR allows readv identifieation of a polyester. The ve;y strong olefinic carhon-earbon stretch in the Raman indicates aromaticity. The conclusion, even without reference spectra, is that the fiber is probably a
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ANALYTICAL CHEMISTRY. VOL 58. NO 3. MARCH 1986
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Flgure 11. IR spectra of two cotton fibers from different sources
Flgw 1 dioxide
I._...pectra
,lticlesof mC
of titanium
terephthalate type (Dacron) synthetic polymer. Further, more detailed interpretation can be carried out on the other spectral features. These methods allow one to differentiate fibers of the same material that have been chemically treated in a different manner. This may be extremely important in determining the identity and source of a fiber contaminant. As an example, Figure 11 shows the IR spectra of two cotton fibers from different sources. The two spectra are obviously different, although clearly both are cotton. These fibers were again about 15 pm in diameter. The spectra were obtained from 300 scans (5min) of an FT-IR instrument. Titanium dioxide is a substance that is widely used in industrial processes and often appears as an unwanted or extraneous microscopic contaminant. The Raman spectra of two small particles of titanium dioxide are given in Figure 12. Each represents one of the common polymorphs of the substance. Note that it would normally be impossible to identify them as Ti02 with an FT-IR, let alone to differentiate the polymorphs, as there are no bands above 700 cm-1. As can be seen, the Raman spectra are quite strong. No discussion of microspectroscopy would be complete without some reference to forensic applications. Physical evidence collected in criminal cases is often very scant. Identification of such small amounts of materials may be an arduous task. The use of IR microspectroscopyallows identification of samples several orders of magnitude smaller than those that can be identified using other “micro” methods. For example, the MML was recently involved in the analysis of a faint black smudge on a shoe sole belonging to one of the occupants of a fatal single-car accident. If the material was the same as that of which the
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Floure 13. iR spectrum of flake of unknown material from shoe sole 4761
ANALYTICAL CHEMISTRY’. VOL. 58. NO. 3, MARCH 1986
Flgure 14. IR spectrum of organic portion of sample in Figure 13
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brake or accelerator pedal of the automobile were composed, it would help to identify the driver. A single flake of the material, about 50 pm in diameter, was removed with a probe and placed on a small KC1 window. Its IR spectrum is reproduced in Figure 13. The three strong bands at 3700-3500 cm-l and the three bands at 1050-900 cm-l are typical of aluminosilicates, the major components of many clays. A drop of methylene chloride was added to the sample and allowed to stand for a few seconds. The KC1 window was then tilted so that the methylene chloride extract would flow away from the clay residue. After evaporation of the methylene chloride, the extract residue gave the IR spectrum shown in Figure 14. This spectrum is quite different from that of brake and accelerator pedal samples. In this way it was established that the pedals were not the source of the black smudge on the shoe sole. Complete identification of the sample was not necessary in this case.
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Selected blbliography There are only a few descriptions of molecular microspectroscopy in the open literature, and many of these are not available in most libraries. Some of the most general and readily accessible ones follow. (1) Dhamelincourt, P.; Wallart, F.; Le-
Title
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The new techniques of Raman and IR microspectroscopy have great potential for the analysis and identification of samples previously impossible to identify. Analysts must use caution, however, in interpreting their results. There seems little doubt that further advances will be made in technique development in the field. In many cases the only requirement is adaptation of techniques already developed for either macro spectroscopy or optical microscopy. The sample size required is only about 5 pm in the IR and perhaps 100-500 nm in the Raman, depending on excitation wavelength. The power of the technique is so great that its future seems very bright indeed.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
ciercy, M.;N'Guyen, A.T.; Landon, D.0. Anal. Chem. 1979,51,414A. (2) Rosasco, G.J. In Advances zn Infrared
and Raman Spectroscopy; Clark, R. J. H.; Hester, R. E., Eds.; Heyden and Son: London, 1980;Vol. 7,Chapter 4. (3) Janssen, R. K.; Krol, D. M.Appl. Opt.
1985,24,275. (4) Carvalho, W.; Dumas, P.; Delhaye, M.; Corset, J.; Levy, Y.; Imbert, C. Appl. Opt. 1984,23,4197. (5) Bullock, K.R.;Trischan, G. M.; Burrow, R. E. J. Electrochem. SOC. 1983, 130,1283. (continued on p. 481 A)
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
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(8) Cournoyer, R.; Shearer, J. C.; Anderson,D. H. Anal. Chem. 1977.49.2275. (9) Shearer, J. C.; Peters,D.C.; Hoepfner, G.; Newton, T. Anal. Chem. 1983.55, 874 A. (IO) Bartiek, E. G. Appl. Speetrosc. 1985, 39,885. (11) ASTM is planning to publish a special
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Gilbert E. Pacey is an associate professor of chemistry and associate director of the MML. He received a B.A degree in chemistry at Bradley University and a Ph.D. from Loyola University of Chicago i n 1979. He joined
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Jerome O'Keefe received his A.B. from the University of Pennsylvania in 1973. After serving as an officer in the U.S. Army for four years he completed his Ph.D. i n inorganic chemistry at Pennsylvania State Uniuersity in 1984. He taught at Duke Uniuersit y before joining the MML at Miami Uniuersity in 1984 as a postdoctoral associate. His current research interests include characterization of molecular vibrations and applications of vibrational spectroscopy.
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
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