edited by FRANKA . SETTLE,JR.
ch&ical in~trument~tion
v i ~Lexington, i ~ ~ l i w nVA s t i24450 tute
Fourier Transform Infrared Spectroscopy Part Ill. Applications W. D. Perkins The Perkin-Elmer Corporation, 41 1 Clyde Avenue, Mountain View, CA 94043
In the two previous parts of this paper ( I , 2) we have discussed the design of FT-IR
spectrometers, the computation of spectra from interferograms, and the advantages of FT-IR spectroscopy over dispersive t e c h niques. While many if not most samples can he run quite satisfactorily on either type of instrument, the FT-IR spectrometer becomes preferable when we are energy limited and when we need increased signal-tonoise ratio. With the availability of FT-IR, many analyses that we used t o avoid have now become routine. In addition, over the last several years a whole new group of sampling accessories has been developed in order to take advantage of the capabilities of FT-IR spectroscopy. Let us now examine some of these applications. Aqueous Solutions In the past, the infrared spectroscopist has tried to avoid working with aqueous solutions. Liquid water has very strong ahsorption bands centered a t about 3400 cm-' (OH stretch) and 1640 cm-' (OH bend) and is completely opaque below about 800 cm-I. Figure 1, scan A, is the spectrum of water taken in an 0.015-mm-thick cell fitted with barium fluoride windows. Barium fluoride is an excellent choice of window material for aqueous solution studies because its long wavelength cutoff is well matched with the
cutoff of liquid water. Spectrum B in Figure 1 is the superimposed spectrum of an aqueous solution of a water-soluble aspirin tahLet. The problems are obvious. To obtain the spectrum of the aspirin we must subtract the spectrum of the water from that of the solution. This can he done in a double-beam spectrophotometer by placing a cell filled with the solution in the sample beam and then using a matched (thickness) cell filled with water in the reference beam. Conventional difference snectroseonv has been donein precisely this way since the development u l double-beam instruments. Today thedifference is more conveniently calculated by running the solution and the solvent separately (there are also instrumental advantages) and then by doing a computer subtraction of their absorbance spectra. The problems are (1) We are confined to the spectral region between 3WO and 800 em-1. Even with very thin cells the OH absorption around 3400 cm-I is total and very broad. (2) Overall transmission in the spectral region that is available is significantly reduced. Even a t high concentrations (not always possible) the absorbance of the solute will he small because of the cell thickness. Hence to obtain a difference snectrum with acceptable signal-to-noiseratio will require exceptionally high signal-to.noise ratio in the two Bpeetra that are subtracted. ~~~~~~~
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(3) The water band a t 1640 cm-' is especially troublesome. It lies at the mid to low end of the carbonyl region, and important bands in the solute could lie beneath it or along its steep sides. Furthermore, it is totally absorbing at thickness of 0.025 mm and has very little transmission even in a 0.015-mm cell. FT-IR spectroscopy cannot solve the first problem of limited spectral range. Total absorption is total absorption, and taking the difference between two snectra in reeions where they both have zerdtransmissioi will not produce spectral data. But the extremely high signal-to-noise ratios obtained so readily by the FT-IR technique make spectral subtraction and subsequent ordinate expansion of the difference spectrum quite feasible, even over the region of the 1640 cm-I OH bending mode. Note spectrum C in Figure 1, which is the ordinate expanded difference spectrum obtained by absorbance subtraction of the water spectrum from that of the solution. Sipal-to-noise is high and spectral detail is easily observed even in the 1640 ern-' region. FT-IR spectroscopy has now made working with aqueo w solutions quite feasible if not mutine.
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Figure 1. (1eft)Transmissionspectra run in a 0.016mm-thick barium fluoridecell. Scan A is liquid water; scan B is an aqueous solution 01 a water soluble aspirin t a b let: and scan C is lh8 ordinate expanded difference spectrum, lhat is. solution minus water. Figure 2. (right)Spectra run in a circular internal reflection cell wilh a zlnc selenide internal refledion element.The two lower spectraare of water (a)and of a water concentrate of a commercial soil fumigant (b).The upper spectrum (c)is the difference spectrum, (b) minus (a), presented in an ordinate expanded absorbance format.
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Circular internal Reflection Despite the success just described in obtaining spectra from aqueous solutions, two problems persist. To work effectively aver the entire 3000-800 cm-I window, we must operate with a cell thickness that transmits reasonable energy across the 1640 cm-' water hand. From Figure 1 it can he seen that even a 0.015 mm cell barely meets this requirement. Commercially available cells can vary from their nominal thickness by ar much as 10% and, if one has a cell whose thickness varies on the high side, the task of getting a good difference spectrum becomes increasingly difficult. It would he preferahle to use an even thinner cell, hut a quick perusal of infrared accessory catalogs discloses that cella thinner than 0.015 mm are not offered for sale. The other problem will he familiar to anyone who has ever filled a 0.015-mm amalgam sealed cell and, even more so, to anyone who has had to clean it out after using it. The procedure is tedious at best, and the more viscous the solution, the more impossible the task becomes. The problem can he avoided in qualitative applications by using a demountable cell and working with a capillary film, hut if quantitative results are required, controlled and repeatable cell thicknesses are essential. A solution to hoth of these prohlems is the use of internal reflectance (sometimes referred to as attenuated total reflection, m d tiole internal reflection. or frustrated multiteehnioue oie internal reflectioni. In this ~~~~rhe s&plebeam of the spectnmetcr is'dLreckd into one end of what is usually a long, thin crystal of high refractive index. KRS-5, germanium, and zinc selenide (Irtran-4) are among the commonly used crystals. The accessory is designed so that the beam inside the crystal strikes the crystal interface a t an anele ereater than the critical anele. Total &nil reflectron occurs, and thi beam is reflected hark and forth ~nsidethe crystal untrl it evenrually emerges at the far end and continues on through the spectrometer. The sample is now placed in clme optical contact with the exterior surface of the crystal. At frequencies where the sample has no infrared absorption, t h e beam passes through the accessory essentially undiminished. But a t those frequencies where the sample has infrared absorption hands, there is interaction sample a t each reflection along the cryatal+ample interface, and the intensity of the internally reflected beam is attenuated. (This is the origin of the term attenuated total reflection, or ATR.) The resulting sneetrum. whose aooearance is eavernei h; the nh&ics of reflkEtion theorv and not by ;he lakr;~fabsorbanre,neverthee less bears a strong resemblance to a conventional transmission spectrum and is often compared usefully with reference spectral libraries generated by transmission spectroscopy. Another verv imnortant characteristic of internal reflection &trosconv in that onlv the surface layer of the sample contactink the crystal participates in this process. Penetration depths into the sample vary with wavelength, angle of incidence, and refmetive index, hut a realistic average value is
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perhaps about 8 to 10 rrm. In other words, if we scan a liquid sample hy internal reflection, the spectrum we get has band intensities comparable to those that can be obtained by transmission using a 0.010-mmthick cell. In particular, the absorption hand of water at 1640 cm-' now transmits several percent permitting good spectral subtraction of the water hand from solute bands that are coincident with it. Figure 2 is typical of the spectra we can obtain in this manner. It shows (a) the internal reflection spectrum of water, (h) the spectrum of a commercial soil fumigant that is marketed as an aqueous solution concentrate, and (c) the difference spectrum, plotted this time in absorbance. The absorbance data were used in a quantitative method to determine the strength of the concentrate. The spectra were run in a device known as a circular internal reflection (CIR) cell. A diagram of the cell can be seen in Figure 3. The internal reflection crystal is now a rod (with a circular cross section) of zinc selenide (Irtran-4) approximately 'I8 in. in diameter and, in this particular cell, about 2 in. long. The ends of the crystal are conical so that the beam of the spectrometer can he brought in and out using two Cassegranian collecting optics in an in-line configuration. The rod is surrounded hv an onen tub (clobed flow-through designs of the eell are alaomarketedj intowhich thesample can be easily poured. The volume of the ruh for the eell used in these measurements was ahout 2 mL. Teflon O-rings seal the rod into the surrounding sample tub and account for the weak hands hetween 1000 and 1200 cm-I observed in the water spectrum. They cancel out when the difference spectrum is computed. The device does not have a high optical efficiency-transmission through the empty accessory is of the order of 10% or less, depending on the matching of accessory optin to the spectrometer optics, and also depending very strongly on the condition of the internal reflection crystal itself. Far these reasons the cell has been designed orimarilv for use in FT-IR soectrometers where good-quality spectra can he routinely obtained evcn with energy thn,ughpurs of this magnitude. Thedesign of theeell also responds well to the two problems outlined a t the beginning of this section-the need for a shorter pathlength, paticularly for aqueous solutions, and the difficulty in filling thin sealed cells. The shallow penetration depth in the internal reflection process gives a liquid water spectrum of ideal intensity for subsequent subtraction. Since liquids will provide reproducihly good optical contact with the crystal, and since the design ensures that the sample-crystal contact area is constant for esch filling, we have the equivalent of a fixed-thickness sealed cell and can use the device for single-hand quantitative analysis. Finally, the open tuh design makes filling and cleaning the cell hoth rapid and efficient. The cell can he readily washed out usingsolvents in squeeze bottles. Even highly viscous, almost gelatinous samples (polyaerylamide solutions for example) have been run routinely in the device (3).Thecell is ideal for aoueous solution work where it has found itsgreatest application, hut it can of course also be used with other liquids when the relatively short parhlength ir appropriate.
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Figure 3. Circular internal refraction (CIR) cell. The tap figure (a) is a cutaway view of Its Mmplete accessory and shows the construction of lhe "tub" or "open boat." me lower figure (b) is an optical schematic.
Samples with Low Transmission Sometimes we encounter samples with very high optical densities. Even when overall transmission is only a fraction of a percent, FT-IR techniques can aften produce usahle, interpretable spectra. Figure 4 shows a series of spectra run on a used engine oil with an excessively high soot loading. Figure 4a, run at 4 cm-' resolution, is a single sample scan ratioed against a single background scan using the TGS detector and a (normal) mirror velocity of 0.5 cmls (optical path difference). The spectrum appears rather noisy until one notes that the full-scale ordinate is only 0.3% T.Even so, the signal-to-noise ratio (SNR) leaves a great deal to he desired. There are two instrumental techniques to improve SNR. Spectral averaging will reduce noise in proportion to the square root of the number of scans averaged. Reducing the velocity of the moving mirror will also enhance SNR, hut now in direct proportion to the reduction in velocity. Obviously there is a time advantage to slowing dawn the mirror as opposed to spectral averaging, hut the former is not always an option because detectors can he saturated if the mirror velocity is too slow. With very low transmission samples, however, detector saturation does not become a problem, and we can now use mirror velocity to improve the SNR. The spectrum in Figure 4h is again a single scan hut made with a mirror velocity of 0.05 cmfs. SNR is improved 10-fold, and scan time is increased 10-fold. Using a lower mirror velocity does not preclude us from seeking an even further improvement by signal averaging. The spectrum in Figure 4c was obtained by averaging 100 scans still using the 0.05-cmls mirror velocity. This gave an additional 10-fold improvement in SNR but a t the expense of a 100-fold increase in scan time. Even so, the spectrum was obtained in a matter of minutes. Improvement i n S N R can also be achieved by switching from the normal TGS room-temperature detector to a liquid-ni-
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Figure 4. Spectra ofa heavily soot-loaded, used engine oil scanned under various instrumentalconditions. Maximumtransmission is 0.30%. Scan (a) isa singlescan. TGS detector, 0.6cmIs 0.p.d. velocity. Scan (b) is a single-scan. TGS detector, 0.05-cmls 0.p.d. velocity. Scan (c) is a 100-scan average, TGS detector. 0.05-cmls 0.p.d. velocity. Each successive scan shows a 10-told Improvement in signal-to-noise ratio. Scan (d) is a single scan made using a cooled MCT detector, 3.0-cmls 0.p.d. velocity. These spectra illustrate the trade-offs between mirror velocity, signal averaging, and choice of detector.
trogen-cooled MCT (mercury-cadmiumtelluride) detector. The spectrum in Figure 4d is again a single scan made using the MCT detector a t its normal mirror velocity of 3.0 emls, and took only a sixth as much time to run as the spectrum in Figure 4a. Figure 5 is a spectrum run using the MCT detector, a reduced mirror velocity, 113scan averaging, and ordinate expansion. Full-scale ordinate is only 0.05% T and yet the hands on the Law frequency side of the G H stretching vibration are clearly discernible above the noise Level. Dlfluse Reflectance Diffuse reflectance measurements, which for many years have been routine in the visible region of the spectrum, have always heen difficult in the mid-infrared because the amount of energy that is reflected is so small. As a fraction of the incident energy. the diffusely reflected beam, collected over the largest solid angle practicable, seldom exceeds lo%, is often of the order of 5 4 % and with some samples can he as small as 1% or less. While diffuse reflectance can he done with disoersive instruments 14). the sneetroseonv mike its advantams of h-IR uae pref&le, andloday diffuse reflectance infrared Fourier transform (acronym DRIFT) spectroscopy (5, 6 ) is an estahlished technique. Figure 6 shows the layout af a typical commercial accessory. The sample is usually contained in a small cup about 10-15 mm in diameter and 3-4 mm deep. Micro cups, about 2-3 mm in diameter may also he used in sample limited applications. Energy is focused onto the surface of the sample, and the diffusely reflected beam is then collected with a large aperture mirror, usually an off-axis ellipsoid, and passes on to the detector. The actual process taking place at the samole is comolex and involves oenetration into'the hulk phase followed h i panial shsorption, multiple scattering, and re-entry through the sample surfacc0. ~
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Fiaure 5. Soenrum of same s o w . used emine oil as in Fiaure 4. One-hundred-scan averaoe. MCT dstenw, 1.i-cmlso.p.d. wloc3ty.~xpandedto0.05% full-&le ordinate to showreadability~ofsmall side bands on the low frequency side of the C-H stretching vibration. The resultant spectrum is one that bears a close similaritv to the corresnondine transmission meetrum with marked differences , ~~~in band intensities. Conversion uf the reflectance spectrum to Kubelka-Munk format (8) produces a spectrum that is equivalent toalinear absorbance plot and that can then he used for quantitative as well as qualitative purposes. For best results the samole should be finely ground and mixed witl; a nonabsorbing diluent such as KBr or KC1 Sampleconcentrationa of 1%or less are preferred, and, when the concentration rises above the 51% level, distortions often occur especially in the stronger hands. These statements notwithstanding, useful spectra are frequently obtained at much higher sample concentrations and even on undiluted samples. Figure I is the spectrum of an ABS polymer ground to 60 mesh and run neat. The spectrum is ratioed against a hackground scan of pure potassium bromide. Spectra have also been obtained on coal samples, foam rubbers, textiles, and adsorbed species in situ on their supporting suhstrates. Soectrahave evenbeen obtained directlv from messed oharmaceutical tablets, h"t the smooth-prbssed surface usually gives e strong specular component that ran severely distort the appearance of the spectrum. An unusual sampling approach has been described by Spragg (9)that isapplrcable to vew hard materiala that resist rrindina and alternate methods of sample prepar&ion. Its usefulness is illustrated hv the soectrum in Figure 8. The sample was a cured epoxy resin that was so hrictle that attempts to grind it resulted in more material being splintered and ejected from the mortar than remained behind. With great patience and diligence, enough of the resin was finally ground 8 0 that it could he mixed with KBr and run in the reflectance accessory. A good speetrum was produced. The spectrum in Figure 8, however, was ohtained much more easily. The resin was simply rubbed against apiece of emery paper until enough of it had abraded and stuck to the paper. A small patch was then cut from the paper with a
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Figure 6. Optlcal schematic of a diffusereflectance accessory. A large-apenure elllpsoldal mirrw is wed first to focus energy onto G?e sample and G?en to collect the diffuselyreflected beam over a large solid angle.
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pair of scissors. ohced in the reflectance eccessory samplecup, and scanned directly aenaisoectrum shown. The to oroduce the ~~~, ~~~. - ~ ..~.~ .. . tivity of the diffuse reflectance technique i3 such that spectra can be dnained even on microgram and, with care, submicrogram amounts of sample. As is usually the ease in microsampling, the problem of manipulating and transferring minute quantities can be greater than the task of obtaining spectra once they are in position in the speetrometer. A common technique is to dissolve the sample in a small amount of solvent. The resulting solution is usually easier to transfer than the original sample, and a variety of infrared microsampling procedures utilize solutions in intermediate steps. The spectrum in Figure 9 was obtained by first packing the diffuse reflectance microsample cup loosely with KBr. The cup, ahout 3 mm in diameter and 3 mm deep, holds approximately 10 mg. Neat, two drops (measured from a micropipet) of an LSD (lysergic acid diethylamide) solution were added directly to the KBr in the microcup; the solvent was allowed to evaporate, and the spectrum then run. The amount of LSD added in this instance was approximately 30 pg. This procedure, automated and done more elegantly, has also been applied to the identification of HPLC fractions. The reasons for wing diffuse reflectance usually come down to the ease of sample preparation. Powdered substances can sometimes he runas received, and if they do need to he ground, the degree of grinding is far less severe than that required for preparing mulls or pressed pellete-and takes less time and effort. In addition, the polymorphic changes that may be induced in the pressing of KBr pellets can be avoided. Other samples just seem to give better spectra with less effort when run by diffuse reflectance. The technique has heen particularly successful with adsorbed species and with coals and carbon blacks. ~
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Photoacoustlc Spectroscopy Another sampling technique that benefits from the energy advantages of FT-IRis pho-
Figure 7. (left)Diffusereflectance specbvm of an ABS polymer ground to 60 mesh and run neat. Figure 8. (right)Diffusereflectance spectrum of a cured epoxy resin obtained horn the residue abraded onto a piece of emery paper.
Flgure 9. (1en)Dlffusereflectance spectrum obtained from 30 irg of LSDspnted horn aoiutlon antoapproximately 10 mg of KBr contained In DRlFTacceasory miwosample cup. Figure 10. (right)Photoacoustic spectra of a pulverized medium-volatility 1toacoustic spectroscopy (PAS). The effect itself was recognized and studied as early as the last century, but it seems to have generated little interest until the early 1910's when experiments were carried out using hieh-intensitv. tunable laser sources. In the la& 1970's it k s recognized that the energy characteristics of the Michelson interferometer made it well suited for photoacoustic measurements and since then, the technique has evolved from a laboratory curiasity of limited utility to become a standard, albeit somewhat expensive, detector accessory for FT-IR spectroscopy. A useful review paper has been written by Vidrine (10). The sample analyzed is placed in a small cup (typically 3-4 mm on a side) inside a small chamber containing a nonabsorbing gas such as helium or nitrogen. The infrared beam from the interferometer is focused onto the sample, heating its surface and causing it to expand. The gaslayer above the samnle is also heated and ex~ands.and all this occurs with a modulation frequency corresponding to the product of the interferometer's OPD (optical path difference, velwity (proportional to mirror velocity) multiplied by wavenumher. A very sensitive microphone in the gas chamber is used to detect the acoustic signal, and the entire device may be considered as yet another type of detector for the FT-IR spectrometer. The advantages of PAS are that sample
(top)and of an extruded polymer pellet (bottom)run as received.
size and shape are not critical. Samples may consist of powders, odd-shaped chunks or shavings, pharmaceutical tablets, single fibers, fragments of polymer foams, and single crystals, to list a few. Since grinding and dilution are not required, not only is sample ore~arationmeatlv simnlified if not totallv eliminated, but the morphological changes associated with grinding and pressing are also avoided. PAS spectra are usually ratioed against background spectra run on carbon black, which is totally absorbing and which gives a relatively flat baseline. As a consequence the technique often gives good spectra on carbon-filled polymers and other carbonloaded samples. Finally, the depth that the radiation penetrates into the sample is proportional (among other things) to modulation frequency, itself the product of OPD velocity (mirror velwity) and wavenumber. The wavenumber dependence has an effect on relative hand intensities. makine" the lower frequency bands somewhat more incense than in the corresponding transmiasion spectrum. The OPD velocity dependence can be used to advantage in an instrument where this parameter can he varied in order to do a degree of depth profiling. A PAS spectrum looks somewhat like an absorbance spectrum and is usually presented i n s format with the bands going upscale. It also bears a close resemblance to a
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diffuse reflectance spectrum displayed in Kubeh-Munk format. Figure 10 shows photoacoustic spectraof apulverized, medium-volatility coal sample and of an extruded polymer pellet, run as received. Thespectrain Figure 11are both of caffeine; the top soeetrum was m neat hv PAS and the hot&mspectrum wasrun bidlffuuse reflectance and replotted in Kubelka-Munk format.
Infrared Ernlsdon The ability of the FT-IRspectrometer to detect very weak signals makes it possible to study infrared emission, even when the emitting sample is a t a relatively low temperature. Figure 12 shows the spectrum from a polymeric coating used to line the inside of a soft drinkcan. This spectrum was obtained by placing a section cut from the can just in front of the FT-IR source with the coated side facing the interferometer. The sample was warmed only by the radiant heat from the source, and the spectrum is a single-beam scan. One can observe the hroad black hody shape of the underlying background with the characteristic emissions of the polymer superimposed on it. The black hody maximum appears to occur somewhere between 1500 and 1000 em-', indicating a relatively low sample tempera-
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Figure 11. (lell) Spectra of caffeine.The top spectrum was run on the neat sample using a photoacoustic detector; the bouom specbum was run by diffuse reflectance On a 1% mixture of caffeine in KBr. The spectrum is displayed in Kubelka-Munk format. Figure 12. WgM) Spectra of the polymer-coated inside surfaceof a solt drink can. Tha top specbum was run using a specular reflectance sccesaory. The bonom Spectrum is an inhared emission spectrum run by placing a section cut hom the can in front of Wm FT-iR source. ture, and the emission a t this temperature is too low to permit observation of spectral features in the 4OM)-2000-crn-' region. For comparison an absorption spectrum of the same sample (run using a specular reflectance accessory) is also shown. Tbis was a relatively simple experiment, done without any special equipment. Amessory optics are available for some commercial instruments that will permit emission measurements to be made from the sample compartment using a split aperture. In a more sophisticated experiment, the sample scan might have been ratiaed against a scan from a true hlnck body radiator a t the same temperature in order to give a purer spectrum of the emission. Other applications of infrared emission have involved the remote sensing of smokestacks to assess pollutants. Special long-focus collecting optics have been designed to being energy from the plume into the interferometer.
The infrared Microscope Ordinarily one can obtain an infrared spectrum using standard macro accessories on samples ranging in size from a fraction of a milligram to a few milligrams. When smaller samples are encountered, a variety of microsampling techniques and accessories must beemployed. Invariably this leads to the use of a microscope or a beam condenser to reduce the size of the beam at the sampling point so that the instrument's full beam energy can be passed through decreasingly smaller sample areas. A commercial infrared microscope utilizing a Cassegranian optical system was marketed in the early 1950's (I I) but was never used very extensively, probably because of its cost and because it was tedious to use. Instead we saw the development of various beam condensers using a t first refractive optics (KBr lenses) and then, as instrument scanning ranges increased, all-reflective optical systems. Magnifications ranged from 4X to 8X depending on optical quality and cost of the accessory, and the majority of mierosampling work was done using pressed KBr pellets. Micro and ultra-micro dies A302
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were manufactured for pressing pellets with diameters ranging from 1.5-2 U mm to as small ay 0.5 mm. With s hrgh-performance dispersive spectrophotometer and a good beam condenser, spectra can be obtained routinely on as little as 1-2 rrg of sample in an 0.5-mm pellet, aod under favorable eonditions. such as strone " absorbers or the use of computer s v e r a p g , these limits can sometimes be extended downward by one to two orders of magnitude. As FT-IR spectrometers came into increased use, the limitations of microsampling were re-examined, and it was quickly established that with the higher signal-tonoise ratios available in an FT instrument one could often obtain FT soectra without beam condensers that were as good as those run on dispersive instruments using beam condensers. Not long thereafter the infrared microscope was "rediscovered" and by the ~
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mid-1980's it was available commercially from a variety of vendors. Figure 13 shows the optical path through a simple but typical microscope accessory. The beam passing through the spectrometer sample compartment is diverted upward in a vertical direetion after which it passes through the sample. The sample is situated on a horizontal stage that can be translated in two directions to permit positioning in the field of view. Demagnification a t the sample position is achieved by an all reflectiveobjective, most commonly a 15X Cassegranian system. The beam continues on to a movable mirror assembly that can he positioned to send it either to the infrared detector far measuring spectra, or upward through an ocular for operator viewing and positioning of the sample. The full field of view at the sample is generally about 500 rrm in diameter, and with the normal 10X ocular the sample can
Figure 13. Optical schematic of an infrared microscope. The diagram on the lelt (a) shows Wm accessory inthe viewing mode. Note thatthe incoming beam Isblocked just belwrthe sample pasnlon to prevent the laser component horn being seen by the observer.A small incandescent bulb illuminates the sample.The ocannirq mDde is shown at the right (b). Energy passes Ihrough the sample and is collected by the Cassegrain objective alter whidl it is diverted to the detector.
be viewed a t 150X maenification. A fourblade independently a&stable and rutatable aperture, located at a point optically conjugate with the sample, can be set to further Limit the portion of the sample being examined toany rectangular area within the 500-pm field of view. With some microscope desians the FT-IR s~ectrometer'sown detectors are used. and a well-desiened svstem will nrodnce e&d soeetra with &e standard D T ~ Sdetecror at iarger apertures For the smallest apertures or the beat signal-tonorse ratloand shortest datacollection pen. ods a liquid-nitrogen-cooled MCT detector is preferable. Some microscope accessories are built only in s configuration that includes a fully dedicated MCT detector. Most microscopes are also designed so that they can be used in either transmission or reflection mode, thus permitting the examination of samples on infrared opaque substrates. More sophisticated designs are also available in wbicb dual Cassegranian elements, independently adjustable and independently focusable, are located hoth before and after the sample, thus allowing better compensation for transmitting suhstmtes used to support minute samples. Typical samples requiring the use of the
microscope include single tlbers, corrosion specks, occlusions in polymers, laminates,
paint chips, and minor impurities imbedded in hulk samples. The spectra in Figures 14 and 15 illustrate a problem occuring in paper manufacture. Tiny specks, barely the size of a pinpoint, were observed imbedded in the finished paper product. A small piece of the paper was taped over the open transmission stage of the microscope and the stage was then positioned while viewing through the ocular until one of the impurity spots was located within the field of view. The four-blade rectangular aperture was then inserted into the system and the blades adjusted until they delimited the area of interest. The microscope was then switched from the viewing mode to the scanning mode, and a transmission spectrum taken through the impurity and the paper in which it was imbedded. The mechanical stage was then translated (again nsing the viewing mode) until an unblemished section of the paper was in view and a second spectrum was taken through the same aperture. These two spectra are shown in Figure 14. The difference soectrum. ahtained bv subtracting (m absorhanee) the spectrum of the paper from that of spot plus paper, is shown
in Figure 15. It appears to be the spectrum of a csrboxylic acid salt. leading to the n ~ n jectnre that the spots were probably caused by grease from the machinery~. cettinp.into the paper during processing. In another instance a forensic laboratom had a samnle of a snsoected drue that eonsisted uf only a few grains of a powder, h u e ly visibleto the oakedeye. A 13-mm-diameu r KBr window wasplaced in the horizontal sample stage of the microscope a s s supporting substrate, and the sample was deposited onto the window. Using the viewing mode, the stage was moved ahout until one of the larger grains of the sample came into the field of view. This was then masked off with the variable aperture-it had an area of approximately 200 X 400 fim--and the spectrum shown in Figure 16 was run using the FT-IR's own DTGS detector. A computer search was carried out on the spectrum, and the results of searching a reference library that included a number of controlled substances are shown in Figure 17. The two best matches, both with significantly higher scores that other compounds on the "hit
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Figure 14. (left) Spectra of a paper sample with an impurity speck imbedded in its surfsce. The scans were made using an infrared mlnoswpe. Scan A was made in situthroughW impurity speckandthe paper in which it was imbedded.Scan B was madethrough Wsamedelimitingapertureon asection oftheunblemished paper adlacent to the spot. Figure 15. (right)Differencespectrum obtained fromthe two spectra shown in Figure 14. The spemum of the adjacent paper was subtracted ham the specbum of the speck imbedded in the paper. The difference specbum appears to be that of a carboxyllc acid san.
Figure 16. (islt)Specbum of a single grain of a suspected drug scanned through an inhared microscope using a 200- X 400-pm viewing aperture Figure 17. (right)Search "hit list" showing ranked best matches between suspected drug spectrum in Flgure 18and a reference library of drug spectra. Comparison of spectra cOnfiRned that the sample was cocaine hydrochloride.
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inftr urnentotion list," were both cocaine hydrochloride, and comparison with reference spectra canf i i e d the identification. The role of the infrared microscope needs to he kept in perspective. It is an energy inefficient accessory and needs the signalto-noise advantages of FT-IRspectroscopy for viability. Energy througb the full aperture system varies from manufacturer to manufacturer hut is usually below 1m of the unohstructed single-beam level. When the system is apertured down to accommodate the smallest of samples, working energies can be reduced to a small fraction of a percent of the unohstructed single-beam level. Just how small a sample can we examine? The answer deoends on how low a sipnal-to-noise ratio we are willing to accept, but very usable spectra have been obtained using an MCT detector through apertures as small as 10 pm in diameter and under conditions where the total working energy was as low as 0.0018% of the unobstructed single-beam level. One of the greatest advantages of the microscope, however, relates not to optical performance, hut to manipulative capability. So often the biggest problem in micrmmpling is in isolating the sample in the spectrometer beam. With the microscope we are able to view the sample under magnification, move it about in the field of view, and isolate that portion of the sample which we wish to examine. At times these capabilities are even more important tban the degree of magnification that is involved. ~
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GCIFT-IR Spectra on tho Fly A final example of an application uniquely suited to the capabilities of FT-IR spectroscovv is the identification of eas chromatograsy fractions as they elute from the column, that is, on the fly. For a capillary column, the amount of eluant in a given GC peak may vary from as little as a few nanograms for a narraw-bore column, lightly loaded, to as much as 5 to 10 pg for a widehore capillary with a thick film c o a t i , ap-
erating near maximum capacity. The halfwidth of the GC peak will also vary with conditions but can be as narrow as a few seconds oer wan. The heart of any GCIFT-IR accessory is the sample cell itself. The cell needs to have a long pathlength and a small volume, and it must he maintained a t a temperature high enough to prevent condensation of the sample in the cell. These requirements are met hy a device known as a light pipe, a piece of glass tubing 10-20 cm long, a few tenths of a millimeter inside diameter, gold coated on the inside, with infrared transmitting windows on each end, and contained within a heating jacket or mantle. The light pipe is connected to the effluent port of the gas chromatograph by a heated transfer line. Since GC detectors tend to be much more sensitive than infrared detection systems, it is common practice to split the flow coming from the chromatograph, sending typically 10% to the GC's own flame ionization (or other) detector and the remaining 90% to the light pipe. Thus it is possible togenerate a normal chromatopam from the GC simultaneously with the collection of the infrared dam. In some GCFT-IR svatems the lieht beam, after passing through the light p&, goes to the F T instrument's own built-in detectors; other systems have separate dedicated MCT detectors for the GC accessory. While acceptable spectra can be generated using a DTGS detector, the use of a cooled MCT detector with its higher sensitivity is much to he preferred since energy througb the light pipe is seldommore than 10-15%of normal sinele-beam enerw and GC meek3 are usuall~alwaysweak.rnother advantage in using an MCTdetector is speed. Not only can it be scanned faster than a DTGS detector; its sensitivity actually increases with sampling frequency. Typical scanning rates for collecting GCIFT-IR data vary from a fraction of a second to as many as 20 scansls. Data are usually collected at 8 cm-1 resolution desoite a recommendation for 4 cm-1 reao~utidnmade by a Coblentz Society subcommittee (12).The lower resolution correspunds to s shorter mirror travel and hence to faster scan times. Data collection and post-run processing are done under the control of software writ-
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seconds or as wide as 10to 20 a. The problem is to ohtain spectra of micr~lamplesof hot -eases in a matter of no more tban a few ten specifically for the CC/PT-IR mode of uperation. Details vary from vendor to vendor, hut moat software packages have more similarities than differences. During the GC run an independent chromatogram is usualIv aenerated from the infrared data and -is displayed on the computer m e e n of theFTIR soectrometer in real time. Sinre the ...-rri-.. reria for infrared absorption vary from those for GC detection (flame ionization detection for example) it is not uncommon to see intensity and relative intensity differences hetween the two records. Some systems also present real time infrared spectra on the computer screen during the course of the run. The number of spectra generated during a GC run can be impressive. Even at one scan per second, a 20-minute run would generate 1200 spectra, most of which would be taken during time periods when only carrier gas was passing through the light pipe. In order to conserve data storage space, most software systems allow the user to set a threshold so that spectra are stored in memory only when the intensity of the chromatogram exceeds this preset value. Data are usually collected on hard disks or magnetic tape and a t the end of the GC run are available for review and for additional data processing. A typical system will allow the user to position a cursor on the infraredgenerated chromatogram displayed on the comnuter screen and then brine to the screen the spectrum corresponding ta that point in time. One may then view precediw or succeasive spectra, one at a time and, if they appear to he from the same species, computer average them t o provide enhanced signal-to-noise ratio. Since this procedure allows viewing individual spectra as we move across the chromatographic peak, it is also possible to determine haw well the chromatography was done, that is, whether the GC peak was a single constituent or whether two componenta co-eluted. In the latter case, taking computer differences beT scan8 at the beginning and tween the F end of the chromatographic peak can often yield good spectra of individual components ~~
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in the vamr Dhaselbonoml. Noticethe shin in ths heouencvotme stretchino Figure 18. (IefiIComwisonof thss~ecbaofmethanol in the liouid ~, - O-H . .ohaseItooIand . .. . &~on in going h& the hydrogenionded form WOOcm- ' n ths liqdgto free 0 1 1(3879 cm-' in the vspar'phass) hole alsothe P - G R band envelops eontow ii the C-0-11vibration near 1050 cm-', discernible In me vapm phase spectrum but washed out In me specbm of the liqbia. Y
Figure 19. (right)Vapor phase GCIFT-IR specbvm of cocaine. A304
Journal of Chemical Education
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even when the chromatogram itself is incompletely resolved. The bottom line, of course, is the positive identification of the fractions eluted from the GC column. This is usually accomplished by computerized spectral search and retrieval programs. Search programs are quite common today and their effectiveness will depend, among other things, on both the quality and the size of the reference library that is searched. An advantage in searching the spectra of GC fractions as compared to searching the spectra of miscellaneous unknowns is that the GC fraction is usually a pure substance. The chromatomaoh has in fact served as a seoaration device that greatly facilitatevinfrared identification, which is always much easier for pure substances than ior mixtures. Indeed, the infrared spectroscopist often views the GC as just t h a e a convenient separation devirp-while the chromatographer 5,iews the FT-IR spectrometer as merely another, albeit soohistirated. detector for his GC. Roth viewpoints have their merit. When camparing an unknown spectrum with reference library spectra it is important to remember that the GC/FT-IR spectrum is a high-temperature vapor-phase spectrum, and that vapor-phase spectra often show significant differences from spectra of the corresponding condensed phase. Two examples will serve to illustrate this mint and will also demonstrate the ouslitv If the spectra that can he obtained by thi GCiFT-IR technique Figure 18 show two spectra of methanol, one taken in the vapor phase by G C m - I R and the other as a liq-
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uid using a circular internal reflection cell. Notice the absorption in the OH-stretching region. The spectrum of the liquid shows considerable hydrogen bonding-the hand around 3300 em-' is broad and intense. In thevapor phase, where the molecules are too far aoart to hvdroeen bond. we see the at a sha& -~~~~~ free OH a b k t i o n &urine significantly higher frequency centermg at 3679 cm-l. Note also the shape of the C-OH hand near 1050 cm-'. In the vapor phase, where the molecules are free to rotate, we observe the typical P-Q-R band structure. In the condensed phase, where free rotation is no longer possible, this structure is lost. Fieure 19 is the GC/FT-IR soectrum of cocaine, obtained by chromatographing a solution of cocaine and piperoeaine. Compare this spectrum with that in Figure 16, which was obtained from a grain of the crystalline solid. The importance of vapor-phase libraries for the identification of spectra from GC fractions is obvious. ~ortunatelyan excellent vapor-phase library does exist and is eammerciallv available. It was beeun in the late 197U's under an ~nvironmencalProtection Agency contract and bas now grown tn over 9.W.0 compounds.
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nmtinely. New accessories have been developed just to take advantage of the capabilities of the IT technique. The future of in. frared spectroscopy, looked upon only recently a s a m a t u r e d a n d somewhat uninteresting technique, has never seemed brighter than today.
Literature Cited 1. Perkina, W. D. J. Chem.Edm. t98$63.A5. 2. Petins, W. D. J. Chem.Edu. 1981,M.A269.
3. Oxlcy. J. C.; Perkins, W. D. Infmrod Bulletin 113; Perkin-Elmer Carp.: Nomalk, CT, 1986. 4.
H-ah.R.
W.;Anae~eon,R.E.Appl.Sp~rlmae. t983,
dl 7 5
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533.
Wendlandt, W. W.; Heeht, H. 0.Refkctame Spectmcopy:Inte-ems: NaaYork, 1966:Chapter 111. 8. Kubdha,P.;MunL, F. 2.Toeh.Phys. 1931,12,593. 9. Spragg, R. A. Appl. S p l m s c . 1981,38,604. LO. Vidrine, D. W. In Fourier T m ~ f o r m Infrared Sprctroscopy;Fm, J. R.;Basiie,L.J., Eds.;Aeademie: I.
New York. 1962; Voi. 3, Chapter 4.
11. Coa~,V.J.;Offn~.A.;Siegier,E.H.,Jr. J.Opt.Sac. Am. 1953.43.984. 12.
Griffiths,P.R.;kanwa,L.V.;deHaKt4J.;Hd, 8.W.; Jakobsen. R.J.:Ennie,M. M.Appl. Sp@etmsc.
Summary FT-IR spectroscopy, with its energy advantages, has made it possible for us to ohtain spectra under conditions previously considered difficult to imoossible. Soectra of aqueous solutions, micrometer-sized particulates, and samples wlth incredibly low transmissionsaw now run hoth rapidly and
Volume 64
Number 12
December 1987
A305
