Anal. Chem. 1988, 60, 1908-1910
1908
(32) Sanders, M. J.; Cooper, R. S.; Small, G. J.; Helslg, V.; Jeffrey, A. M. Anal. Chem. 1885. 57, 1148. (33) Randerath, E.; Reddy, M. V.; Gupta, R. C. Roc. Natl. Acad. Sci. U . S . A . 1981, 78, 6126. (34) Harris, C. C.; Yolker, R. H.;Hsu, I. C. Roc. Natl. Acad. Sci. U . S . A . 1979, 7 6 , 5336. (35) Day, E. D. I n Advanced Immunochemistry; Williams and Wllklns: Baltimore, MD, 1972. (36) Pecht, I. In The Antigens; Sela, M., Ed.; Academic: New York, 1982; VOl. 6, pp 1-68. (37) Nisonoff, A.; Hopper, J. E.; Spring, S. B. The Antibody Molecule; Academic: New York, 1975; Chapter 2.
RECEIVED for review February 18, 1988. Accepted April 25,
1988. This research was supported by the National Institutes of Health under Contract GM 34730 with the University of Tennessee, Knoxville, and the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. B.J.T. acknowledges support from the U.S. Department of Energy/Oak Ridge Associated Universities predoctoral fellowship program administered a t Oak Ridge National Laboratory. R.M.S. acknowledges support from NIH and NIEHS under Contract CA 21111 and ES03881, respectively.
Fourier Transform Infrared Spectra of Organic Compounds in Solution and as Thin Layers Obtained by Using an Attenuated Total Internal Reflectance Fiber-optic Cell Shimon Simhony and Abraham Katzir School of Physics a n d Astronomy, Sackler Faculty of Exact Sciences, Tel-Aviv University, Ramat-Aviv, Tel- Aviv 69978, Israel
Edward M. Kosower* Biophysical Organic Chemistry Unit, School of Chemistry, Sackler Faculty of Exact Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel
A cell lined wlth Teflon fluorocarbon resin, contalnlng a silver halide (AgBr/AgCI) Infrared fiber, allows convenient and reproducible loadJng of organic solutions and sdlds (amorphous or crystalllne) derlved by evaporatlon of the solvent. Attenuated total Internal reflectance (ATR) measurements wlth a Fourler transform Infrared (FT-IR) spectrometer are reported for organlc compounds. Known spectroscoplc features can be recognlzed with quantltles as small as 6 ng (ca. ‘/20th of a monolayer). Usable spectra are obtalnable wlth 1OO-ng quantities (1 monolayer), and high-quality spectra are measurable wlth 10-pg quantltles. Absorbance Is linear up to ca. 600 monolayers (0.55-pm layer thlckness), the timlt of absorptlon belng ca. 0.9-pm layer thickness (about 1000 molecular layers).
Infrared absorption spectra are useful for obtaining structural information about organic compounds. Substantial concentrations of material are normally used with suitable path lengths to compensate for the low absorption coefficients in the infrared region. Polar solvents require the use of special cell materials (usually expensive) and often have absorption bands in interesting regions. These difficulties may be overcome by the measurement of spectra of thin-layer films (I, 2) by using attenuated total reflection (ATR) (3), but the currently available cells utilize costly crystals (ZnSe, Si, Ge, KRS-5, for example) and may not be convenient for samples with unusual physical characteristics. We recently reported the development of a silver halide (AgBr/AgCl) infrared fiber-optic cell for aqueous solutions ( 4 ) . A different design was introduced for protein solutions and suspensions (5). We now describe a moderate-cost cell suitable for a variety of infrared measurements with organic materials; of especial
interest are measurements on small amounts of solid deposited on the fiber by evaporation of a solution.
EXPERIMENTAL SECTION A specially designed cell (Figure 1)was used to measure spectra with a Nicolet 5DX Fourier transform infrared (FT-IR) spectrometer. The cell contains a 0.9-mm silver bromide/silver chloride polycrystalline fiber of 100-mm length, sealed into a Teflon-lined well with solvent-resistant gaskets. In our experiments, the FT-IR source radiation was focused on the input end focal length, 25-mm of the infrared fiber with a ZnSe lens (25” diameter). The radiation was collected from the exit of the fiber and collimated with a similar ZnSe lens. It was then introduced into the detector section of the FT-IR spectrometer, which incorporates a pyroelectric detector (deuteriated triglycine sulfate), The silver halide fibers are fabricated by extruding a pure crystalline mixture of AgClxBr,,, with x between 0.5 and 0.95, through a die at a temperature close to the softening point of the halide (6-9). The fiber inside the cell is readily removed and replaced. Exposure of the fiber to strong fluorescent or UV light should be avoided due to darkening. However, no noticeable degradation of fiber transmission was observed after prolonged exposure to incandescent lamp light or infrared radiation. With a mount, the cell (illustrated in Figure 1)can be removed and replaced without affecting optical alignment. Solvent or solution may thus be introduced into the cell at locations away from the FT-IR spectrometer,a precaution necessary for avoiding contamination of the light path with solvent vapors. After a background spectrum is measured with the clean fiber, about 250 p L of solvent is introduced into the cell, covering the fiber. The spectrum of the solvent is then measured. The solvent is removed, a solution of sample is introduced, and a spectrum is measured. The difference between the spectra is the spectrum of the sample in solution. The solvent may then be carefully evaporated with a gentle stream of dry nitrogen, leaving a thin solid film on the fiber, and the spectrum of the solid sample may now be measured. About 5 min (300 scans) is required for each spectrum of small amounts of material, but only 50 scans (less
0003-2700/88/0360-1908$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 18, SEPTEMBER 15, 1988
1909
Syn- (ETHYL, METHYL) B I M A N E
FIBER s/\ FACE AND SEALING GASKET
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ZnSe
LENS
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J Figure 1. Schematic drawing of the silver halide fiber-optic cell used
for organic compound solutions and solids. Syn- B
h g B r + AgCl
F I B E R CTR
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: 1400
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Figure 3.
Infrared spectra of syn -(ethyl,methyl)bimane (EMB) betweeen 2000 and 500 cm-' on a siliver bromide/silver chloride fiber with loadings of 58.75 pg (lower curve), 93.5 ng (middle curve), and 11.75 ng (upper curve). The last two spectra were measured with nitrogen purging. The bar is equivalent to an absorbance of 0.06 for the 59-1.18 spectrum or 0.003 for the 93- and 12-ng spectra.
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Flgure 2. A comparison of the infrared spectrum of a syn-(carbeth-
0.004 0.00s 0.006
oxy,chloro)blmane (CCB) solution in dichloromethane with that of CCB as a microcrystalllne film using attenuated total internal reflectance on a silver bromide/silver chloride infrared fiber. The solution used contained about 1 mg of CCB in 0.3 mL of solvent (spectrum labeled "Solution"). The spectrum labeled "Solid" was obtained wlth the cell after evaporation of the solvent from the solution, The absorbance axis is different for each spectrum, as shown by the numbers next to the bar on the figure.
than 1 min) are sufficient for good spectra with microgram amounts of materials. Spectra were measured with 4 cm-' resolution. RESULTS AND DISCUSSION We report here two types of experiments with bimanes (10) as typical organic compounds. The first type (with syn(carbethoxy,chloro)bimane (CCB)(11))illustrates how easily the infrared spectra of a solution and a solid may be compared (Figure 2). To illustrate the quality of the spectra obtainable with the fiber-optic cell, no averaging of the data was performed. The second type of experiment (with syn-(ethyl,methy1)bimane (EMB)(12))demonstrates that spectra may be acquired with amounts of material between 6 ng and 100 pg. (Figure 3 shows spectra of E M B solid films of 11.75 and 93.5 ng and 58.75 pg.) The intensities of absorption are reasonably linear over the range 0.1-40 pg (Figure 4). CCB shows similar behavior.
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Assuming uniform distribution of material over the surface of the fiber, the coverage by E M B corresponds to about 5%
1866 P u y
Figure 4. Plots of the absorbance of syn-(ethyl,methyl)bimane (EMB) at 1265 cm-' versus weight (in micrograms) introduced into the cell: A, 0-4 pg; B, 0-200 pg.
of a monolayer for the smallest amount and about 1000 monolayers for 100 pg. The absorbance becomes almost constant a t coverages corresponding to ca. 0.6 pm from the
1910
Anal. Chem. 1988, 60,1910-1914
surface, the limit imposed by the penetration depth of the evanescent wave (3). The linearity of absorbance with weight at low concentrations is particularly striking and demonstrates that the intensities of selected peaks over certain concentration ranges can be measured with good accuracy. The fact that the plot line goes through zero suggests that absolute quantitation is possible, although caution requires the statement that what is constant is the distribution of the solute after evaporation between the fiber and the cell. Indications are that most of the material is deposited on the fiber. The coverage of the fiber by the solid must be reasonably uniform on the basis of the linearity of absorbance with concentration. Solvents may be readily exchanged by evaporating one solvent and introducing a second solvent. Dynamic changes in the solutions are readily followed. The continuing modest rise in absorbance for amounts greater than the limit suggests that “thin spots” in fiber surface coverage are being filled. Some distortion of the spectrum at the limit should be expected from the different penetration depths for different wavelengths; the phenomenon will be examined in another connection and reported elsewhere. The present silver halide fiber-optic results compare favorably with other ATR experiments such as the ATR study of Badilescu et al. (13). Between 100 and 600 pg of a transretinylidene tert-butylimine (RB) was placed on thallium bromide iodide (KRS-5), zinc selenide, germanium, or silicon ATR crystals. The area of surface covered by the RB was between 2.5 and 5.0 cm2 (assuming that one side was used), and 100 scans were used to acquire the spectra. The area of the silver halide fiber used in the present work was 3 cm2. Assuming that the syn-(ethy1,methyl)bimanefits into a lo-A cube, ca. 110 ng is needed to form a uniform monolayer. Saturation of the absorption occurs at a loading of ca. 75 Fg, corresponding to 680 layers or a layer thickness of 0.68 Fm. CONCLUSIONS Evanescent wave fiber-optic spectroscopy is particularly convenient for the study of small quantities of a large variety of materials in various forms (3). Silver halide fibers are fairly useful as ATR elements because they are nontoxic and insoluble in water and have a particularly wide infrared wavelength transmission range (2-25 pm). This work represents
their first application to quantitative infrared spectroscopy. The fiber-optic cell makes it possible to examine both solutions and solids derived from evaporation of the solvent in a single set of experiments, which might be of great advantage if only small samples are available. In principle, silver halide fibers are inexpensive enough to be used for materials like adhesives, which cannot be readily removed from the fibers and might require replacement of the fibers after a single use. Otherwise, the cell and fiber are easily cleaned by ordinary solvents after an experiment. Further applications of the cell to infrared spectroscopy of monolayers will be reported in due course. ACKNOWLEDGMENT We wish to thank A. Levite, A. Yekuel, B. Bar, B. Agam, and N. Lavie for their enthusiastic and effective cooperation in various technical aspects of the research. Registry No. CCB, 79746-74-0; EMB, 99240-32-1;AgCl,Br,, ( X = 0.5-0.95), 115419-70-0. LITERATURE C I T E D Fourier Transform Infrared Spectroscopy: Appllcafions to Chemical Systems; Ferraro, J. R., Basile, L. J., Eds.; Academic: New York, 1979; Vol. 2, pp 321. Analfllcal Applications of FT-IR to Molecular and Bioiogical Sysfems ; Durig, J. R., Ed.; NATO Advanced Study Institute; Reidel: Dordrecht, The Netherlands, 1980; Vol. C-57, pp 607. Harrick, N. J. Infernal Reflection Spectroscopy; Wiley: New York, 1967. Simhony, S.; Kosower, E. M.: Katzir, A. Appl. Phys. Len. 1988, 4 9 , 253-254. Simhony, S.; Kosower, E. M.; Katzir, A. Biochem. Biophys. Res. Commun. 1987, 742, 1059-1063. Bendow, B.; Rast, H.; El-Bayoumi, 0. H. Opt. Eng. 1985, 2 4 , 1072-1 080. Katzir, A.; Arieli, R. J . Non-Cryst. SolMs 1982, 4 7 , 149. Sair, A.; Moser, F.; Akselrod, S.; Katzir, A. Appl. Phys. Left. 1986, 4 9 , 305-307. Sa&, A.; Katzir, A. J . Opt. Soc. Am. A , in press. Kosower, E. M.; Pazhenchevsky, B. J . Am. Chem. SOC. 1980, 102, 1983-4993. Kosower, E. M.; Ben-Shoshan, M.; Faust, D.; Goldberg, I. J . Org. Chem. 1982, 4 7 , 213-221. Kosower, E. M.; Qoldberg, I . ; Zbaida, D.; Baudh, M.; Marclano, D., Tel-Avlv Universlty, unpublished results, 1988. Badilescu, S.; Lussier, L. S.; Sandorfy, C.; Le Thanh, H.; Vocelle, D. Chem. Phys. Len. 1987, 133, 63-66.
RECEIVED for review February 8, 1988. Accepted April 26, 1988. Partial support from the Israeli Fund for Higher Education allowed the purchase of the FTIR spectrometer.
Examination of Textural Differences between Polymeric and Brush Phases C. H.Lochmuller* a n d M. T. Kersey Department of Chemistry, Duke University, Durham, North Carolina 27706 I n this work steady-state and time-dependent luminescence data for polymeric and brush phase samples In contact with 100% hexane and methanol from 0.17 to 1.08 pmd/m2 have been examined. The resuits Indicate that polymeric phases are more motlonally constrained than brudr phases, exhiblling longer average lifetimes for simllar surface coverages. Despite their relative rigidity, polymeric phases exhibit textural changes between hostile and good solvents. The tlmadependent lumlneecence behavior of poiymerk phases, while similar to that of the brush, suggests that polymeric surface layers are more complex than the brush phase surface.
A concerted effort has been made to better understand the
role of covalently bound ligands in determining solute selectivity in liquid chromatography (1-4). Our laboratory introduced the use of luminescence spectroscopy to study common liquid chromatographic stationary phases and to determine mobile-phase-induced effects on the bound ligands (5). The results suggest a liquidlike environment in monomeric alkyl bonded phases, and these brush phases undergo dynamic conformational changes in response to changes in contacting solvent. Steady-state and time-dependent luminescence experiments suggest that monofunctional silane/surface silanol reactions with silica result in brush phases composed of bound ligands clustered predominantly into regions of high local density (6, 7). Recently, a unified molecular theory for selectivity based
0003-2700/88/0360-1910$01.50/00 1988 American Chemical Society
