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Anal. Chem. 1907, 59, 1632-1636
studies are under way in the authors' laboratory.
ACKNOWLEDGMENT The authors thank S. G. Schulman of the School of Pharmacy, University of Florida for the phenytoin. Registry No. PABA, 150-13-0;uracil, 66-22-8; uridine, 5&96-8; 6-aminouracil, 873-83-6;naphthalene, 91-20-3; 2-naphthol, 13519-3; 2-aminonaphthaline, 91-59-8; 2-aminofluorene, 153-78-6; phenytoin, 57-41-0; silver, 7440-22-4. LITERATURE CITED Flelschmann, M.; Hendra, P. J.; McOuillan, A. J. Chem. Phys . Lett, 1074, 26, 163-166. Jeanmarie, D. L.; van Duyne, R. P. J. Nectroanal. Chem. Interfacial Electrochem. 1077, 8 4 , 1-20. Aibrecht, M. G.; Crelghton, J. A. J. Am. Chem. SOC. 1077, 9 9 , 5215-5217. Surface Enhanced Raman Scattering; Chang, R. K., Furtak, T. E., Eds.; Plenum: New York, 1982. Furtak, T. E.: Reyes, J. Surf. Sci. 1080, 9 3 , 351-382. Otto, A. Appl. Surf. SCi. 1080, 6 , 309-355. Saki, H. J. Electron. Spectrosc. Relat. Phenom. 1886, 3 9 , 289-310. Pemberton, J. E.: Buck, R. P. Anal. Chem. 1981, 5 3 , 2263-2267. Tran. C. D. Anal. Chem. 1084, 56, 824-826. Vo-Dinh, T.: Hlromoto, M. Y. K.; Begun, G. M.; Moody, R. L. Anal. Chem. 1084, 56, 1687-1670. Vo-Dinh, T.; Meier, M.: Wokaun. A. Anal. Chim. Acta, in press. Me&, M.: Wokaun, A.; Vo-Dlnh, T. J. Phys. Chem. 1085, 8 9 , 1843-1846. Enlow, P. D.; Bunclck, M.; Warmack, R. J.; Vo-Dinh, T. Anal. Chem 1086, 58, 1119-1123. Jennings, C.;Aroca, R.; Hor, A.-M.: Low,R. 0. Anal. Chem. 1084, 56, 2033-2035. Gautner, E.; Steinert. D.; Reinhardt, J. Anal. Chem. 1085, 5 7 , 1658-1662. Sheng, R.-S.; Zhu, L.; Morris, M. D Anal. Chern. 1986, 5 8 , 1116-1 1 19. Crelghton, J. A.: Biatchford, C. G.; Albrecht, M. G. J. Chem SOC., Faraday Trans. 2 , 1070, 75, 790-798.
(18) Koglin. E.; Sbquarls, J. M.; Valenta, P. J. Mol. Struct. 1080, 6 0 , 421-425. (19) Ervin, K. M.; Koglin, E.; S6quarls. J. M.; Valenta, P.; Niirmberg. H. W. J. Electroanal. Chem. Interfacial Electrochem. 1080, 114, 179-194. (20) Koglin, E.; SOquaris, J. M.; Valenta, P. J. Mol. Struct. 1082, 79, 185- 189. (21) Otto. C.; van den Tureel, T. J. J.; de Mui, F. F. M.; Grew. J. J. Raman Spectrosc. 1086, 17, 289-298. (22) Kogiin, E.; S6quaris. J. M.; Valenta, P. I n Surface Stdies with Lasers; Aussenegg, F. R., Leitner, A., Lippitsch, M. E., Eds.; Springer-Verlag: New York, 1983. (23) Koglin, E.; SBquaris, J. M.; Fritz, J. C.; Valenta, P. J . Mol. Struct. 1984, 114, 219-223. (24) Sgquaris, J. M.; Koglin, E. fresenius' Z . Anal. Chem. 1085, 321, 758-759. (25) Tsuboi. M.; Takahashi, S.; Harada, I. I n Physiochemical Properties of Nucleic Acids: Duchesne, J., Ed.; Academic: New York, 1973. (26) Lord, R. C.: Thomas, G. J., Jr. Spectrochim. Acta, PartA 1067, 23A. 255 1-259 1. (27) Dollish, F. R.; Fateley, W. G.; Bentley, F. F. Characteristic Frequencies of Organic Compounds ; Wiley-Interscience: New York, 1974. (28) Suh, J. S.;Di Lelia, D. P.; Moskovits. M. J. Phys. Chem. 1083, 8 7 , 1540-1544. (29) O'Neal. J. S.;Sloan, K. 6.; Schulman, S. G. J. Pharm. Biomed. Anal. 1086. 4 . 103-106. (30) Clarke, E. G. C. Isolation and Identification of Drugs; The Pharmaceutical Press: London, 1969. (31) The Sadler Standard Spectra ; Sadier Research Laboratories: Phildeiphia, PA: Raman Number 2583. (32) Lippitsch, M. E. Chem. Phys. 1080, 74, 125-127. (33) Lee, P. C.; Meisei, D. Chem. Phys. Lett. 1083, 9 9 , 262-265. Bumm, L. A.; Callaghan, R.; Blatchford, C. G.; Kerker, M. (34) Siimian, 0.; J . Phys. Chem. 1083, 8 7 , 1014-1023. (35) Heard, S.M.; Grieser, F.; Barraciough, C. G. Chem. Phys. Lett. 1983, 9 5 , 154-156. (36) Heard, S. M.; Grieser, F.; Barraclough, C. G. J. Phys. Chem. 1085, 8 9 , 389-392. (37) Laserna, J. J.; Torres, E. L.; Winefordner, J. D. Anal. Chim. Acta, in press
RECEIVED for review December 29, 1986. Accepted March 5, 1987. This research supported by NIH-GM-11373-23.
Simultaneous Laser-Based Refractive Index and Absorbance Determinations within Micrometer Diameter Capillary Tubes Darryl J. Bornhop' and Norman J. Doviehi* Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
A high-sensltivity, small-volume refractive index detector has been reported, whlch Is based upon the diffraction of a probe laser beam by a Capwaary tube. HIgh-semHMty thermooptkal absorbance measurements may sknultaneausly be made wlth this detector if a second, modulated pump laser beam is focused into the capillary. The modulated component of the probe beam Intensity is related to the sample absorbance whereas the umnodulated component Is related to refractive index. I n this caplilary refractive Index and absorbance detector, a l-mW hellun-neon probe laser and a 2 m W Miumcadmlum pump laser are employed to generate refractive index detection limits of 4 X lo-' A R I and absorbance detection limits of 6.3 X loT6, both measured across a 50-ymdlameter cuvette. SlmUar results were obtained for 100- and SOO-pmdIameter capbrles. The dead volume of the system, 50 pL for the 50-ym caplllary, Is limited by the tube radius; 6 mllilon amaranth molecules are present wlthln the detector volume at the absorbance detection ilmlt. Linearity In both refractive Index change and absorbance extends for at least 3 orders of magnitude.
'Present address: Lee Scientific, 4426 South Century Dr., Salt Lake City, U T 84123.
The problem of analytical measurements within smallvolume, cylindrical samples is of particular importance in capillary separation techniques. Both open tubular capillary liquid chromatography and electrophoresis require low dead volume detectors with good detection limits to preserve separation efficiency ( I , 2). A good concentration detection limit, better than M, is required because dilute analyte must be analyzed to prevent column overload. Low dead volume, less than L, is required to minimize peak spread. The combination of good concentration detection limit with small dead volume produces heroic requirements for mass detection; femtomole or better detection limits are required for the capillary separation methods. Lasers are convenient light sources for optical analysis within capillary tubes. Laser beams with good spatial coherence may be focused to small spots with no loss of power. For example, laser-induced fluorescence has been applied to conventional capillary tubes with excellent detection limits for chromatographic detection ( 3 , 4 ) . Also, the sheath flow cuvette has been combined with laser-induced fluorescence to produce excellent detection limits within micrometer diameter sample streams (5, 6),approaching single molecule detection in favorable cases (7,8). Laser-based light scatter measurements within the sheath flow cuvette have been ap-
0003-2700/87/0359-1632$01.50/0 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59,
plied to size qnalysis of nanometer diameter particles within picoliter probe volumes (9-13), including capillary hydrodynamic chromatographic detection (13). Photothermal refraction, based upon the crossed-beam thermal lens, uses laser excitation to measure very weak absorbance within micrometer square-bore tubes to praduce excellent mass detection limits for neat samples (14) and capillary liquid chromatography (15, 16). Lastly, a simple, rugged laser-based refractive index detector has been developed for measurement of refractive index changes on the order of 5 X within micrometer diameter tubes (17,18). In the determination of the refractive index of solutions contained within capillaries, a laser beam is focused into the cylindrical tube. Interaction of the laser beam and the capillary produces a fan of scattered light in the plane perpendicular to the capillary tube axis (3). The distribution of scattered light is complex, consisting of many light and dark fringes (19,ZO). Although the fringe pattern may be described by conventional light scattering theory, evaluation of the theory is impractical for tubes much larger than the wavelength of light. Instead, the fringes are treated phenomenologically. The position of some of these fringes is observed to change with the refractive index of the solution. A small area photodiode is located at the border of a fringe; a change in the position of the fringe results in a change in intensity monitored by the photodiode. This capillary refractive index detector has been demonstrated for nanoliter, neat samples. Detection limits of 6 X hRI were obtained within a 500-rm-diameter cuvette (17). The capillary refractive index detector has also demonstrated detection of nanogram quantities of sugars separated with a 250 pm diameter packed chromatographic column and a 100 pm diameter detector cuvette (18). As in other refractive index detectors, the primary noise source in these measurements appears to be associated with temperature fluctuations. Since the refractive index of most liquids changes by several parts per ten thousand with a one degree temperature change, detection limits of lo4 ARI correspond to temperature fluctuations of a few millidegrees. The variation in refractive index with temperature of most solvents suggests that the capillary-refractive index detector may be applied for thermooptical absorbance measurements (2,22). In thermooptical techniques, absorbance of a pump laser beam, followed by nonradiative relaxation of the excited states, produces a temperature rise within the sample that is proportional to both absorbance and pump laser intensity. Since the refractive index of the solvent changes with temperature, the heated region can act as an optical element to perturb the propagation properties of a probe beam. In this paper, a capillary thermooptical absorbance measurement is described in which a modulated pump laser beam is tightly focused at right angles to the sample capillary and to the probe laser beam. Absorbance of the pump beam produces a periodic temperature rise and refractive index change within the sample, which periodically deflects the probe beam. Lock-in detection of the modulated component of the probe beam intensity is used to measure absorbance within the capillary tube, The unmodulated component of the signal remains proportional to refractive index. The capillary absorbance measurement is quite similar to two thermooptical techniques: photothermal refraction, based upon the crossed-beam thermal lens (23-28), and photothermal deflection, based upon a crossed-beam thermal prism (29,30). In photothermal refraction, a cylindrically symmetric heated region is produced by absorbance of a pump laser beam. This heated region acts to defocus the probe beam out of the plane containing the pump and probe beams. Photothermal deflection acts similarly to deflect the probe beam
1633
NO. 13, JULY 1, 1987
P HOT0 DIODE
PROBE
-
LASER Q18X PUMP LASER
I
1
Figure 1. Experimental diagram. The probe laser is a 1-mW, polarized helium-neon laser. The probe beam passes through a polarizer and '1, wave plate, used to minimize retroreflections. The beam is focused with a 1OX microscope objective into the capillary tube. Upon traversing the capillary, the probe beam propagates to a 1-mm2 photodiode. The pump laser is a 2.8-mW, linearly polarized helium-cadmium laser, operating at 442 nm. The pump beam is modulated in a symmetric square wave with a chopper, reflected from a mirror, and focused into the capillary with a 18X microscope Objective. The output of the photodiode is conditioned with a current-to-voltage converter and sent to both a 4'/* digit dc voltmeter and a vector-sum lock-in amplifier. The reference signal for the lock-in amplifier is generated within the chopper head.
out of the plane containing the two beams. Defocusing or deflection of the probe beam is detected by measuring a change in the probe beam center intensity or position. Since the pump of probe beams only interact in their intersection region, very small samples may be probed with tightly focused laser beams. However, the analytical signal is obtained by monitoring the probe beam intensity so that the optical quality of the probe beam must be preserved through the optical train. Flat optical windows are required to preserve the probe beam quality. For chromatographic detection, small cross-section, square bore capillary tubes are glued to the end of the column. The use of flat windows is a definite annoyance since these square tubes are fragile, have mediocre optical quality, and are tedious to align perpendicularly to the beam axes. The capillary thermooptical absorbance measurement appears to differ from both photothermal refraction and deflection. The photothermal techniques produce an optical element, which acts to defocus or deflect the probe beam out of the plane containing the pump and probe beams. Instead, the capillary thermooptical absorbance measurement produces a shift of the probe beam in the plane containing the two beams. This shift occurs because the refractive index of the solvent at the cylindrical wall changes with the temperature, which changes the deflection angle of the probe beam. The modulated component of the probe beam intensity measured by the photodiode is related t o sample absorbance whereas the dc or unmodulated component is related to the refractive index of the sample. Simultaneous measurement of absorbance and refractive index within micrometer diameter tubes should prove useful in chromatographic detection since both selective (absorbance) and universal (refractive index) information is produced.
EXPERIMENTAL SECTION Apparatus. A block diagram of the simultaneous refractive index and absorbance detector is shown in Figure 1. A heliumneon laser produces a linearly polarized, 1-mW probe beam with a wavelength of 632.8 nm. The beam passes through a polarizing filter and a quarter-wave retardation plate. The quarter-wave plate is rotated to extinguish light retroreflected from other optical components. A lox, 16 mm focal length microscope objective is used to focus the probe beam into the capillary tube. After traversing the capillary tube, the beam propagates about 20 cm to a 1-mm2silicon photodiode. The output of the photodiode is
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
conditioned by a current-to-voltage converter with a 1-MQ feedback resistor in parallel with a 47-pF capacitor. The dc component of the signal is displayed upon a 4l/, digit voltmeter. The mean and standard deviation are estimated from 15 data points recorded at 1-9 intervals. A helium-cadmium laser produces a linearly polarized, 2-mW pump laser beam with a wavelength of 442 nm. A mechanical chopper modulates the pump beam in a square wave, typically at 12.5 Hz. This beam is focused with a 18X, 10 mm focal length microscope objective into the capillary tube at right angles to both the tube and the probe laser beam. The interaction of the pump laser beam with the sample results in a refractive index change within the sample. This refractive index change produces a deflection of the probe beam profile and a change in the probe beam intensity which is synchronized with the pump beam modulation functon. The modulated component of the probe beam intensity is measured by using a vector-sum lock-in amplifier with a 3-s time constant. A reference signal for the lock-in amplifier is generated within the chopper head. The mean and standard deviation of the lock-in signal are estimated from a set of 15 points recorded at 5-s intervals. Alignment. A significant effort was taken to minimize vibration of optical components. For example, the optical components are held with massive fixtures to a well damped optical table. The optical components for the probe beam are fixed in space and not held with translation stages. A three-axes translation stage holds the pump beam lens to provide complete freedom in the location of the pump beam waist, a two-axes stage is used to move the sample cuvette in the plane formed by the two laser beams, and a one-axis stage moves the detector across the probe beam profile. The interaction of the probe beam and the fluid-filled capillary tube produces a complicated beam profile, consisting of a set of diffraction fringes. One set of fringes undergoes translation upon change in solvent composition, apparently by an amount proportional to the change in the refractive index of the solvent. A 1-mm2photodiode detects the fringe movement. The photodiode is located near the center of a fringe; movement of the fringe produces a corresponding change in the photodiode signal. As a general rule of thumb, the photodiode is first located in the most intense portion of the probe beam profile. Then the photodiode is translated perpendicularly to the laser beam axis and away from the unperturbed beam path a distance such that the photodiode intensity is approximately 0.37 (l/e) times the maximum intensity. This position of the photodiode results in both good sensitivity and reasonable linearity. Higher sensitivity is produced if the photodiode is located near the most intense portion of the beam profile; however, linearity suffers at that position. It may be necessary to translate slightly the capillary tube perpendicularly to the probe beam axis and iterate the detector alignment to generate good refractive index sensitivity and linearity. Large thermooptical signals are observed for a number of positions of the photodiode across the probe beam profile. The position of the photodiode which generates good sensitivity and linearity for refractive index measurements also produces good sensitivity and linearity for the thermooptical capillary absorbance measurement. Chemicals. All chemicals are reagent grade or better. Methanol is used as the solvent. Refractive index standards are 0%, 0.2%,0.4%, 0.6%, O B % , and 1.0% (v/v) glycerol in methanol. A stock absorbance standard 2.2 X M amaranth dye is prepared in methanol. A number of dilutions are prepared to construct the absorbance calibration curve. For this dye, t(442 nm) = 6000 L M-' cm-'.
RESULTS AND DISCUSSION A calibration curve was constructed for refractive index measurements by using glycerol solutions in methanol within 50-, 100-, and 500-~m-diametercapillary tubes. The results are summarized in Table I. The refractive index calibration curves were linear, r > 0.997, over at least a factor of 500 in refractive index change from the detection limit, about 2.5 x lo4 ARI, to the highest concentration sample analyzed, ARI = 1.2 X Although no decrease in linearity was observed for the highest concentration sample investigated, nonlinear
Table I. Limits of Detection for Absorbance and Refractive Index
cuvette diameter,
refractive index
wm
limit: ARI
absorbance detection limita
50 100 500
4 x 10-6 3 x 10-6 3 x 104
6 X lo* 9 x 104 6X
detection
absorbance per unit length at detection limit, cm-' 1.2 x 10-3 9.0 x 10-4
1.2 x 10-3
Detection limits are two standard deviations larger than the noise in the background signal. behavior is expected a t higher concentrations; the fringe maximum had shifted near the photodetector at the highest concentration sample investigated. Smaller cuvettes produced slightly poorer refractive index detection limits, presumably because of greater temperature drift within the low heat capacity tubing. Detection limits of ARI = 6 X lo-' were reported for our earlier refractive index measurements, obtained within a 500-pm-diameter Pyrex tube and aqueous samples (17). The present refractive index detection limits are a factor of 5 poorer. However, the current results are obtained with methanol solutions. Since the change in refractive index with temperature is also a factor of 5 greater for methanol than water, the refractive index detection limits probably are a result of greater sensitivity to temperature change for methanol compared with water. It appears that the low heat capacity of a thin capillary tube results in significant sensitivity to environmental temperature fluctuations. To produce better refractive index detection limits, it will be necessary to regulate the temperature of the capillary tube. Methanol was chosen as the solvent because it produces greater sensitivity than water for thermooptical absorbance measurements. Absorbance calibration curves were obtained for the three capillary tubes and also are summarized in Table I. The calibration curves were all linear, r > 0.9998, over more than 3 orders of magnitude. For the 5O-gm cuvette, linearity extended from the detection limit, A = 6 X lo4, to the highest concentration sample studied, A = 0.01. Slightly poorer detection limits were obtained for the other cuvettes. No deviation from linearity was observed for any of the samples studied. It is interesting that the concentration detection limits produced by the capillary thermooptical absorbance measurements were virtually identical for the three cuvettes, 2 X lo-' M, and independent of path length. The slope of the calibration curves, about 4 x lo2 V/M, was relatively consistent for each cuvette. A constant sensitivity, independent of path length, suggests that the instrument is not measuring absorbance but rather the absorptivity-concentration product in units of cm-'. I t appears that the signal is generated in a localized region within the cuvette, probably a t the portion of the cuvette nearest the entrance of the pump beam. Noise also is independent of cuvette diameter and appears to be associated with probe beam intensity fluctuations. This independence of detection limit upon path length is very exciting; if the independence holds for smaller diameter capillary tubes, high-sensitivity absorbance measurements may be made for open-tubular capillary liquid chromatography. A finite time will be required to reach thermal equilibrium in the thermooptical absorbance measurement. To characterize the time constant for thermal equilibrium, the lock-in signal was measured as a function of modulation frequency and is presented in Figure 2 in the form of a Bode plot. The signal amplitude demonstrated a relatively modest change with frequency below about 120 Hz. At the highest frequencies
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
*
I 2.50
0
LOO
1.50
I 0
2.00
LOG (Chopping Frequency) Flgure 2. Chopping frequency dependence. The logarithm of the amplitude of the lock-in amplifier signal, in dB, is plotted against the logarithm of the chopping frequency in Hz. A 20 dB/decade roll-off in signal is observed for high chopping frequency, whereas a small change In slgnal is observed at low chopping frequency.
probed, the signal approached a 20 dB decrease in amplitude per decade increase in modulation frequency. The shape of this curve is similar to that observed for other thermooptical absorbance measurements ( 2 3 , 2 4 , 3 1 )with a time constant on the order of 0.2 ms. This time constant is about a factor of 10 faster than that observed for the crossed-beam thermal lens with the same focusing lens and the same pump laser (24). One possible source of the short time constant is enhanced heat flow a t the capillary wall. A short time constant is of value since very fast phenomena may be studied without degradation due to instrumental artifacts. Several alignment constraints were qualitatively investigated for the thermooptical capillary absorbance measurement. As expected, the signal was highest when the pump laser beam waist was centered in the cuvette. Since a tightly focused pump beam also minimizes the system volume, all measurements reported in this manuscript were obtained with the pump waist located in the cuvette. The offset of the pump and probe beam axes turns out to be quite critical. Small changes in alignment of the axes, on the order of 20-pm, produces a significant change in absorbance signal. This alignment constraint probably reflects the very small beam spot-sizes produced by the short focal length microscope objectives used in the experiment. The sample cuvette was located at a number of positions before, at, and after the probe beam waist. No systematic difference in absorbance or refractive index sensitivity was noted a t these positions. To minimize system volume, all measurements were obtained with the probe beam waist also located in the cuvette. The definition of detector volume for crossed-beam instruments is somewhat arbitrary. For example, the intersection volume of the pump and probe beams in this instrument is quite small, slightly less than a picoliter. Instead, we use a much more conservative definition of the detection volume as a cylinder whose radius and height are given by the capillary radius. In a chromatographic application, it is difficult to imagine an eluted peak with smaller volume. For the 50-fim-diametertube, the detector volume was about 50 M, only mol or pL. At the detection limit of 2 X 6 million analyte molecules were present within the detector volume. Of course, a significantly smaller amount of analyte was present within the small intersection volume of the laser beams. It is amusing to extrapolate these detection limits to smaller capillary tubes, higher pump laser power, and more strongly absorbing analyte. Assuming signal linearity with regard to pump laser power and sample absorptivity and independence with regard to capillary diameter, then detection of tens of analyte molecules will be possible with a 100-mW pump laser, 5-fim-diameter capillary tube, and analyte with molar absorptivity of lo5 L M-' cm-I.
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Two other laser-based detectors have been employed for measurement of refractive index and absorbance (32,33). In the first case, the sensitivity of refractive index to temperature change is used to construct a thermooptical absorbance measurement. A very sensitive Fabry-Perot interferometer is used to measure the refractive index of the analyte. A second pump laser beam is introduced along the probe beam path; absorbance of the pump laser beam produces a temperature rise and refractive index shift. Absorbance detection limit of 2 X lo4 and refractive index detection limits of 1.5 X were obtained over a 10-cm path with a 60-mW pump laser. Linearity extended for 2 orders of magnitude in absorbance and the detector volume about 200 pL. A second combination absorbance-refractive index detector has been reported which utilizes reflection near the critical angle to and reproduce absorbance detection limits of 2.1 x within a 1-pL probe fractive index detection limits of 2 X volume. A high-frequency acoustooptic modulator and lock-in amplifier were employed to reduce laser intensity noise. Absorbance was measured by sample transmission and could not be distinguished from refractive index changes. Absorbance detection limits for the capillary instrument are similar to those of the Fabry-Perot interferometer and 2 orders of magnitude superior to those of the internal reflection instrument. Refractive index detection limits reported for the interferometer and the critical angle detector are 2 and 1 order of magnitude superior, respectively, to our results. It appears the large heat capacity of the interferometer and the prisms used in the critical angle instrument generate much lower sensitivity to temperature fluctuations than the micrometer capillary tube used in our instrument. Of course, the sample volume utilized in the capillary detector is over 4 orders of magnitude smaller than the critical angle reflection instrument and 6 orders of magnitude smaller than those of the interferometer. Very low power, relatively inexpensive, and reliable lasers were employed to measure simultaneously absorbance and refractive index within micrometer diameter capillary tubes. The optical and electronic system, minus the optical table, could be duplicated for about US $5000. The combination of relative low cost, high reliability, small volume, and excellent detection limits for both absorbance and refractive index should prove attractive for a number of applications in capillary separation techniques.
LITERATURE CITED Novotny, M. Anal. Chem. 1981, 5 3 , 1294A-1308A. Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222, 266-272. Folestad, S.; Johnson, L.; Josefsson, B.; Galle, B. Anal. Chem. 1982, 5 4 , 925-929. Gluckman, J.; Shelly, D.; Novotny, M. J . Chromatogr. 1984, 317, 443-453. Hershberger, L. W.; Callis. J. B.; Christian, G. D. Anal. Chem. 1979, 5 1 , 1444-1446. Kelly, T. A.; Christian, G. D. Anal. Ghem. 1981, 5 3 , 2110-2114. Dovichi, N. J.: Martin, J. C.; Jett, J. H.: Kelier, R . A. Science, 1983, 219, 845-847. Dovichi, N. J.; Martin, J. C.; Jett, J. H.;Trkula, M.;Keller, R . A. Anal. Chem. 1984, 5 6 , 348-354. Zarrin, F.; Dovichi, N. J. Anal. Chem. 1987, 5 9 , 846-850. Zarrin, F.; Risfelt, J. A.; Dovichi, N. J. Anal. Chem. 1987, 5 9 , 850-854. Zarrin, F.; Bornhop, D. J.; Dovichi, N. J. Anal. Chem. 1987, 5 9 , 854-860. Zarrin, F.; Dovichi, N. J. Anal. Chem. 1985. 5 7 , 2690-2692. Zarrin, F.; Dovichi, N. J. Anal. Chem. 1985, 5 7 , 1826-1829. Nolan, T . G.; Dovichi, N. J. I€€€ Circuits Devices Mag. 1986, 2 , 54-56. Nolan, T. G.; Bornhop, D. J.; Dovichi, N. J. J. Chromatogr. 1987, 384, 189-195. Nolan, T. G.; Dovichi, N. J., submitted for publication in Anal. Chem. Bornhop. D. J.; Dovichi, N. J. Anal. Chem. 1986, 58, 504-505. Bornhop, D.J.; Nolan, T. G.; Dovichi, N. J. J. Chromatogr. 1987, 384, 181-187. Kerker, M.; Matijevk, E. J. Opt. SOC.A m . 1961, 57, 506-508. Watkins, L. S. J. Opt. SOC.A m . 1974, 6 4 , 767-771. Harris, J. M.; Dovichi, N. J. Anal. Chem. 1980, 52, 695A-706A. Dovichi, N. J. CRC Crit. Rev. Anal. Chem., in press.
Anal. Chem. 1907, 59, 1636-1638
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(23) Dovichi, N. J.; Nolan, T. G.; Weimer, W. A. Anal. Chern. 1084, 56. 1700- 1704. (24) Nolen, T. G.; Weimer. W. A.: Dovichi, N. J. Anal. Chem. 1084, 56. 1704-1707. (25) Weimer, W. A.; Dovichi, N. J. J . Appl. phvs. 1086, 59, 225-230. (26) Weimer, W. A.; Dovichi, N. J. Appl. Opt. 1985, 24, 2981-2986. (27) Weimer, W. A.; Dovichi. N. J. Appl. Spectrosc. 1085, 39, 1009- 10 13. (28) Burgi, D. S.;Nolan, T. G.; Risefelt, J. A,; Dovichi, N. J. Opt. f n g . 1084, 23, 756-758. (29) Jackson, W. B.; Amer, N. M.; Boccara, A. C.; Fournier, D. Appl. Opt. 1081, 20, 1333-1344.
(30) Wetsel, G. C.; Stotts, S. A. Appl. Phys. Lett. 1083, 42, 931-933. (31) Dovichi, N. J.; Harris, J. M. Proc. SPIE-Int. SOC. Opt. fng. 1981, 288, 372-375. (32) Woodruff, S. D.; Yeung. E. S. Anal. Chem. 1082, 54, 1174-1178. (33) Wilson, S. A.; Yeung, E. S.Anal. Chem. 1085, 5 7 , 2611-2614.
RECEIVED for review December 23, 1986. Accepted March 2, 1987. This work was funded by the Natural Sciences and Engineering Research Council of Canada.
Fluorescence Emission as a Probe To Investigate Electrochemical Polymerization of 9-Vinylanthracene Prashant V. Kamat
Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556
The cationic polymerization of 9-vinylanthracene can be InC tiated at a transparent Sno, electrode with the application of anodic potentials ( E > 1.1 V vs. saturated sodium chloride calomel electrode) In acetonitrile solutions. The exclmer 500 nm) of poiy(9-vinylemlsslon (emission maxlmum anthracene) whlch is dlstingulshabie from the monomer fluorescence emlsslon (emlsslon maxima 410,430 nm) has been wed to probe the eiectrochemkai polymerization process directly. The in situ spectroelectrochemkai technique, which wouM be useful in obtalning kinetic and mechanistic Information of the electropdymerizatlon process, Is described.
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In recent years, much attention has been drawn to the development and characterization of conducting polymers (1-4). Many aromatic heterocyclic molecules, upon electrochemical oxidation, lead to the formation of an electrically conducting organic polymer film at the electrode surface (1). Recently we reported ( 5 ) electrochemical polymerization of 1-vinylpyrene a t a conducting SnOpelectrode achieved with the application of anodic potentials. Such polymers with aryl molecules as pendent groups often exhibit excimer emission which is distinctively different from the monomer fluorescence emission. It is of interest to see whether one could utilize this photophysical property to probe the polymerization process directly. The purpose of this study is to demonstrate the feasibility of an in situ spectroelectrochemical technique in monitoring the electropolymerization process by using such fluorescence emission as a probe. As shown earlier (610), a technique could be useful for electrochemists to study the electrode surface as well as the transients and products generated in an electrochemical reaction.
EXPERIMENTAL SECTION Materials. 9-Vinylanthracene (Aldrich),acetonitrile (Aldrich, gold label), and tetrabutylammonium perchlorate, TBAP (Alfa), were used as supplied. SnOz electrodes were cut from an antimony-doped NESA glass obtained from PPG industries and were cleaned as described earlier (11). Instrumentation. Electrochemical and spectroelectrochemicd measurements were done with a Princeton Applied Research (PAR) Model 173 potentiostat/galvanostat, a PAR Model 175 universal programmer, and a Kipp and Zonnen X-Y recorder. Experiments were performed in a standard three-compartment
cell with a Pt wire as a counter electrode (CE) and a saturated sodium chloride calomel electrode (SSCE) as a reference electrode (RE). The potentiostat had a provision to compensate for the iR drop and this was employed during the electrochemical measurements. For spectroelectrochemical measurements a poly(tetrafluoroethy1ene) block was machined to accommodate a 0.5 cm X 4 cm SnOz plate (WE), a Pt wire (CE), and an Ag wire (RE) and was inserted into the 1 x 1 x 4.5 cm cuvette (Figure 1). The distance between the SnOP and the front wall of the cuvette, which was about 0.5 mm, allowed a thin layer of the sample. The whole assembly could then be inserted into the sample chamber of the fluorometer such that the excitation beam was at 45' to the SnOz electrode. In principle this spectroelectrochemical cell was similar to a previously described OTTLE-optically transparent thin layer electrochemical-cell (9, IO). Fluorescence measurements were carried out in the front face geometry. A SLM single photon counting fluorescence spectrometer was used t o monitor the fluorescence emission.
RESULTS AND DISCUSSION Electropolymerization of 9-Vinylanthracene. Cyclic voltammograms observed under anodic scans are shown in Figure 2. At potentials around 1.1 V (vs. SSCE) 9-vinylanthracene exhibited an irreversible oxidation and led to a curve crossing in the reverse scan. Successive anodic scans led to the decrease in the peak current and simultaneous growth of the polymer film a t the electrode surface. This behavior was similar to the electrochemical polymerization of 1-vinylpyrene and is induced by the radical cation of 9vinylanthracene formed during electrochemical oxidation The
polymer is insoluble in acetonitrile but soluble in solvents such as T H F or CH2C12. Absorption and Emission Characteristics. The excitation and emission spectra of 9-vinylanthracene in CH&N and poly(9-vinylanthracene) film coated on the SnO, electrode are shown in Figure 3. (The polymer film coated S n 0 2plate was carefully washed with CH&N to remove any traces of the monomer.) The monomer exhibits absorption maxima at 350,368, and 388 nm and emission maxima at 420 and 428 nm. The polymer film has absorption maxima a t 358, 376, and 396 nm which is red-shifted compared to its monomer
0003-2700/87/0359-1636$01.50/00 1987 American Chemical Society
