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Anal. Chem. 1990, 62, 1754-1758
(8) Almenningen, A.; Bastiansen. 0.; Fernholt, L.; Cyvin, B. N.; Cyvin, S. J.: Samdal, S. J. Mol. Shuct. 1985, 728, 59-76. (9) Almenningen, A.; Bastiansen, 0.; Fernholt, L.; Gundersen, S.; KlosterJensen, E.; Cyvin. B. N.; Cyvin. S. J.; Samdal, S.:Skancke, A. J. Mol. Stfuct. 1985, 128, 77-93. (10) Almenningen, A.; Bastiansen, 0.; Gundersen, S.;Samdai, S.; Skancke, A. J. Mol. Struct. 1985, 728, 95-1 14. Samdal, S. J. Mol. Struct. 1985, 128, 115-125. (11) Bastiansen, 0.; (12) Ramming, Chr.; Seip, H. M.; Aanesen Dymo, Acta Chem. Scand. Ser. A . 1974, 2 8 , 507-514. (13) Hargreaves, A,; Rizvi, S. H. Acta Crystal/ogr. 1982, 15. 365-373. (14) Charbonneau, G.P.; Delugeard, Y. Acta Clystallcgr., Sect. 8 : Struct. Ctystallogr. Cryst. Chem. 1978. 32, 1420-1423. (15) Brock, C. P. Acta Crystallogr., Sect. 8 : Struct. Crystallogr. Cryst. Chem. 1980, 36, 968-971. (16) Aimlof, J. Chem. Fhys. 1974, 6 , 135-139. (17) McKinney, J. D.; Gottschalk, K. E.; Pedersen, L. J . Mol. Struct.: THEOCHEM 1983, 104. 445-450. (18) Hafelinger. G.; Regeimann, C. J . Comput. Chem. 1987, 8. 1057- 1065. (19) Janssen, J.; L a k e , W. J. Mol. Struct. 1979, 55, 265-281. (20) Tinland, B. Theor. Chim. Acta 1968, 1 1 , 452-454. (21) Gropen, 0.; Seip, H. M. Chem. Phys. Lett. 1971, 7 7 , 445-449. (22) Casalone, G. L.; Mariani. C.; Mugnoii, A,; Simonetta. M. Mol. phvs. 1968, 15. 339-348. (23) StQlevIk, R.; Thingstad, 0. J. Mol. Struct. 1984, 106, 333-353. (24) Lindner, H. J. Tetrahedron 1974, 30, 1127-1 132. (25) Charbonnler, S.:Beguemsi. S. T.; N'guessan, Y. T.; Legoff. D.; Proutiere. A.: Viani. R. J. Mol. Struct. 1987. 758.109-125. Grant, D. M.; Paul, E. G. J. Am. Chem. Soc. 1984, 86, 2984-2990. Lindeman, L. P.; Adams, J. Q. Anal. Chem. 1971, 43, 1245-1252. Small, G. W.; Jurs, P. C. Anal. Chem. 1983, 5 5 , 1128-1134. Small, G. W.; Jurs, P. C. Anal. Chem. 1984, 56, 2307-2314. Egolf, D. S.; Jurs, P. C. Anal. Chem. 1987, 59, 1586-1593. Egolf. D. S.;Brockett, E. 8.; Jurs. P. C. Anal. Chem. 1988. 6 0 , 2700-2706.
(32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) (47)
(48) (49) (50) (51)
Sutton, G. P.; Jurs, P. C. Anal. Chem. 1989, 61, 863-871. Ranc, M. L.; Jurs, P. C. Anal. Chem. 1989, 61, 2489-2496. Smlth. D. H.; Jurs, P. C. J. Am. Chem. Soc. 1978, 100, 3316-3321. Small, G. W.; Jurs, P. C. Anal. Chem. 1983, 55, 1121-1127. Yanaglsawa, M.; Hayamiru, K.; Yamamoto, 0. Magn. Reson. Chem. 1986, 24, 1013-1014. Brugger, W. E.; Jurs, P. C. Anal. Chem. 1975, 47, 781-783. Stuper, A. J.; Jurs, P. C. J. Chem. Inf. Comput. Sci. 1978. 16. 99-105. Rohrbaugh, R. H.; Jurs, P. C. UDRAW. W E 1988, Program 300. Stuper. A. J.; Brugger, W. E.: Jurs, P. C. Comp& Assisted Studies of Chemical Structure and 8iological Function ; Wlley-Interscience: New York, 1979; pp 83-90. Roberts, R. M. G. M a p . Reson. Chem. 1985, 2 3 , 52-54. Ewing, D. F. Org. Magn. Reson. 1979, 12, 499-524. RandiE, M. J. Am. Chem. SOC. 1975, 97, 6609-6615. Kier. L. 8.; Hall, L. H. J. Pharm. Sci. 1978, 65, 1806-1809. RandiC, M. J. Chem. Inf. Comput. Sci. 1984, 24, 164-175. Burkert. U.; Allinger, N. L. MokukrMechanlcs; ACS Monograph 177; American Chemical Society: Washington, DC. 1982. Yates. K. Huckel Molecular Orbital Theory; Academic: New York, 1978. Del Re, G. J. Chem. SOC. 1958, 4031-4040. Topliss. J. G.; Edwards, R. P. J. Med. Chem. 1979, 22, 1238-1244. Draper, N. R.; Smith, H. Applied Regression Ana/)&, 2nd ed.; WlleyInterscience: New York, 1981. McKinney, J. 0.; Singh, P. Chem.-Biol. Interact. 1981, 33, 271-283.
RECEIVEDfor review March 14,1990. Accepted May 14,1990. This work was supported by the National Science Foundation under Grant CHE-8815785. The PRIME 750 computer was purchased with partial financial support of the National Science Foundation.
Unintensified Photodiode Array Fluorescence Detector for High-Performance Liquid Chromatography Jeff Wegrzyn, Gabor Patonay,' Michael Ford,2and Isiah Warner* Department of Chemistry, Emory Uniuersity, Atlanta, Georgia 30322
An unlntenslfled photodiode array based multichannel fluorescence detector has been developed for use In hlghperformance UquM chromatography. The detector uses a low wattage xenon capillary flashlamp as an excitatlon source s u m hlgbhtensrty radlaUon throughout the UV and VlSiMe reglon. Fluorescence emission Is monltored over a 250-nm range, provldlng on-line spectral lnformatlon of chromatographic effluent. Llnear dynamlc range covers at least 3 orders of rnagdtude, with detection lknns In the low nanogram range for several polycyclic aromatic hydrocarbons.
INTRODUCTION Fluorescence is one of the most sensitive and inherently selective methods of detection available in HPLC. However, conventional single-channel detectors, even in combination with highly efficient analytical columns (1,2),are limited in their ability to completely characterize the column effluent in one chromatographic run. Additional information about Permanent address: D e p a r t m e n t of Chemistry, Georgia State University, Atlanta, GA 30303. *Permanent address: Perkin-Elmer Limited, Beaconsfield, Bucks HPSlQA, Beaconsfield, England. * A u t h o r t o w h o m correspondence should be addressed
the components in the effluent can be gained when fluorescence excitation and/or emission spectra are obtained along with retention data. This has been the incentive for a number of innovative detectors designed to monitor multiple excitation/emission wavelengths. These detectors, when used with the enhanced separation capabilities of modern HPLC, become very powerful tools for analyzing complex mixtures. Several distinct approaches have been taken in the development of multichannel detectors. One configuration is to surround the flow cell with photomultiplier tubes (PMT). Fluorescence emission wavelengths are then selected by using interference filters placed before each P M T (3). This approach retains the sensitivity of a single-channel detector, while providing fluorescence emission intensities a t several different wavelengths to help resolve fused peaks. This design does have some limitations, since it can only accommodate a small number of PMTs. Solute characterization can be improved by fluorescence spectral detectors. An easy method to obtain a complete spectrum is to stop the HPLC flow and scan the effluent in the flow cell ( 4 ) . The technique develops problems when diffusion of components occurs during scanning. This causes a loss of chromatographic resolution and nonreproducible results. The possibility of rapid mechanical scanning (10 nm/s) without altering the flow has been discussed (5). While adequate for a number of applications, some of the spectral
0003-2700/90/0362-1754$02.50/0Q 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO.
resolution is lost in this method. Another approach to mechanical scanning is an instrument for rapid-scanning constant-energy synchronous fluorescence spectroscopy (CESFS) (6). This system is capable of scanning at 200 nm/s. However, a decrease in sensitivity is observed a t these high rates. Loss of resolution associated with mechanical scanning can be overcome by incorporating into the instrument, an optical multichannel imaging device such as a silicon-intensified target (SIT) vidicon, or a linear intensified photodiode arrays (IPDA), (7-10). The video fluorometer, based upon the SIT vidicon, is capable of providing multiple excitation and emission matrixes (EEMs) of the chromatographic stream (11-13). In addition, a number of sophisticated data reduction strategies have been developed for both the qualitative as well as quantitative evaluation of the EEMs (14-16). Intensified imaging detectors provide high resolution and sufficient sensitivity for many analytical applications, but the cost of the instruments may be quite prohibitive. Nonintensified self-scanned linear photodiode arrays (PDA) have been successfully employed in a number of multichannel UV-visible absorbance detectors (17, 18). However, to date very few attempts have been made to use PDAs in lowlight-level applications such as HPLC fluorescence detection. This is due to two important limitations: lower sensitivity and higher noise as compared to photomultiplier tubes. These operational characteristics can often require long integration times and spectral averaging, leading to large detection time constants. There are two components to the electronic noise associated with PDA operation. These are fixed pattern noise and integrated dark current. Fixed pattern noise is produced by spatial variations in the switching transients coupled into the video line through the clocks and the internal multiplex switches. The effects of the fixed pattern noise can be removed from the data through digital background subtraction routines. Dark current is the thermal discharge of reverse bias on each diode. Fluctuations in dark current are directly related to temperature fluctuations and integration time (19). Electrothermal peltier cooling units are used for careful regulation of PDA temperature. However, over the duration of a chromatogram, some drift may occur in the electronic background. This can affect the ability of the PDA fluorescence detector t o quantitate solutes from run to run. Therefore, to minimize the contribution of electronic noise, it is best to obtain and subtract the background throughout the chromatogram. The research reported here focuses on the development of a compact PDA fluorescence spectrophotometer for use as an HPLC detector. The optical breadboard is designed with very short light paths between components improving the instrument's throughput and thus sensitivity. A unique feature of this detector is the use of a xenon capillary flashlamp as an excitation source. Unlike xenon arc lamps operating in direct current (dc) mode, flashlamps produce low heat radiation, while still providing a high-intensity continuous spectrum. Xenon flashlamps also have a spatially stable arc, long operational life, and a geometry compatible with the illumination of sample volumes encountered in HPLC flow cells (20). The use of a flashlamp enables the system to acquire electronic background spectra a t any time during the chromatogram.
EXPERIMENTAL SECTION Instrumentation. A schematic diagram of the detector is given in Figure 1. The optical breadboard of the fluorescence spectrophotometer used in this study was a prototype model supplied by Perkin-Elmer Limited Beaconsfield, England. An 8.3-W xenon capillary flashlamp (Perkin-Elmer part no. L5212-6271),is used as an excitation source. The lamp is under the control of a power supply, designed and constructed in house. The supply has control over lamp operational characteristics such as electrode potential, flash frequency, flash duty cycle, and flash
17, SEPTEMBER 1, 1990 1755
Figure 1. Schematic diagram of the photodiode array HPLC detector: cell; B, circular mirror; C, plane grating; D, 512-element linear photodiode array. A, flow
pattern. Details of its construction is given elsewhere (21). An adjustable ellipsoidal reflector is mounted behind the flashlamp to focus the radiation onto a 0.5-mm slit. Excitation wavelength is selected by placing an interference filter in front of the slit. Two interference fiten, 360 nm, bandpass 10.3 nm, transmittance 2570, and 400 nm, bandpass 11.4 nm, transmittance 40% (Andover Corp., Lawrence, MA) were used in the detector. An ellipsoid mirror collects the light and focuses it onto a square quartz flow cell (1X 1mm2 i.d., -9 pL totalvolume). Since the flash occurs within the lamp capillary, the geometry is particularly suitable for imaging upon the narrow (1 mm) inner bore of the flow cell. Fluorescence emission is dispersed by a 300 line/mm planar grating and focused onto a 512 element EG&G S series photodiode array by a 100-mm-radius circular mirror. Both the Xe flashlamp excitation source and PDA require start signals to initiate their operation. However, due to the relatively short duration of each flash (20 ps), the two start signals must be precisely synchronized to obtain reproducible quantitative results. If the PDA and flashlamp are allowed to operate independently, there is a chance that the number of flashes per integration period would not be constant. Any changes in the amount of excitation light incident on the chromatographic effluent per integration period will produce some variability in the intensity of fluorescence emission. Our detector has been deaigned so that the flashlamp begins pulsing exactly with the start of the PDA integration period. To synchronize both start signals, a 2-MHz crystal establishes a base clock frequency. The clock signal is then altered to the desired flash frequency,and PDA integration period by separate banks of programmable counters. A schematic for the timing circuit that produces the start signals is published elsewhere (21). Use of just one base clock helps simplify the synchronization of the flashlamp and PDA operation, thereby producing consistent results. The analog signal from the PDA is digitized by using an Analog Devices Model ADC 1140 16-bit A/D converter. Conversion time per diode is approximately 80 ps, which means the entire array can be scanned every 40 ms. Data are sent to a Zenith 241 computer through a Metra Byte Model PI012 %bit parallel digital 1/0 interface. To facilitate electronic background subtraction and spectral averaging routines, an Everex EV-125 multifunction card has been added to the computer, providing 2.0 Mbytes of ramdisk storage. Data are stored in the ramdisk under direction of machine language routines developed in house. The speed of the software and ramdisk allows the user to interchange between real-time display of fluorescence spectra, fluorescence intensity at a single wavelength, or total fluorescence chromatograms covering 250 nm of fluorescence emission. All spectra are sequentially stored, enabling the computer to produce three-dimensional chromatograms or contour plots. The temperature of the diode array is regulated by a peltier thermoelectric cooling system. However, the PDA is not enclosed in a vacuum or in a chamber where nitrogen purging can be performed efficiently. Therefore, the cooling system was set to 20 "C. Lower temperatures caused condensation to form on the PDA's quartz window and pins. Excessive condensation can short
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
Table I. Limits of Detection (S/N = 3) and Linear Dynamic Range for PDA HPLC DetectoP
PAH
anthracene
integration period, ms 200
exc wavelength, nm
emission max, nm
limit of detection, ng/20 gL
360
400
11.8
400
43 1
4.9 8.9
400
431
10.5
400
445
3.6 6.9
600
benzo(k)fluoranthene
200 600
9,lO-diphenylanthracene
200 200 600
corr
>219 >414 >221 >442 >201 >332 >204 >390
0.9994 0.9932 0.9994 0.9998 0.9987 0.9990 0.9993 0.9992
3.2
600
perylene
linear dynamic range
2.1
“Measurementconditions: eluent 80/20 (v/v) acetonitrile/H20; 15 cm out the PDA possibly damaging the device. Reagents. The limits of detection (LOD) and the linear dynamic range of the detector were measured by using polycyclic aromatic hydrocarbons (PAHs), anthracene, perylene, and 9,lOdiphenylanthracene, which were purchased from Aldrich Chemical Company (Milwaukee,WI). Benzo(k)fluoranthenewas obtained from Chem Services, Inc. (West Chester, PA). A multicomponent sample also contained fluorene, fluoranthene, and rubrene from Aldrich. All PAHs were used without further purification. The chromatography mobile phase was 80% by volume HPLC grade acetonitrile (Burdick and Jackson), in type I water (Millique 4 cartridge system with 0.2-pm final filter). HPLC System. The HPLC system consisted of a PerkinElmer Model Series 10 isocratic pump and a Rheodyne Model 7510 injector with a 20-pL sample loop. PAH solutions were injected onto a 15 cm X 4.6 mm 10-pm ODS column (Regis Chemical Co.), and eluted isocraticaly with the 80/20% (v/v) acetonitrile-water mobile phase. Flow rates were maintained at 2 mL/min for all experiments. Flashlamp. The xenon flashlamp operational parameters were a flash frequency of 100 Hz and a duty cycle of 20 p s for all the experiments.
RESULTS AND DISCUSSION Spectral Resolution. A PDA-based HPLC detector’s spectral resolution is an important consideration when evaluating its ability to characterize chromatographic effluent. Since the light paths in our detector are short and slits relatively large (0.2 mm), it was feared that poor spectral resolution would be observed. T o measure the polychromator’s resolution, a 0.2-mm slit was placed in the position of the flow cell. The light from a low-pressure Hg pen lamp was then focused onto the slit. The spectrum of the Hg pen is displayed in Figure 2. Peak widths (full widths at half-maximum) for the Hg lines ranged between 8 and 12 nm for the lines measured between 360 and 600 nm. Over the monitored region, the instrument’s wavelength accuracy is within 170based on the Hg lines. The quality of the spectrum is due to highly accurate placement of the detector’s optical components. The position of the grating and PDA are adjusted with 80 turn/in. pm. Once the position of optical components is optimized, any slight alteration in their position will severely degrade spectral quality. Spectral resolution can be improved (average 7 nm) by using smaller slits; however, this would reduce the amount of light incident on the PDA, thereby decreasing the detector’s sensitivity. Detection Limit and Dynamic Range. The PAHs used in this study were selected because they have often been used in the evaluation of other HPLC fluorescence detectors. This allows for more accurate comparisons between systems. Standard solutions of four different PAHs ranging in concentration from 0.01 to 100 pg/mL were prepared to test the detectors linear dynamic range and the limits of detection (LOD). The LOD for each PAH was calculated as the injected quantity that produced a signal to noise ratio of 3 (S/N = 3). The procedure used to calculate the LOD was described by
X 4.6
mm, 10 fim ODA column; 2 mL/min flow rate.
600
564
529
493 457 Wavelength (nm)
421
386
350
Figure 2. Spectrum of Hg pen light dispersed by detector’s polychromator.
“T
I
I
00
340
680
1020
1360
1700
concentration (ng/20u1)
Flgure 3. Calibration curves: (1) anthracene; (2) benzo(k)fiuoranthene; (3) 9, lodiphenylanthracene; (4) perylene.
Miller and Miller (22). Table I lists the LOD for each PAH at the integration periods of 400, and 600 ms. The results are based on seven (n = 7) injections of each PAH solution at each integration period. The detector interference filter that determines the excitation wavelength was selected by first recording an excitation spectrum of dilute PAH solution in mobile phase with a Perkin-Elmer 650-10s fluorescence spectrophotometer. Since the PDA detector monitors 250 nm of fluorescence emission simultaneously the complete PAH fluorescence spectra were stored in the AT’S RAMDISK. From this point, the wavelength of maximum emission intensity was selected, and the corresponding row was extracted from the chromatography data matrix. Calibration curves for each PAH taken at a PDA integration period of 6 0 0 ms are shown in Figure 3. Only the electronic
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17. SEPTEMBER 1, 1990
1757
Peak 7 (Rubrene) 0.5 T
"." 380
384
348 3.32 Wavsismth Inm)
G
3 300
316
300
5
Flgure 4. Chromatogram of a seven-component mixture of polycyclic aromatic hydrocarbons,
Table 11. Elution Order and Concentration of Components in the PAH Mixture Sample
fluorene
43
anthracene 33 fluoranthene 26 benzo(b)fluoranthene 51
perylene
18
benzo(k)fluoranthene 31 rubrene 31
background of the PDA has been removed in real time. No additional post chromatographic background corrections have been performed on the data. Even a t the higher injected concentration no deviation due to inner filter effects were observed. Stability. Stability of the excitation 80urce is a very critical factor in the reproducibility and stability of data from fluorescence detectors. High-wattage Xe arc lamps used in many detectors often use complex feedback regulation in their power supplies and forced air cooling, to minimize power fluctuations due to arc wander. Fluctuations that do occur must be monitored and compensated for by some t y p of ratio circuit. The capillary flashlamp under the control of our power supply proved to be a very stable excitation source. For a 200-m~integration period, lamp intensity at 4M) nm changed less than 1% over 8 h. The flashlamp stability can be attributed to several factors. Over the length of an integration period the lamp will pulse multiple times. Any slight variation in pulse-to-pulse intensity is averaged out over the length of the PDA intregation period. The net effect is a very stable or reproducible amount of excitation radiation incident upon the chromatographic effluent over each integration period. The detectors stability was monitored by injecting standmd samples of perylene and anthracene over 8 b (N = 16). The integration period of the PDA was kept constant at 600 ms. For 16.7 ng of anthracene peak heights varied 1.6%, and for 10 ng of perylene peak heights fluctuated 1.8%. Use of the xenon flashlamp contributes to the consistency of the data, since the flashlamp is operated in cycles that permit collection of an electronic background produced by the PDA. This capability allows the detector to collect backgrounds
throughout the chromatogram, thereby eliminating electronic noise from the fluorescence emission data. Multicomponent Sample. T o illustrate the detector's ability to obtain real-time fluorescence spectra of eluting solutes, a seven-component sample of PAHs in the mobile phase was prepared and injected into the HPLC system. The concentration of each PAH injected is listed in Table 11. The PDA integration period was set t o 600 ms,and the xenon flashlamp was operated 80 that a PDA electronic background was obtained every fourth integration period. The background was subtracted from the previous emission spectra before storing into the computer's ramdisk. The data matrix for the entire chromatogram consisted of 1125 individual spectra. Figure 4 presents a three-dimensional representation of the chromatogram. However, due to software limitations many points in the data matrix are averaged together to produce the 3-D plot. Additional solute spectral information can be gained by plotting the complete spectrum at the retention time of interest. This has been done for peaks 1and 7 representing fluorene and rubrene, respectively. CONCLUSIONS An unintensified photodiode array has been combined with a capillary xenon flashlamp to produce a relatively inexpensive rapid-scanning HPLC fluorescence detector system. While not as sensitive as a single-channel P M T based detector, our instrument exhibits good sensitivity while rapidly acquiring emission spectra without loss of chromatographic resolution. Additional research into improving the light throughput of the emission optics and use of higher wattage Xe flashlamps can further improve the detector's sensitivity. LITERATURE CITED (1) Novotny. M. Anal. Chem. i981. 53,1294A-1308A. (2) Conbn. R. D. Anal. Chem. 1969. 4 1 , 107A. (3) Tanabe. K.: Glick. M.: Smim. 0.; Voigtman, E.: Winetordner. J. D. Anal. Chem. i987. 59. 1125-1129. (4) Harano. H.: Yamamlo. Y.: Sailo. M.; Mochida. E.: Watanabe. S. J. Chromtogr. 1983. 83. 373. (5) Pellirrari. E. D.;Sparacino. C. M. Anal. Chem. 1973, 45, 378. (6) Kerkofl. M. J.; Wintordner, J. D. Anal. Chlm. Acta 1985. 175, "C7
O f 7
L*I-LY..
(7) Vc-Dinh. T.; Johnson. D. J.: Winefordner, J. D. SpecbOdIh. Acta. Part A 1977. 33A. 341. (8) Ryan. M. A,: Miller. R. J.: Ingle. J. D. Anal. Chem. 1978. 50, 1772.
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(9) Gluckmann, J. C.; Shelly, D. C.; Novotny, M. V. Anal. Chem. 1965, 57, 1546-1552. (10) Coomy, R. P.; Vc-Dinh, T.; Winefordner, J. D.Anal. Cbim. Acta 1977, (11) Johnson, D. W.; Gladden, J. A.: Callis. J. B.; Christian. G. D. Rev. Sci. Instrum. 1979, 5 0 , 118. (12) Hershberger, L. W.; Callis, J. B.; Christian, G. D. Anal. Chem. 1961,
(19) Talmi, Y. Appl. Spectrosc. 1962, 36, 1-18. (20) Slavin, W.; Rhys Williams, A. T.; Adam, R. F. J . Chromatogr. 1977, 134, 121-130. (21) Wegrzyn, J.; Patonay, G.; Ford, M.; Warner, I. M. Rev. Sci. Instrum., in press. (22) Miller, J. C.; Miller, J. N. Statistics for Ane/yt/cal Chemistty, 2nd ed.; Ellis Horwood: Chichester, 1988; pp 115-1 17.
(13) Warner, I . M.; Forgarty, M. P.; Shelly, D.J. Anal. Chim. Acta 1979, 109, 361. (14) Johnson, D. W.; Callis, J. B.; Christian, G. D.ACS Symp. Ser. 1979, No. 102, 97-114. (15) Warner, I. M.; DavMson, G. D.;Christian, G. D. Anal. Chem. 1977, 49, 564. (16) Forgarty, M. P.; Warner, 1. M. Anal. Chem. 1981, 53, 259. (17) DesSy. R. E.; Nunn, W. G.; Titus, C. A. J . Cbromatcgr. Scl. 1976, 14, 195-20 1. (18) Milano, M.; Lam, S.;Grushka, E. J . Chromatogr. 1976, 725315-326.
RECEIVED for review January 22,1990. Accepted May 14,1990. The research described in this article was supported by grants from the Perkin-Elmer Corp., Norwalk, CT, the National Science Foundation (CHE-8609372), and the Office of Naval Research. I.M.W. also acknowledges support from a Presidential Young Investigator Award (CHE-8351675).
RO -. P --,
53. .., 971-975 .. . ..
Optical Detection of Cationic Surfactants Based on Ion Pairing with an Environment-Sensitive Fluorophor Ziad M. Shakhsher and W. Rudolf Seitz*
Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824
The environment-sensitive anlonic fluorophor 5-[(2-amlnoethyl)amlnoJnaphthalene-l-wlfonate(AEANS) has been c& valently lmmobiked on glycophase-controlled pore glass, cellulose, and poly(vlny1 alcohol). Maxlmum emlsslon shlfts to shorter wavelength upon knmoMllzatkm due to hydtophoblc lnteractlon wlth the hnmoMlitatlon substrate. When the solid phase indicator k exposed to aqueous soiutlons of catlonlc surfactants, fluorescence Intendties and undergoes a further hypsochromic shlft. The magnitude of the shlft can be used to sense the concentratlon of catlonic surfactant. The dynamlc range Is approximately 30. The mlnlmum detectable concentration Is between 0.1 and 1 mM, wlth lower values observed for catlonic surfactants wRh longer hydrocarbon talk. Cationlc Wactants can be contkruously and reversibly sensed wlth a two-wavelength-lntendty ratlo measurement that Is Insensitive to instrumental drlft, quenchers, and slow loss of Indicator.
INTRODUCTION The coupling of immobilized fluorigenic indicators with fiber optics offers new opportunities for in situ spectroscopic analysis (1-3). Here we describe a new type of fluorigenic indicator designed to sense cationic surfactants. The indicator is a covalently immobilized, anionic derivative of 5-(dimethylamino)-1-naphthalenesulfonicacid, which forms an ion pair with added cationic surfactant. Interaction with the hydrophobic part of the surfactant reduces the polarity of the indicator environment, causing its fluorescence to intensify and shift to shorter wavelength. This effect has been demonstrated previously by using the anionic fluorescent probe formed by reacting 5-(dimethylamino)-l-naphthalenesulfonyl chloride with glycine (4). Similarly, addition of an organic polyelectrolyte has been observed to cause "dansyl" countenons to undergo spectral shifts (5). Other dansyl derivatives have been used to probe the effect of solvent composition on hydrocarbon bonded silica surfaces (6-8). The sensing chemistry is analogous to ion-pair solvent extraction (9),which may be represented 0003-2700/90/0362-1758$02.50/0
A-(aq)
+ C+(aq)
-
-
A'-C+(aq)
+ organic solvent
(1)
(2) where A- is a lipophilic anion, C+ is a lipophilic cation, and A%+ is an ion pair. The driving forces for solvent extraction include (1) electrostatic interaction between the negative charge on A- and the positive charge on C+, (2) hydrophobic interaction between the lipophilic parts of A- and C+, and (3) lipophilic interaction between the ion pair and the organic solvent. In this paper we demonstrate that cationic surfactants may be reversibly sensed via fluorescence changes accompanying ion pair formation with an anionic environment-sensitive fluorophor. The effects of experimental variables on response to cationic surfactants are consistent with a model based on ion pair solvent extraction. The immobilized indicator is combined with fiber optics to reversibly sense cationic surfactant in solution. A'-C+(aq)
A'-C+(org)
EXPERIMENTAL SECTION Reagents. Glycophase-controlledpore glass (G-CPG)with an average pore diameter of 460 A and particle sizes of 37-74 pm was obtained from Pierce Chemical co. Powdered cellulose (microcrystallinefor TLC through 60-mesh sieve) was purchased from Baker. Poly(viny1 alcohol) (PVOH),100% hydrolyzed with an average molecular weight of 14000, and cyanuric chloride were purchased from Aldrich. Glutaraldehyde, 50% (w/w) in water, was obtained from Fisher Scientific. Surfactants dodecyl-, tetradecyl-, and cetyltrimethylammoniumbromide, sodium dodecyl sulfate, and poly(oxyethylene(23) lauryl ether) were obtained from Aldrich. They are designated DDTAB, TDTAB, CTAB, SDS, and PELE, respectively. The fluorophor 5-[(2-aminoethyl)aminolnaphthalene-1-sulfonate(AEANS) was obtained from Molecular Probes, Inc. Its structure is known in Figure 1. Apparatus. Emission spectra were measured with an SLM 8OOO spectrofluorometer with the excitation wavelength set to 370 nm. Fiber optic measurements were made with excitation and emission arms of a bifurcated fiber optic bundle (3/le-in.diameter at the common end) coupled to source and detector lens housings in the sample compartment of the SLM spectrofluorometer using light-tight aluminum fittings. Procedures. (A) Immobilization: AEANS was immobilized on G-CPG by the reactions shown in Figure 2a ( I O ) . Aldehyde 0 1990 American Chemical Society
