Anal. Chem. 1987, 59, 572-576
572
orifices may be obtained by inserting a pulsed valve in the sheath gas line. A wider range of orifice diameters and backing pressures would allow more flexibility in achieving the proper gas flow for expansion cooling. In the current apparatus, fluorescence detection is limited by the amount of light collected. Fluorescence collection could be increased by a factor of 40 by using an elliptical reflectance mirror in the vacuum chamber (19). Therefore, we conclude that low-picogram detection limits should be achievable with this method for compounds that absorb strongly and possess reasonable fluorescence quantum yields. Absorption (ZO), resonant two photon ionization (21),and phosphorescence (22) detection have also been reported in supersonic jets. Each of these methods can be interfaced to a gas chromatograph through the sheath flow nozzle. Other forms of chromatography may be coupled as well. In a separate publication (23),we describe the application of this technique to supercritical fluids. Finally, we note that the selectivity of supersonic jet spectroscopy dramatically reduces the demand placed upon a chromatographic separation. In this regard, short open tubular columns (24) can be used to provide crude separations for rapid and sensitive analysis of samples.
ACKNOWLEDGMENT The authors acknowledge many helpful discussions with N. S. Nogar and R. A. Keller and the assistance of B. A. Anderson in modeling the rotational contours of aniline.
LITERATURE CITED (1) Levy, D. H. Science 1981, 2 7 4 , 263. (2) Warren, J. A,: Hayes, J. M.; Small, G. J. Anal. Chem. 1982, 5 4 , 138.
Amirav, A.; Even, U.; Jortner, J. Anal. Chem. 1982, 5 4 , 1666. Lubman, D. M.; Kronick, M. N. Anal. Chern. 1982, 5 4 . 660. Tembreull, R.; Lubman, D. M. Anal. Chem. 1984, 5 6 , 1962. Tembreull, R.; Sin, C. H.; Li, P.; Pang, H. M.; Lubman, D. M. Anal. Chern. 1985. 5 7 . 1186. Tembreull, R:; Sin, C. H.;Pang, H. M.;Lubman. D. M Anal. Chem 1985. 5 7 . 2911. Imasaka,'T.; Fukuoka, H.; Hayashi, T.; Ishibashi, N. Anal. Chim. Acta 1984, 756, 111. Imasaka, T.; Hirata, K . ; Ishibashi, N. Anal. Chem. 1985, 5 7 , 59. Hayes, J. M.; Small, G. J. Anal. Chern. 1982, 5 4 , 1204. Keller, R. A.; Nogar, N. S.Appl. Opt. 1984, 2 3 , 2146. Melamed, M. R.; Mullaney, P. F.; Mendelsohn, M. L. Flow Cyfometry and Cell Sorting; Why: New York, 1979. Guyer, D. R. Ph.D. Thesis, University of Colorado, Boukler, 1983; p 48. Levy, D. H.;Wharton, L.; Srnalley, R. E. I n Chemical and Biochemical Applications of Lasers; Academic Press: New York, 1977; Vol. 2, Chapter 1. Amirav, A.; Even, U.; Jortner, J. Chem. Phys. 1880, 5 1 , 31. McClelland; Saenger, K. L.; Valentini. J. J.; Herschbach, D. R. J . Phys. Chem. 1979,8 3 , 947. Amirav, A.: Even, U.; Jortner, J.; Birss, F. W.; Ramsay. D. A. Can. J . Phys. 1983, 6 1 , 278. Oikawa, A.; Abe, H.; Mikami, N.; Ito, M, J . Phys. Chem. 1983, 8 7 , 5083. Spangler, L. H.; Pratt, D. W. J . Chem. Phys. 1988, 8 4 , 4789. Amirav, A.; Even, U.; Jortner, J. Chem. Phys. Lett. 1981, 8 3 , 1. Dietz, T. G.; Duncan, M. A.; Llverman, M. G.; Smalley, R. E. J . Chem. Phys. 1980, 7 3 , 4816. Abe, H.; Kamei, S.; Mikami, N.; Ito, M. Chem. Phys. Lett., 1984, 109. 217. Anderson, B. D.; Stiller, S. W.; Johnston, M. V., submitted for pubiication. Trehy, M. L.; Yost, R. A.; Dorsey, J. G. Anal. Chem. 1988, 58, 14.
RECEIVED for review February 28, 1986. Accepted November 3, 1986. This research was supported by the National Science Foundation under Grant No. CHE8308049. S.W.S. acknowledges support through a Graduate Student Foundation Award from the University of Colorado.
Fluorescence Spectra and Lifetimes of Several Fluorophores Immobilized on Nonionic Resins for Use in Fiber-optic Sensors Wayde A. Wyatt, Greg E. Poirier, Frank V. Bright, and Gary M. Hieftje* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Fluorescence emlssion spectra and lifetimes are reported for 0.01-1.0 mM samples of rhodamine B, rhodamine 6G, eosln Y, and fiuoresceln each adsorbed on XAD-2 and XAD-4 resins. Trends are discussed and comparisons made between bound fluorophore and fluorophore In solution. The systems appear to be useful In fiber-optlc-based sensors.
The development of fiber-optic sensors is one of the fastest growing new areas in analytical chemistry and is driven largely by the ability to use traditional spectroscopic techniques to make real-time, on-line measurements of a selected chemical species. Most of these sensors do not involve direct analyte determination, but instead measure the optical properties of a reagent attached to the fiber's distal end. This reagent is chosen so that one or many of its optical properties are changed upon interaction with the target substance. To date, fiber-optic sensors utilizing immobilized reagents have been developed based on reflectance (1-3),fluorescence complexation (4-7), and dynamic fluorescence quenching (8-10). As work continues in this area, fundamental knowledge of each
optrode component (optical design of probe, immobilization method, membrane choice, etc.) is becoming more important. In sensors based on fluorescence or dynamic fluorescence quenching, both fluorescence lifetimes and excitation/ emission spectra can be affected by immobilization in a way that depends upon the immobilization method (adsorption or covalent bonding), the type of solid support, and the amount of reagent present in or on the support medium. When a dynamic fluorescence quenching scheme is being used as a probe, it is desirable to choose the reagent concentration and solid support in a way that will minimize any decrease in the fluorescence lifetime. If the fluorescence lifetime is shortened greatly by immobilization, the probability of excited-state quenching by an analyte molecule is greatly reduced (11). Consequently, sensitivity is similarly reduced. Of course, the lifetime of a fluorophore can also increase upon immobilization. In such cases, the amount of analyte needed to achieve a particular quenching level will decrease and will result in improved dynamic range, sensitivity, and detection powers. In the present study, two solid supports have been characterized (XAD-2 and XAD-4 nonionic polymeric resins); both have earlier been utilized in fiber-optic sensors reported in
0003-2700/87/0359-0572$01.50/0Q 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 4. FEBRUARY 15. 1987
Double Monochromator
Computer
B Rerh
Figure 1. (A) Schematic diagram of instrument for collecting flwrescence emission spectra utilizing optical fibers: M1, M2. minors; L, lens (7.0 cm focal length); PMT, photomultiplier tube; PA, picoam meter. (E) Schematic diagram of instrument for fluorescence-lifetime measurements utilizing optical fibers: L. lens (7.0 cm focal length); PMT, photomutliplier tube; PD. photodiode.
the lierature (2,3,9). However, spectral or temporal changes in the immobilized reagent’s fluorescence emission were not discussed. Here, fluorescence lifetimes and emis2ion spectra were obtained for 0.01-1.0 mM concentrations of rhodamine B, rhodamine 6G, eosin Y, and fluorescein adsorbed on these resins. In general both parameters (lifetime and spectrum) were strongly dependent on the nature of the solid support and on the flnorophore concentration. At higher fluorophore concentrations, emission spectra became progressively more red-shifted. Lifetimes of some fluorophores were shortened by immobilization and to a degree that depended on concentration; other fluorophores exhibited longer lifetimes when immobilized. Possible reasons for this discrepancy in behavior are offered. EXPERIMENTAL S E C T I O N Immobilization of Fluorophores on Nonpolar Resins. Individual solutions of rhodamine B (Exciton Co.), rhodamine 6G (Exciton Co.), eosin Y (Aldrich Chemical Co.), and the disodium salt of fluorescein (Sigma Chemical Co.) are prepared a t concentrations of 1.0,0.1,0.01, and 0.001 mM in reagent-grade ethanol. XAD-2 and XAD-4 polystyrene resin beads (Rohm and Haas Co.) are immersed for 24 h in each fluorophore solution at the ratio of 0.1 g of resin beads to 1.0 mL of fluorophore solution. The supernatant fluorphore solutionsare then removed by vacuum filtration and the heads washed with distilled-deionized water and stored in disposable cuvettes (Fisher Scientific Co.) under distilled-deionized water. Determination of Emission Spectra Using Remote Fiber Spectrometry. A schematic diagram of the instrument used for the measurement of emission spectra is shown in Figure 1A. The 488.0-nm line of an argon-ion laser (Spectra Physics, Inc., Model 171) operating a t a plasma current of 30 A is focused onto one end of a 2-m optical fiber (Valtec 240-pm core diameter). Radiant power measured a t the fiber’s distal end is 47 mW. The suhsequent fluorescence is collected by a bundle of five 2-m fibers arranged concentrically about the input fiber and delivered to a double monochromator (Spex, Inc., Model 1680 Spectramate). The spectral hand-pass used for obtaining all spectra is 9 nm. The
573
fluorescence is detected by a photomultiplier (RCA R928) operated at a biasing voltage of -900 V dc. The output from the photomultiplier tube (PMT) is connected to a picoammeter (Keithley Instruments Model 414s) employed as a current-readout device. A computer (Digital Equipment Corp. MINC 11-23) drives the monochromator from 500 to 700 nm in 1-nm increments and collects the output from the picoammeter. The entire system is controlled by an interactive BASIC routine. Determination of Lifetimes Using a Sampling Oscilloscope. A schematic diagram of the instrument used for the determination of fluorescence lifetimes is shown in Figure 1B. The 488.0-nm line of a mode-locked argon-ion laser (Spectra Physics, Inc., Model 171laser, Model 342 mode locker, and Model 452 made-locker drive) is mechanically chopped and focused into one end of the optical fiber described in the previous section. The mode-locker frequency is between 40.9900 and 40.9940 MHz, the laser plasma current is 35 A, and the radiant power measured at the distal end of the fiber is 9 mW. The optical fiber delivers excitation radiation to the sample holder and a bundle of five fibers collects the resulting fluorescence and sends it to a fast photomultiplier tube (RCA 31024) operated at a biasing voltage of -3500 V dc. A longpass filter which rejects light at wavelengths below 520 nm is located in B sliding mount and inteqmsed between the return fiber bundle and the PMT. The filter is placed into or out of the path of the radiation depending on whether a fluorescence decay curve or instrument response curve is heing collected, respectively. The output from the PMT is directed via a 50-R coaxial cable to a sampling oscilloscope (Tektronix, Inc.: Model 7844 mainframe, Model S4 sampling head, and Model 7Sll sampling unit) which is triggered by the synchronous output of the mode-locker driver. The output from the oscilloscope is connected to a lock-in amplifier (EG&G Princeton Applied Research, Model 5101, time constant set tQ 0.1 s),which is referenced to the mechanical chopper frequency. The lock-in amplifier reduces additive noise introduced after the chopper (12). The output from the lock-in amplifier is sampled hy a computer (DEC, MINC 11-23)which collects 128 points across B 12-nstime window. A total of 128 points were chosen for convenience in the event that a fast-Fourier digital filter would need to he employed. Operation of the Time-Resolved Fluorescence Instrument. Depending on the required signal-to-noiseratio, between 200 and 500 data points are collected and averaged at each time increment of a fluorescence decay curve. An instrument response curve is measured before and after each fluorescence decay curve by collecting and averaging 200 data points of scattered radiation with the long-pass filter displaced from the light path. The lifetimes are extracted from fluorescence decay curves using a convolute-and-compare algorithm (13-15) on a VAX 11-780 computer. RESULTS AND DISCUSSION S p e c t r a l Effects. It was found that the fluorescence spectra of the immobilized fluorophores became progressively red-shifted with increasing fluorophore concentration. Typical fluorescence spectra for two of the immobilized fluorophores are shown in Figure 2. Figure 3 shows the shift in peak emission wavelength for each immobilized fluorophore as a function of its initial concentration in immobilizing solution. The shift toward longer wavelength upon immobilization indicates an average decrease in energy of the radiative decay from the excited electronic state (SI)to the ground electronic state (So). Electronic spectral shifts related to chemical environment have been reported in the literature (16-18). In our case the spectral shifts can he broken down into three categories: solvent effects, adsorption effects, and spectral reabsorption. Dissolving a fluorophore in increasingly polar solvents increases the degree of fluorescence red shift (19). Waggoner and Stryer (18)found that fluorophores entrapped in a nonpolar lipid bilayer exhibited a blue shift in fluorescence peak relative to those in a methanol solution. They found also that the degree of blue shift was directly proportional to the depth the fluorophore was buried in the bilayer. Thus, a fluorophore close to the more polar surface of the bilayer was red-shifted
574
ANALYTlCAL CHEMISTRY, VOL. 59, NO. 4, FEBRUARY 15, 1987
Table I. Maximum Intensity and fwhm of Immobilized Fluorophore Fluorescence Spectra intensity and fwhm at the following concns' 1.0 mM 0.1 mM 0.01 mM 0.001 mM Maximum Intensity (Relative)
____
L p -p -
500
540
580
rhodamine B XADB XAD4 rhodamine 6G XAD2 XAD4 fluorescein XADZ XAD4 eosin Y XAD2 XAD4
~
660
620
700
I
B
16.24 8.46
7.14 10.65
1.90 1.84
0.54 0.39
20.29 5.30
8.28 2.43
2.15 2.55
0.74 0.52
8.99 8.05
5.92 5.16
2.89 1.15
0.69 0.33
1.39 0.70
1.79 1.32
0.72 1.04
1.35 0.39
Spectral fwhmb (nm)
500
540
580 620 Wavelength (nm)
660
i o 0
Figure 2. Typical fluorescence emission spectra of bound Huorophore. The concentrations listed on the figure refer to those of the solution which was used to immobilize the fluorphore on the XAD-2 resin. The H,O designation represents 0.01 mM fluorophore in aqueous solution: (A) XAD2 immobilized rbodamine B; (B) XAD2 immobilized fluorescein. 600,
I
I
I
I
I -6
I
I
I
-5
J
-4
-3
-2
I
I
I
I
1
1
h
E
Y
fui
3 01
I
01
600
Eoaln Y
1
1
rhodamine B XADZ XAD4 rhodamine 6G XADB XAD4 fluorescein XAD2 XAD4 eosin Y XAD2 XAD4
-6
-5
-4
-3
-2
LOGCConc., M ) Flgure 3. Fluorescence emission peak maxima for the fluorophores bound to (A) XAD-2 and (B) XAD-4 resins. (See Table I1 for peak maxima of the fluorophores in solution.)
from one nearer to the nonpolar center. In our case, if the fluorophores immobilized at low concentration preferentially occupy nonpolar sites deeper in the polystyrene resin's porous structure while those at higher concentrations must occupy sites closer to the polar aqueous environment, then the
40 38
36 36
40 42
52 57
47 41
39 39
41 45
41 40
35 33
23 24
22 27
17 79
66 73
35 33
35 37
Concentration of initial solution used in immobilization. width a t half maximum of emission spectrum.
Full
fluorophores immobilized at higher concentrations would be red-shifted from those a t lower concentrations. Peak broadening should also occur at the higher concentration as the total fluorescence emission is integrated over the range of environments. This behavior was observed (Table I). Spectral shifts can arise also from changes in the equilibrium distance (uRF) between the resin and the fluorophore in the excited state (17), from reabsorption of the short-wave portion of a fluorescence band, and from the aggregation of dye molecules. Franck-Condon considerations require that changes in uRF result in shifts in the absorption and emission lines. Similarly, short-wave absorption cannot be ruled out as a cause of the observed spectral shift, since no convenient method is available for measuring the absorption spectrum of the immobilized dye. The existence of simple first-order excited-state decay (see below) argues against this cause and against the aggregation of dye molecules, however. Temporal Effects. The fluorescence decay curve for rhodamine 6G (immobilized on XAD-4 resin from 1.0 mM solution), the instrument-response function, and the same instrument response function convoluted with the calculated 4.98-ns lifetime (calculated decay curve) are shown in Figure 4. The ringing observed on the instrument response function was due to an impedance mismatch between the photomultiplier tube and the oscilloscope sampling head. The excellent fit between experimental and convoluted decay curves was found for the other immobilized fluorophores as well. The quality of this single-exponential fit is somewhat surprising in view of the apparent range of microenvironments in which the fluorophores are immobilized. However, a range of hydrophobicity does not always produce a commensurate shift in fluorescence lifetime (20);presumably, if such a shift occurs, it lies below the time resolution of our measurements. The fluorescence lifetimes obtained for each fluorophore in ethanol and water are shown in Table I1 and are in good agreement
7
500-7
49 54
ANALYTICAL CHEMISTRY, VOL. 59, NO. 4, FEBRUARY 15, 1987
575
INSTRUMENTAL RESPONSE
CALCULATED DECAY CURVE
i i
\
EXPERIMENTAL DECAY CURVE
? A
- 02
'3,
5 5 ,
!
i
\
\
I i '
I
3
2 5-15
I
12ns
Flgure 4. Comparison of typical experimental (dotted line) with cal-
culated (solid line) decay curves (4.98-ns lifetime) for rhodamine 6 0 immobllied on X A D 4 resin. Ringing is due to an impedance mismatch between the photomultiplier tube and oscilloscope sampling head. Table 11. Lifetimes (ns)of Fluorophores in Solution lit. value
e x p t l value fluorophore rhodamine rhodamine fluorescein eosin Y
deionized water
B
1.59 f 0.05"
6G
4.23 f 0.05 3.64 f 0.02
1.18 f 0.05
ethanol 3.00 f 0.03 4.44 f 0.04 4.03 0.03 3.01 f 0.07
*
deionized water
ethanol
2.0b 4.1d 4.0'
3.2c 3.7'
5.0b
l.gb
2.788
'Standard deviation o f three replicant samples. * F r o m r e f 24. c F r o m r e f 21. d F r o m r e f 26. e F r o m r e f 22. ' F r o m r e f 23. g F r o m r e f 25.
with literature values (21-26). Plots of fluorescence lifetime as a function of initial concentration of immobilizing solution are shown in Figure 5. I t is clear that, for the same initial solution concentration, fluorphores immobilized on XAD-4 resin have longer lifetimes than those immobilized on XAD-2. Interestingly, lifetimes of rhodamine B and rhodamine 6G became longer with increasing fluorophore concentration whereas lifetimes of fluorescein and eosin Y became shorter. Time-resolved emission spectroscopy studies by Badea et al. (27) of 2-anilinonaphthalene (2-AN) in a dimyristoyllecithin single-bilayer liposome showed that the deconvolved decay curves a t the red end of the emission spectrum had a longer initial rise. This behavior indicates that some of these lower-energy emissions were from species generated during the lifetime of the excited state. That is, 2-AN self-absorption was occurring. Self-absorption will increase the radiative lifetime because emitted radiation can be reabsorbed and reemitted many times before finally reaching the detector (28). Self-absorption is a concentration-dependent process. In our case, because the adsorbed fluorophores undergo self-absorption, the radiative lifetime increases with concentration. Because XAD-4 has a smaller pore diameter and larger surface area than XAD-2, it should be more prone to self-absorption of radiation from the immobilized fluorophore and should exhibit longer lifetimes as was observed. The decrease in lifetime with increasing concentration of fluorophore, which was observed for eosin Y and fluorescein, could be attributable to concentration quenching of luminescence (CQL) (29,30). This CQL occurs at high concentration when the dye molecules form nonluminescent aggregates which absorb energy from the luminescent monomers. In CQL the luminescence intensity will decrease with the excited-state lifetime. This behavior was observed for eosin Y but not for fluorescein.
CONCLUSIONS Each fluorophore was observed qualitatively to bleed from the resins. Since adsorption is an exothermic process, heating
-5
~i
i O o ( c o n r , MI
4; LOG(C6"C
u1
Figure 5. Fluorescence lifetimes of XAD-2-and XAD-4-immobilized fluorophores vs. concentration of initial solution used for immobilization.
of the sample by laser irradiation would further cause the fluorophores to be desorbed. There was a direct correlation between the hydrophilicity of the fluorophore and the degree to which it bled from the resin. The fundamental differences between the two resins studied are their surface areas and pore diameters. The surface areas of XAD-2 and XAD-4 are 300 and 725 m2/g, respectively, with corresponding average pore diameters of 900 and 400 nm. Among the fluorophores studied, a greater fluorescence intensity is observed from those immobilized on XAD-2 than on XAD-4 resin (see Table I). This finding would indicate that pore size is more important than surface area in determining the magnitude of fluorescence emission from resinbound fluorophores. From this work it is clear that the optical properties of the fluorophores that were examined are significantly altered upon adsorption to the resins studied. It appears that these systems might be useful for remote-fiber-optic chemical sensors which utilize an immobilized reagent. However, the pore size of the solid support and the concentration of the reagent should be coordinated in order to optimize sensitivity.
LITERATURE CITED (1) Peterson, J. I.; Goidstein, S. R.; Fitzgeraid, R. V.; Buckhold, D. K. Anal. Chem. 1980, 52, 864-869. (2) Kirkbright, G. F.; Narayanaswamy, R.; Weiti, N. A. Analyst (London) 1984, 705,15-17. (3) Edmonds, T. E.; Ross, I.D. Anal. R o c . (London) 1985, 22, 206-207. (4) Freeman, T. M.; Seitz, W. R . Anal. Chem. 1978, 5 0 , 1242-1246. (5) Saari, L. A,; Seitz, W. R. Anal. Chem. 1983, 55,667-670. (6) Saari, L. A.; Seitz, W. R. Analyst (London) 1984, 709, 655-657. (7) Zhujun, Z.;Seitz. W. R. Anal. Chlm. Acta 1985, 177,251-256. (8) Saari, L. A.; Seitz, W. R . Anal. Chem. 1982, 54,823-824. (9) Peterson, J. I.; Fitzgeraid, R. V.; Buckhold, D. K. Anal. Chem. 1984,
56,62-67. (IO) Urbano, E; Offenbacher, H; Woifbeis, 0.S. Anal. Chem. 1984, 56, 427-429. (11) Hieftje, G.M.; Haugen, G. R. Anal. Chim. Acta 1981, 723,255-256. (12) Vickers, G. H.; Miller, R. M.; Hieftje, G. M., submitted for publication in Anal. Chim. Acta.
(13) Ware, W. R . Transient Luminescence Measurements ; Marcel Dekker: New York, 1971;Voi. I A , Chapter 5. (14) Rarnsey, J. M. Ph.D. Dissertation, Indiana University 1979. (15) Demas, J. N. Excited State Lifetime Measurements; Academic: New York, 1983;pp 128-129. (16) Longuet-Higgins, H. C.; Pople, J. A. J . Chem. Phys. 1957, 27, 192. (17) McCarty, M.; Robinson, G. W. Mol. Phys. 1959, 2 , 415. (18) Waggoner, A. S.;Stryer, L. Proc. Natl. Acad. Sci. U.S.A. 1970, 67,
579-589. (19) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum: New York, 1983;pp 189-225. (20) Bright, F. V.; McGown, L. B. Talanta 1985, 32, 15. (21) Beriman, I. B. Handbook of fluorescence Spectra of Aromatic Molecules; Academic: New York, 1965. (22) Kubin, R. F.; Fletcher, A. N. J. Lumh. 1982, 27,455. (23) Bright, F. V. Ph.D. Dissertation, Oklahoma State University, 1985. (24) Gavioia, E. Z . Phys. 1927, 42,853. (25) Gati, L.;Szalma, I . Acta Phys. Chim. 1988, 1 4 , 3. (26) Harris, J. M.; Lytle, F. E. Rev. Sci. Instrum. 1977, 4 8 , 1469. (27) Badea, M. G.; DeToma, R. P.; Brand, L. Siophys. J. 1978, 24,
197-212. (28) Chandler, D. W.; Ewing, G. E. Chem. Phys. 1981, 5 4 , 241-248.
576
Anal. Chem. 1907, 59,576-581
(29) Levshin, L. V.; Yuzhakov. V. I. Opt. Spektrosk, 1974, 3 4 , 503-508. (30) Zemshii, V. I.; Meshkovskii, I. K.; Sokolov, I . A. Opt. Spectrosc. ( ~ n g i r. m s i . ) 1985, 59(2), 197-199.
RECEIVED for review August 18,1986. Accepted October 27,
1986. Supported in part by the Office of Naval Research, The Upjohn Co. and the National Science Foundation through Grant CHE 83-20053. We also thank the National Science Foundation for its support of the VAX 11-780 used in this work (Grants CHE 83-09446 and CHE 84-05851).
Identification of Mixture Components in Organic Waste Materials by Carbon- 13 Nuclear Magnetic Resonance David A. Laude, Jr., a n d Charles L. Wilkins*
Department of Chemistry, University of California, Riverside, Riverside, California 92521
Carbon-13 nuclear magnetlc resonance ("C NMR) is used to determine mixture component ldentltles and relatlve concentratlons In organic waste materials wlthout prior separatlon. A quantltatlve 13C NMR spectrum provides the data used In an algorithm to extract subspectrum resonances speclflc to each mlxture component. The subspectra are then subjected to reverse searches of elther a 13C NMR slmulated spectrum llbrary or a 13C NMR chemical shlft library. The quantitative 13C NMR mlxture analysis algorithm Is appHed to seven waste solvent mlxtures procured from analytlcal and organlc laboratories. The 34 subspectra comprised of NMR resonances above the 0.5 % level In the mlxtures yleid 31 unambiguous identlflcatlons for the simulated library search and 27 unamblguous Identifications from a search of the chemical shlft library.
Characterization of bulk components in organic mixtures is essential to the management of hazardous waste materials. Ideally, any method developed for mixture analysis should permit the rapid identification and quantitative measurement of all components in the mixture, with minimal sample preparation. Although gas chromatography/mass spectrometry (GC/MS) is presently the separation/characterization method of choice for unknown mixtures, the uncertainty associated with chromatographic separation of all sample components, the nonuniform detector response of the mass spectrometer, and the need for sample volatility all impose constraints on the general application of the technique. In two recent publications ( I , 2 ) , we advocated the use of interpretation schemes for mixture analysis which incorporated 13Cnuclear magnetic resonance (NMR) data. NMR is unique among the major molecular spectrometry techniques in that quantitative conditions can be achieved so that signal response is equivalent for all nuclei; coupled with a large spectral bandwidth relative to line width for 13C NMR, it becomes possible to extract the resonances specific to each compound in a mixture without prior separation. The peak intensity data from this quantitative data may be used to determine directly the relative concentrations of each compound. In addition, the well-defined relationship between a 13C NMR spectrum and chemical structure permits the implementation of elaborate computer-aided identification methods for unknowns. These properties of 13C NMR previously were exploited in procedures which utilized quantitative and multiplicity 13CNMR data to characterize petroleum distillate fractions ( I ) and to improve the reliability of GC!MS search results ( 2 ) .
In the present work quantitative 13C NMR data facilitates the isolation of pure-component subspectra from the mixture spectrum in an algorithm denoted as Q13CNMR; subsequent identification procedures include two independent reverse search algorithms: (1)comparison of subspectrum chemical shift data with a library of 13CNMR chemical shift values (3); and (2) comparison with a library of simulated 13C NMR spectra created from quantitative, multiplicity, and functional group chemical shift ranges. Selection of any identification procedure is ultimately dependent upon the compounds to be analyzed and the availability of appropriate spectral libraries and computer software. Although the preponderance of mixtures would be conveniently analyzed with algorithms employing large chemical shift library data bases, the reliability of the library search applied to unknown mixtures is suspect because of potential solvent and pH effects on chemical shift data. It is therefore of interest to contrast the search results from a chemical shift 13CNMR library with a simulated library created specifically for the mixtures to be analyzed.
EXPERIMENTAL SECTION A Nicolet NMC 300 NMR spectrometer operating at 75.497 MHz for 13Cnuclei was used for all measurements. Two commercial Nicolet probes, a 5-mm fixed-frequency 13Cprobe and a 12-mm wide-band (*H to 31P)probe, were used without modification. Processing of spectral data was performed on a Nicolet 1280 computer with Nicolet-developed software. Quantitative, multiplicity, and chemical shift data extracted from the spectra were used in Fortran programs written for the Q13CNMR analysis and executed on a Digital Equipment Corp. MicroVAX 11. Implementation of the mixture analysis algorithm requires the acquisition of quantitative 13C NMR data for the tabulation of relative peak areas. The quantitative NMR measurement is achieved when the recycle time between scans exceeds five times the longest spin-lattice relaxation time (I",-) in the sample and the nuclear Overhauser effect (NOE) is quenched (4). In order to satisfy these conditions in an efficient manner, chromium triacetylacetonate (Cr(acac)dwas added to each mixture to achieve a 0.05 M concentration. A delay of between 5 and 15 s, depending upon the sample, was imposed between scans to ensure a leveling of T,and NOE for each nucleus. Reliable determination of relative peak areas from quantitative spectra is essential to the success of the algorithm. To ensure that measurement errors are minimized (less than 2%),adequate peak definition is required. These requirements were satisfied by choosing data acquisition parameters such that at least 8 to 10 points defined each peak with signal to noise (S/N) in excess of 20 to 1. Transients (32K and 64K) were acquired for spectra in which resonances encompassed a 200 ppm (k7500 Hz)region. The application of a line-broadening function (0.5-3.0 Hz) also increased the number of data points defining each peak for situations when maximum spectral resolution was not essential.
0003-2700/87/0359-0576$01.50/0G 1987 American Chemical Society
