Anal. Chem. 1983, 55, 1340-1344
1340
anthracene, 963-82-6; 8-hydroxy-7-methylbenz[a]anthracene, 85337-41-3;9-amino-3-hydroxyphenanthrene, 85337-42-4;oxygen, 7782-44-7.
LITERATURE CITED (1) Orlmsrud, E. P.; Stebbins, R. G. J. Chromafogr. 1978, 155, 19-34. (2) Grimsrud, E. P.; Miller, D. A. Anal. Chem. 1978, 50, 1141-1145. (3) Miller, D. A.; Grimsrud, E. P. Anal. Chem. 1979, 57,851-859. (4) mlmsrud, E. P.; Miller, D. A.; Stebbins, R. G.; Kim, S. H. J . ChromafOgr. 1980, 797, 51-58. (5) Miller, D. A.; Skogerboe, K.; Grlmsrud, E. P. Anal. Chem. 1981, 5 3 , 464-467.
(6) Grlmsrud, E. P. I n “Electron Capture Theory and Practlce in Chromatography”; Zlatkls, A., Poole, C. F., Eds.; Elsevler: Amsterdam, Oxford, New York, 1981; Chapter 5, pp 91-117. (7) Campbell, J. A.; Grimsrud, E. P. J . Chromafogr. 1982, 243, 1-8. (8) Grlmsrud, E. P.; Warden, S. W.: Stebblns, R. G. Anal. Chem. 1981, 53, 716-718.
(9) Karr, C.; Chang, T. L. J . Jnst. Fuel 1958,
37,522-527.
(IO) Wilson, B. W.; Pelroy, R. A. Presented at the 25th Annual Conference
of Mass Spectrometry and Allied Topics, Seattle, WA, June 1979, Paper No. MAMOA-5. (11) Wilson, B. W.; Pelroy, R. A.; Cresto, J. T. Mufat. Res. 1980, 79, 193-202. (12) Later, D. W.; Lee, M. L.; Wilson, B. W. Anal. Chem. 1982, 5 4 , 117-123. (13) Later, D. W.; McFall, T.; Booth, G. M.; Lee, M. L., Brigham Young University, Provo. UT, personal communication Nov 1982.
(14) Grlmsrud, E. P.; Kim, S. H.; Gobby, P. L. Anal. Chem. 1979, 57, 223-229. (15) Gobby, P. L.; Grlmsrud, E. P.; Warden, S. W. Anal. Chem. 1980, 52, 473-482.
RECEIVED for review November 22, 1982.
Accepted March 18,1983. This work is supported by the National Science Foundation under Grant No. CHE-8119857.
Matrix Isolation Fluorescence Spectrometric Detection in Gas Chromatography Vincent B. Conrad,‘ William J. Carter, E. L. Wehry,” and Gieb Mamantov” Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996
The use of matrix isolation fluorescence spectrometry for detectlon in the open tubular column gas chromatography of polycyclic aromatic hydrocarbons is described. Chromatographic column eluents are deposlted dlrectly on a movable 12-sided depositlon surface positloned In the head of a closed-cycle cryostat. The chromatographlc carrier gas serves as the matrix In the cryogenlc deposit. An undispersed xenon lamp source and silicon intensified target vldicon detector are used; thus, fluorescence spectra can be obtained of sample constltuents without prior knowledge of thelr identitles. Use of the fluorometric detector to distinguish between members of a 12-component mixture of anthracene derivatives is considered, and the quantltative vaildity of the technique Is examined via determlnation of the pyrene content of three Natlonai Bureau of Standards reference materials.
The development and application of substance-selective detectors ( I ) for gas chromatography(GC) continues to attract considerable attention. Molecular fluorescence spectrometry offers a number of potential advantages for detection in GC, including very high sensitivity (for fluorescent compounds), high selectivity, fast response, freedom from influence of column temperature, flow rate, or column bleed on the detector signal, and the nondestructive nature of the measurement (2). Although the earliest fluorometric GC detector functioned by trapping the column eluent in a liquid solvent followed by measurement of room-temperature solution fluorescence (3), virtually all subsequent designs for fluorescence GC detectors have entailed measurement of gas-phase fluorescence, with heated transfer lines utilized to convey the GC effluent to a heated flow cell for measurement of fluorescence (4-9) or modification of conventional GC detectors (e.g., a flame photometric detector) for molecular fluorescence measurements (10). Such detectors are capable of achieving subnanogram limits of detection for fluorescent sample constituents. Their principal shortcoming arises from Present address: Conoco, Inc., Library, PA. 0003-2700/83/0355-1340$01.50/0
the fact that molecular fluorescence spectra of hot gases at relatively high temperatures tend to be broader and more congested than their counterparts measured in liquid solution media. Hence, gas-phase fluorescence detectors are not necessarily capable of identifying unknown sample constituents or distinguishing between fluorescent species which are not separated by the column. An obvious approach for achieving improved selectivity in fluorometric GC detection is to employ a low-temperature measurement technique, so that relatively high resolution can be achieved in both emission and excitation spectra (11). For example, an interface for a gas chromatograph to a supersonic nozzle system for the measurement of rotationally cooled fluorescence in the gas phase recently has been described (12). Although the instrument as described utilized a fixed-wavelength fluorescence detector, and thus characteristic fluorescence spectra of column eluents were not obtained, the possibilities inherent in this technique for very high-resolution fluorometric GC detection are quite promising. We have adopted a different approach to this problem, by utilizing matrix isolation (MI) fluorescence spectrometry as the detection technique. Because preparation of a sample in MI proceeds by mixing solutes in the gas phase with a diluent (”matrix”) gas (11, 13, 14), MI appears well suited to GC detection if the carrier gas is used as the matrix. Indeed, the use of MI in GC detection by Fourier transform infrared spectrometryhas been described in considerable detail (15-17). We now report the application of matrix isolation fluorometry in GC detection, employing open tubular columns and polycyclic aromatic hydrocarbons (PAHs) as analytes. EXPERIMENTAL SECTION Instrumentation. A diagram of the optical instrumentation is shown in Figure 1. The output of a 300-W “Eimac”xenon lamp was passed through a 9-cm water-jacketed solution filter (comprising an aqueous solution 1.90 M in NiS04.6H20and 0.27 M in CoSO4.7H2O),the purpose of which was to remove the near-IR and much of the visible output of the lamp. Use of this filter limited the wavelength range for excitation of samples to 220-345 nm, though the lamp provided useful output at wavelengths below 220 nm. No other excitation-wavelength discrimination was employed. 0 1983 Amerlcan Chemlcal Soclety
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
1341
IR-Visible Filter
CRYOSTAT “COLD-FINGER”
Sample Disc
1
-
1
Sphetica> I,’.
Polychromator
Flgure 1. IDbgram of optical system (top view) for MI fluorometric GC detection. The GC effluent enters through the transfer line at the upper right of tho cryostat head.
The filtered beam from the lamp was focused to a 3-mm diameter spot on one face of a movable 12-sidedgold-plated copper deposition surface mounted in the head of a closed-cycle helium cryostat (.Air Products CSW-204E “Displex”). Fluorescence was collected via right-angle geometry and focused onto the entrance slit of a 0.:!5-m grating polychromator (PrincetonApplied Research No. 1225). The polychromator dispersed an 86-nm segment of a fluorescencespectrum onto the faceplate of a silicon intensified target (SIT) vidicon (PAR No. 1554);a PAR “OMA 2” detector controller and microcomputer system was employed. The deposition surface and associated hardware are shown in greater detail in Figure 2. Each in' 9-PHA 425
445
365
385
nrn
405
nm
425
445
' 365
'
385
405
'
425
'
445 '
nrn
Figure 4. Comparison of the fluorescence spectrum of a three-component mixture of anthracene derivatives deposited on "surface4" (top)with those of the indlvldual pure compounds (bottom). surface is properly aligned with respect to the column effluent stream. Such alignment can be accomplished visually for the first face (prior to injection of a sample), and alignment of the other faces by the stepping motor then occurs without difficulty. Characterization of a Synthetic Mixture. To evaluate the capabilities of the MI fluorometric GC detector for mixtures of structurally similar compounds, a synthetic mixture containing 500 ng of each of the 12 anthracene derivatives listed in Table I has been examined. The chromatographic retention times for these compounds are such that the most realistic procedure is subdivision of the eluent into four fractions. The appearance of the resulting fluorescence spectra is exemplified by Figures 3 and 4, which compare the "mixture" spectra of the f i a l two GC fractions (each consisting of three components, deposited on faces 3 and 4) with those of the pure compounds obtained under identical conditions. For all six compounds, identification in the respective fractions is based upon observation of at least two well-defied spectral features not exhibited by any other component. This con-
dition is satisfied for 11of the 12 anthracene derivatives in the sample, the exception being 2-ethylanthracene (which deposited on face 2, and the presence of which was manifested only by a shoulder in the four-componentmixture spectrum). Differentiation of the various anthracene derivatives in the MI fractions is based solely upon differences in their fluorescence spectra. Because undispersed continuum radiation was used for excitation, selective excitation (which has previously been shown to be extremely useful for resolution of individual fluorescent constituents in mixtures) was impossible. This approach has been used because, in a mixture of unknown compounds, intelligent decisions as to optimal excitation wavelengths cannot be made solely on the basis of chromatographic retention data. With undispersed lamp excitation, it is possible to obtain the MI fluorescence spectrum of a deposited GC fraction in less than 1s. Under these conditions, the slow step in the procedure is the chromatographic separation. Such a technique therefore is appropriate for "rapid screening" of the fluorescent constituents of a sample.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
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Table I. Components of Synthetic Mixture of Anthracene Derivatives
a
compound
abbreviation
deposition time, a s
deposition surface
anthracene 2-methylanthracene 9-methylanthracene 2-chloroanthracene 2-ethylanthracene 1-chloroanthracene 9 , lO-dimethylanthracene 9-bromoanthracene 2-tert-butylanthracene 9-phenylanthracene 9,10-dibromoanthracene 9,10-diphenylanthracene
A 2-MA 9-MA 2-CLA 2-ETA 1-CLA 9,:lO-DMA 9-BRA 2-TBA 9-PHA 9,lO-DBRA 9 ,lO-DPHA
05-105 12 0-1 40 140-1 55 150-175 160-175 160-190 2 05 -21 5 20 5-2 20 2 3.0-2 55 375-400 420-440 1380-1500
1 1 2 2 2 2
3' 3 3
4 4 4
For isothermal chromatography at 290 "C. -
Table 11. Determination of Pyrene in NBS Standard Reference Materials concn of pyrene, ppm NBS this work standard reference material PAH mixture (SRM 1647) shale oil (SRM 1580) urban dust (SRM 1649) a
9.84 -t 0.10 104f 18 6.3 f 0.4
% deviation from
NBS value
9.5 c 0.5a 113 c 20 5.8 f 0.7
-2.0
+ 8.7 -7.6
95% confidence interval.
Pure Pyrene
,
Pyrene in
t i
Shale Oil
I
!i
380
400
nm
11 I
1:
NBS Shale Oil
Pyrene in Urban Dust Extract
140
380
400
nm
140
380
1
1, e,
100
440
nm
LJ'
10
20
30
40
min.
SO
60
70
Flgure 6. M I fluorescence spectra of (left) pure pyrene, (center) pyrene In NBS SRM 1580, and (right) pyrene in NBS SRM 1649. The , background in the center and right spectra is produced by other (un0 lo 20 identified) fluorescent compounds deposlted on the same face. min.
Flaure 5. Gas chromatoarams of [left) NBS SRM 1580 and frlaht) e i r a c t of NBS SRM 16i9. For SRM'1649, a linear temperature program (8 'C/min) from 100 to 290 OC was used. No distinguishable feature for pyrene is present in either chromatogram. .
"
I
Determination of Pyrene in NBS SRMs. As a test of the quantitative capabilities of MI fluorometric GC detection, the pyrene content of three NBS SRMs of quite diverse composition was determined. The chromatograms of shale oil (SRM-1580) and the urban dust (SRM-1649) extract are exceedingly complex (Figure 51, and no GC peak assignable to pyrene can be identified from the FID chromatograms. Nevertheless, recognizable MI fluorescencespectra of pyrene can be obtained for each SRM (Figure 6). For quantification, benzo[a]pyrene (BaP) was used as the internal standard. The results obtained in this study are compared with those reported by NBS in Table 11. These data indicate the ability of MI fluorometric detection to achieve accurate quantification of an individual component of a very complex sample, under conditions in which the chromatographic separation fails to isolate that component from other fluorescent sample constituents. Precision, Limits of Detection, and Linear Dynamic Range. The precision of GC detection by MI fluorometry is exemplified by an examination of six successive injections and deposits of anthracene, which exhibited a relative standard deviation of 10.7% in peak area. Under the same conditions, the relative standard deviation (RSD) for GC detection using
the FID was 6.6%, presumably attributable to imprecision in injection of microliter samples. The increased RSD for MI fluorometric detection is due principally to slight irreproducibility in the fractions of analyte and internal standard which are deposited on a given face. The linear dynamic range for the detection system typicstlly is in excess of 3 decades. For example, an analytical calibration curve for anthracene was linear from the detection limit (1 ng, obtained by averaging 50 vidicon scans of 17.5 ms duratiion for a total integration time of 0.875 s) to a maximum quantity of 5 bg; the correlation coefficient for the calibration line was 0.99. These results are consistent with the linear dynamic range of 500-1000 typically observed for SIT vidicon tubes (18). The linear range also is affected by the relatively high limit of detection. When a photomultiplier (PMT) is usbed for detection in lamp-excitedMI fluorometry,detection limits of 50 pg are typical (14), and subpicogram detection limits are occasionally encountered when laser excitation is utilized (19). In the present apparatus, as in a gas-phase fluorometric GC detector previously described by Cooney and Winefordner (9),scattered source radiation contributes a significant background. In addition, the limits of detection attainable with the present instrumental system are adversely affected by use of the vidicon detector. Cooney et al. noted that detection limits for gas-phase fluorometric GC detection increased by factors ranging from 5.0 to 7.5 when a PMT detector was replaced by a SIT vidicon. Moreover, Howell and
1344
Anal. Chem. 1983, 55, 1344-1340
Morrison have reported that, although the two detectors exhibited comparable detection power in the visible, a SIT vidicon is about a factor of 50 less responsive than a conventional 1P28 PMT in the ultraviolet region (20). The present instrumental configuration has been designed deliberately to sacrifice both spectral resolution and detectivity for speed of operation. Because no source, monochromator, or detector settings need be altered from sample to sample, no prior knowledge of the identities of fluorescent sample components is required. Of course, use of an undispersed continuum source sacrifices the principal attribute of lowtemperature fluorescence spectrometry: the ability to excite selectively the fluorescence of individual components of a mixture (11). There is little doubt that use of an excitation monochromator or (preferably) a tunable laser would effect substantial improvements in both detection limits (9) and selectivity (19,21). However, the practicality of using a dye laser for routine use in GC detection is highly questionable. Unless the identities of fluorescent sample constituents already are known or strongly suspected, or it is desired to identify and/or quantify only a small number of preselected compounds, the need to retune a laser to obtain fluorescence spectra of many different sample constituents in different MI fractions exacts a substantial time penalty, especially if dye changes are required. From the standpoint of time of analysis, it appears most logical to use either gas chromatographic separation or selective laser excitation of fluorescence (but not both) in conjunction with MI fluorometry. In addition to improving detection limits, use of a PMT rather than an SIT vidicon would offer enhanced spectral resolution, because both “blooming” and “veiling glare” artifacta are likely when a SIT vidicon is used to measure spectra possessing sharp features (18). In addition to speed, however, the SIT vidicon offers other relevant advantages, such as the relatively facile acquisition of “excitation-emissionmatrices”
(18), which could be quite useful in conjunction with lowtemperature fluorometry (22) for identification of specific components in GC eluents.
LITERATURE CITED Ettre, L. S . J. Chromatogr. Scl. 1978, 16, 396. Froehllch, P.; Wehry, E. L. I n “Modern Fluorescence Spectroscopy”; Wehry, E. L., Ed.; Plenum: New York, 1981; Vol. 3, p 79. Bowman, M. C.; Beroza, M. Anal. Chem. 1988, 4 0 , 535. Freed, D. J.; Faulkner, L. R. Anal. Chem. 1972, 4 4 , 1194. Burchfleld, H. P.; Green, E. E.; Wheeler, R. J.; Bllledeau, S. M. J. Chromatogr. 1974, 99, 697. Roblnson, J. W.; Goodbread, J. P. Anal. Chim. Acta 1973, 66, 239. Mullk, J.; Cooks, M.; Guyer, M. F.; Semenluk, G. M.; Sawlckl, E. Anal. Left. 1975, 8 , 511. Cooney, R. P.; Vo-Dlnh, T.; Winefordner, J. D. Anal. Chlm. Acta 1977, 8 9 , 9. Cooney, R. P.; Wlnefordner, J. D. Anal. Chem. 1977, 49, 1057. Thomas, L. C.; Adams, A. K. Anal. Chem. 1982, 5 4 , 2597. Wehry, E. L.; Mamantov, G. I n “Modern Fluorescence Spectroscopy”; Wehry, E. L. Ed.; Plenum: New York, 1981; Vol. 4, p 193. Hayes, J. M.; Small, G. J. Anal. Chem. 1982, 5 4 , 1202. Wehry, E. L.; Mamantov, G. Anal. Chem. 1979, 51, A643. Stroupe, R. C.; Tokousbalides, P.; Dlcklnson, R. B., Jr.; Wehry, E. L.; Mamantov, G. Anal. Chem. 1977, 49, 701. Reedy, G. T.; Bourne, S.;Cunningham, P. T. Anal. Chem. 1979, 51, 1535. Bourne, S.;Reedy, G. T.; Cunningham, P. T. J . Chromatogr. Sci. 1979, 17, 460. Hembree, D. M.; Garrlson, A. A.; Crocombe, R. A,; Yokley, R. A,; Wehry, E. L.; Mamantov, G. Anal. Chem. 1981, 53, 1783. Christian, G. D.; Callis, J. 8.; Davidson, E. R. I n “Modern Fluorescence Spectroscopy”; Wehry, E. L., Ed.; Plenum: New York, 1981; Vol. 4, p 117. Maple, J. R.; Wehry, E. L.; Mamantov, G. Anal. Chem. 1980, 52, 920. Howell, N. G.; Morrison, G. H. Anal. Chem. 1977, 4 9 , 106. Maple, J. R.; Wehry, E. L. Anal. Chem. 1981, 53, 266. Corfleld, M. M.; Hawkins, H. L.; John, P.; Soutar. I. Analyst (London) 1901, 106, 188.
RECEIVED for review January 7, 1983. Accepted March 29, 1983. This work was supported in part by the National Science Foundation (Grant CHE-8025282) and the Electric Power Research Institute (Contract RP-1307-1).
Organization and Distribution of Molecules Chemically Bound to Silica C. H. Lochmuller,” A. S. Colborn, and M. L. Hunnlcutt P. M. Gross Chemical Laboratoty, Duke University, Durham, North Carolina 27706
J. M. Harris Department of Chemistty, Unlverslty of Utah, Salt Lake City, Utah 84 1 12
A study of the lumlnescence of pyrene sllane molecules chemically bonded to mlcropartlculate slllca gel at several surface concentrations was undertaken to assess the proxlmlty and dlstrlbutlon of chemlcally bound molecules on the natlvb slllca gel. The fluorescence lntensltles of the monomer and exclmer emlsslon were measured as a functlon of the surface concentration of the chemically bonded pyrene silane. The fluorescence lntensltles and surface concentratlon data Indicate that molecules chemically bound to mlcropartlculate slllca are not evenly dlstrlbuted but rather are clustered Into reglons of high density.
Although there has been much speculation concerning the effect of the bonded phase on solute retention in reversed-
phase liquid chromatography, construction of a model describing the mechanism of solute retention in the bonded phase has been hampered by an incomplete knowledge of the physical nature of the chemically modified surface. A characterization of the surface properties of the microparticulate silica substrate is critical to such an understanding and it is not surprising that the nature of the silica surface has been extensively studied by thermogravimetric analysis, selective silanization, and infrared spectrometry (1-3). Much of the work to date has been focused on determining the total surface density of silanol groups and the differentiation between the types of silanols and their reactivity, but there has been little quantitative discussion concerning the proximity and distribution of silanols on the surface. The work reported here presents data obtained with steady-state luminescence spectroscopy from the examination of [3-(3-pyrenyl)propyl]di-
0003-2700/83/0355-1344$01.50/00 1963 American Chemlcal Society
