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an SEC experiment. We have carried out many variations of flow perturbations during typical analyses and have yet to find conditions that the flowmeter cannot accommodate.
LITERATURE CITED Bly, D. D.; Stoklosa, H. J.; Kirkland, J. J.; Yau, W. W. Anal. Chem. 1975, 4 7 , 1810-1813. "Flow Precislon in a New HPLC Pumping System"; Du Pont Liquid Chromatograph Technical Report; Analytical Instrument Division, Concord Plaza, McKean Bldg.: Wllmlngton, DE. Schuiz, W. W. J . L i 9 . Chromatogr. 1980, 3, 941-952. Letot, L.; Lesec, J.; Qulvoron, C. J . L l 9 . Chromatogr. 1980, 1637-1655 Baker, D.-R.; George, S. A. Am. Lab. (Falrfield, Conn.) 1980, 42-46. Mori, S.; Suzukl, T. J . Li9. Chromatogr. 1980, 3, 343-351. , Miller, T. E., Jr.; Small, H. Anal. Chem. 1982, 5 4 , 907-910. (8) Yau, W. W.; Kirkland, J. J.; Bly, D. D. "Modern Size-Exclusion Liquid Chromatography"; Wiley: New York, 1979; p 294. (9) "Scientific Subroutines Proarammer's Reference Manual". AA-1101D. Digital Equipment Corp., h h n l c a i Documentation Center, Cotton Road, Nashua, NH 03060, revised, June (1980). ~
Flgure 10. Elution profiles for dramatically changing flow rates, the
flowmeter-derived profiles.
are shown in Figures 8-10, The use of the flowmeter for elution volume measurement comdetelv accommodates the dramatic flow changes occurring d&ng these experiments and also quite adequately accounts for any concievable (and even some inconcievable) variations in flow rate occurring during
RECEIVED for review October 15, 1982. Accepted December 3, 1982.
Peroxyoxalate Chemiluminescence Detection of Polycyclic Aromatic Hydrocarbons in Liquid Chromatography Kenneth W. Slgvardson and John W. Birks" Department of Chemistry and Cooperative Institute for Research in Environmental Sciences (CIRES), University of Colorado, Boulder, Colorado 80309
Peroxyoxalate chemlluminescence is applied to the hlghperformance llquld chromatographlc detectlon of polycyclic aromatlc hydrocarbons (PAH). PAH are excited by energy transfer from the decomposition products of the reactlon between hydrogen peroxide and bls(2,4,6-trichlorophenyl) oxalate. These reagents are Introduced by postcolumn mlxlng, and the emission Is observed by uslng a conventlonal fluorescence detector wlth Its source turned off. The method yields llnear reponses to PAH over 3 orders of magnltude, and In some cases detectlon llmlts are better than those determlned by fluorescence uslng the same fluorometer. Chemllurnlnescence detectlon Is compared to ultravlolet absorbance and fluorescence detectlon of PAH In terms of sensltlvlty and selectlvlty
the most complex PAH mixtures. HPLC detection of PAH, on the other hand, is aided by the selectivity of ultraviolet absorbance and/or fluorescence detectors. In this paper the application of a new chemiluminescence method to HPLC analysis of PAH is described and evaluated. Peroxyoxalate chemiluminescence is the most efficient nonenzymatic chemiluminescent reaction known, having quantum yields as high as 25% (2). The chemiluminescent reaction is illustrated by the reaction of an oxalic ester, such as bis(2,4,6-trichlorophenyl) oxalate (TCPO), with hydrogen peroxide and a fluorescent compound:
.
Polycyclic aromatic hydrocarbons (PAH) represent the largest class of chemical carcinogens occurring in the environment. The sources of environmental PAH are due to a variety of processes-both natural and anthropogenic. High-temperature combustion processes have become the major contributors in most urban areas (1). Due to the large number of chemical events which can take place in a combustion process, the mixtures of PAH produced are extremely complex. Currently, there is no single analytical method capable of providing the complete analysis of complex PAH mixtures. High-resolution gas chromatography and highperformance liquid chromatography (HPLC) are the two most commonly used methods of separating PAH mixtures. High-resolution gas chromatography provides the highest separation efficiencies and is necessary in the separation of 0003-2700/83/0355-0432$01.50/0
FLUOROPHOR*+
FLUOROPHOR r'
hu
Hydrogen peroxide and the oxalic ester react, forming intermediates capable of transferring as much as 106 kcal/mol of energy to the fluorescent acceptor (3). The postulated key intermediate is 1,Z-dioxetanedione ( 4 ) . Once excited, the fluorescer emits its characteristic light, as it does in fluorescence detection. The first analytical application of peroxyoxalate chemiluminescence was reported in 1976 by Seitz and co-workers (5). Hydrogen peroxide was measured in a flow injection system and was found to have a linear working range of more than 4 orders of magnitude. This system has since been improved C 1983 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. SOLVENT
VALCO
IYJECTOR
0 5ml /min.
COLUMN
FLUORESCENCE DETECTOR
TCPO SmM/acetone REAGENT
~~0~ 08M/ni:etone MOBILE PHASE RESERVOIR 7 5 % AN 0 8 m M Tris-nilrole (pH 74)
KEL-F
YMIXER
Figure 1. Schematic diagram oi the HPLC apparatus with chemilu-
minescence detection. and is reported in a more recent paper (6). Peroxyoxalate chemiluminescence detection of hydrogen peroxide has been used in the measurement of reduced nicotinamide adenine dinucleotide (NADH) (7). In that system NADH in the presence of methylene blue reduces oxygen to hydrogen peroxide which is subsequently detected by using the peroxyoxalate reaction. MLore recently, Curtis and Seitz applied peroxyoxalate chemiluminescence to the detection of dansyl amino acids separated by thin-layer chromatography (8). Peroxyoxalate chemiluminescence recently was applied to the HPLC detection of dansyl amino acids and fluorescamine derivatives of catecholamines (9, IO). In those experiments mol), respectively, detection limits of 10 and 25 fmol were reported. The chemiluminescence responses of 11polycyclic aromatic hydrocarbons have been measured in a static system (11). In that work concentrations as low as lo-' M were measured in a cuvette. Here we report the chemiluminescence detection limits for 18 PAH separated by HPLC. Under the same chromatographic conditions, detection limits were also determined by using ultraviolet absorbance at 254 nm and 280 nm and fluorescence excited a t 280 nm. Finally, a coal tar extract was analyzed by HPLC using all three means of detection, and the results compared. EXPERIMENTAL SECTION Chemicals. All PAH standards were obtained from either Aldrich Chemical Co. or Analabs. TCPO was prepared as described in the literature (12),further recrystallized in spectrograde ethyl acetate, and then washed with spectrograde hexanes. Reagent grade acetone was used as the solvent for TCPO and HzOP TCPO stock solutions (5.0 mM) were stored in borosilicate glass bottles. The solution was found to be stable over 2 months with no signs of decomposition at room temperature. However, when stored in amber soda-lime glass bottles, the half-life of TCPO was found to be only 6 h. A coal tar extract wa5 obtained from the pharmaceutical preparation Zetar (Dermilr Laboratories, Inc. Bluebell, PA) which contains whole coal tar. A 0.5-g sample was dissolved in 50 mL of methylene chloride and partitioned with three 200-mL portions of distilled water. The methylene chloride phase was dried with magnesium sulfate and diluted with acetonitrile. HPLC Apparatus. Chromatography was carried out with a Model 6000A solvent pump, a Model U6K injector, and a Resolve 5-wm C-18 column (3.9 mm X 15 cm), all manufactured by Waters Associates (Milford, MA). Sample volumes in the range 5-25 pL were utilized in this work. An Altex Model 152 dual wavelength UV detector and a Kratos Model FS 970 fluorescence detector were used for all UV absorbance and fluorescence work. The detector cell volumes for these detectors are 8 and 5 wL, re. spectively. The fluorescencedetector was operated with a Corning 7-54 prefilter and the appropriate emission cutoff filter. The chromatographic solvent was 75%)acetonitrile delivered at a flow rate of 0.5 mL/min. Chemiluminescence Detection. Figure 1shows the HPLC apparatus with chemilumiinescence detection. Solutions of 5.0 mM TCPO and 0.8 M H z 0 2 were delivered to a Kel-F, Y-type
55, NO. 3, MARCH 1983
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mixing cell using two Sage Model 341A syringe pumps. The mixing cell was attached to the solvent inlet of a Waters Model 6000A solvent pump. The optional high sensitivity pulse dampener was used to provide a smooth, pulseless flow. The reagent mixture was passed through a mixing cell consisting of a 3.3-m length of 0.25 mm i.d. stainless steel tubing coiled with a diameter of 1 cm. The effluent of this mixing cell was mixed with the column effluent in a Valco low dead volume mixing tee. The outlet of the mixing tee was connected to the Kratos FS 970 fluorescence detector via a 26 cm length of the same stainless steel tubing also coiled to a diameter of 1cm. The detector was operated with its light source off and with no emission filter. Tris nitrate buffer (pH 7.4) was added to the chromatographic solvent to give a final concentration of 0.8 mM. This buffer provided the necessary base catalysis for the peroxyoxalate reaction. All chromatography was conducted with 75% HPLC grade acetonitrile and 25% water at a flow rate of 0.5 mL/min. A nominal flow rate of 0.34 mL/min was used for the syringe pumps, and the Waters pump delivering the reagent mixture was operated at 0.6 mL/min. The dead volume from the point of mixing (TCPO and HzOz)to the Valco mixing tee was about 2 mL. PAH Assignments. A variety of methods were used for the assignment of PAH components in the coal tar extract. The relative responses of the components at 254 and 280 nm in absorbance and fluorescence (excitation) together with retention data were used to assign peaks resolved by liquid chromatography. A Hewlett-Packard Model 5982 GC-MS with a direct interface was used for a more thorough identification of sample components. Splitless injections were used on a J&W DB-5 fused silica capillary column programmed from 40 to 350 "C at 8 OC/min. The column was 30 m X 0.32 mm i.d. and had a film thickness of 0.25 pm. The temperature program was begun at injection and the final temperature was held for 6 min. PAH having up to five aromatic rings were observed under these conditions. R E S U L T S AND DISCUSSION The intensity of peroxyoxalate chemiluminescence has been shown to be linear with fluorophor concentration over about 3 orders of magnitude ( 9 , I O ) . The concentration of the energy transfer intermediate present in the detection zone can be widely manipulated by the amount of base catalyst utilized. Also, the detection is concentration dependent, and lower reagent flows result in less dilution of analyte in the detector cell. The detection scheme described earlier was designed with these considerations in mind. The detector response was optimized by adjusting the concentrations of TCPO and H,Oz and the catalyst contained in the HPLC solvent. Acetone was selected as the reagent solvent because it sufficiently dissolves TCPO while being miscible with water. When the system is operating, a background emission of about 300 nA above the photomultiplier dark current is observed. Imai e t al. (10) attributed this emission to fluorescent impurities in the solvents. This background limits the detector sensitivity, making pulse-free mixing essential. Our experiments suggest that the background signal occurs as a direct result of the reaction of TCPO with H,Oz. All reagents were scanned separately in a Perkin-Elmer MPF-2A spectrofluorimeter, and no detectable fluorescence was observed. However, when TCPO and HzOpare added to the cuvette together with the source off, a weak emission spectrum is obtained. This spectrum was found to be identical in acetone, acetonitrile, and ethyl acetate. The emission spectrum was also the same in reagent grade solvents as in spectrograde solvents and is shown in Figure 2. To compare chemiluminescence detection of PAH to conventional detectors, we have obtained detection limits for these detectors under identical chromatographic conditions. For the fluorescence detection limits, 280-nm excitation was selected because it has been shown to provide the highest sensitivity for most of the PAH studied using the Kratos FS-970 detector (13). Absorbance detection limits were measured at both 254 and 280 nm. Detection limits are defined as that
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
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Table I. Detection Limits for PAH in Reversed-Phase Liquid Chromatography
compound
retention vol, mL
anthracene benz [ a ]anthracene benzo[ b Ifluoranthene 1,2-benzofluorene benzo [ghilperylene benzo[ alpyrene coronene dibenz[a, clanthracene dibenz[a, hlanthracene 9,lO-diphenylanthracene fluoranthene fluorene indeno [ 1,2,3-cdIpyrene perylene phenanthrene pyrene tetracene triphenylene dansylphenylalanine
4.42 7.19 9.60 6.30 15.56 11.08 15.70 11.96 12.86 19.00 5.25 3.80 15.28 9.82 4.26 6.00 8.45 6.55 2.45
detection limit, picograms UV absorption peroxyoxalate fluorescence chemiluminescence 254 nm 280 nm 280 nm/>370 nm 130 300
96 630 380 325 2000 670 13000 1200 3800 490 910 600 7 50 940 350 1500 1500 330
10000
3000 360 45 2600 3000 3400 20 25000 7600 830 0.77 72000 7300 10 >200000 1.8
27000 220 310 710 940 420 1500 310 300 6400 340 800 760 16000 1300
1000 1.5 43 100 57 1.9 2200 3.5 59 120 29 6000 145 80 490 240 57 610
1100
370 1000
--
Table 11. PAH in Decreasing Order of Chemiluminescent Response
I
corrected retention volume V'idetection ( V ' ) , mL limit, mL/ng
compound perylene 9,lO-diphenylanthracene tetracene benzo[a]pyrene benzo [ghilperylene anthracene benz[a]anthracene indeno[ 1,2,3-cdIpyrene coronene dibenz [a, c ]anthracene dibenz[a,h ]anthracene 1,2-benzofluorene benzo[ b Ifluoranthene pyrene fluoranthene phenanthrene fluorene
8.72 17.90 7.35 9.97 14.46 3.32 6.09 14.18 14.60 10.86 11.76 5.20 8.50 4.91 4.15 3.16 2.70
I
11300 89 5 735 222 40.2 25.6 20.3
1
5
45
I 400
I
420
IO
15
20 25 30 35 40 1
4BSORBANCE
ABSORBANCE 254 nlp
I
5.62 3.62 3.46 1.73 0.850 0.672 0.166 0.044 0.036
TIME
(min)
Flgure 3. Chromatograms of coal tar extract at 254 and 280 nm (UV absorbance and fluorescence detection): fluorescence emission filter passes wavelengths longer than 370 nm; 80% acetonitrile at 0.5 ml/min on Waters Resolve 5-pm '2-18; 1 = indene, 2 = naphthalene, 3 = fluorene, 4 = phenanthrene, 5 = anthracene, 6 = fluoranthene, 7 = pyrene, 8 = 1,2-benzofluorene, 9 = benz[a]anthracene, 10 = benzo[b]fluoranthene, 11 = perylene, 12 = benzo[a]pyrene, 13 = 9,lO-diphenylanthracene.
I 440
5
17.1
2
0 380
15 20 25 30 35 40
280nm
\ ? I
IO
460
I
I
I
480
500
520
1
540
I 560
I
580
600
X (nrn) Flgure 2. Background emission due to the reaction of TCPO with H202. Band-pass = 30 nm.
mass of analyte that provides a signal equal to two times the peak-to-peak noise. The statistical probability that a peak is real is greater than 0.9999 by using this criterion (14). The detection limits for 18 PAH are shown in Table I. The chemiluminescence detection limit of dansylphenylalanine is included to allow comparison to previously published work. The sensitivities of PAH toward peroxyoxalate chemiluminescence can be compared by correcting for chromatographic band broadening. In short, the corrected retention
volume divided by the detection limit provides an index that can be used to rank the PAH in order of sensitivity to peroxyoxalate chemiluminescence. The PAH are listed in order of decreasing chemiluminescence sensitivity in Table 11. These results correspond identically with the work of Sherman and Ryan using a static system (11).Lechthen and Turro have shown that the chemiluminescence intensities, when corrected for fluorescence quantum yield differences, vary with singlet excitation energies (between 52 and 105 kcal/mol) in an inverse manner for various PAH (3). The work of Rauhut and co-workers further indicates that as the singlet energy decreases below 50 kcal/mol, corrected chemiluminescence intensities will also decrease (15). This indicates that the energy transfer efficiency has an optimum for compounds whose first excited singlet state is about 50 kcal/mol above the ground state. The selectivity of chemiluminescence detection was further demonstrated by the analysis of a coal tar extract. Figure 3
ANALYTICAL CHEMISTRY, VOL.
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-
Table 111. Comparison of Optimized Fluorescence Detection Limits with Those of Peroxyoxalate Chemiluminescence chemiluminescence optimized fluorescence fluorescence detection limit, detection limit, conditions (excitation, emission) co'mpound Pi2 Pg 252 nm, >370 nm 2.3 0.77 perylsne 260 nm, >370 nm 5.1 20 9,lO-diphenylanthracene 333 nm, >370 nm 10 1.8 dansylph~enylalanine 275 nm. >370 nm 10 10 tetracene '
1
FLUORESCEhCE
i
I
5
IO
I5
20
25
3C
35
40
I
> I
t (01 W z
1
CHEMILUMINESCENCE
21
I
emission. Unfortunately, the major part of the background emission occurs where most PAH themselves emit. In recent work we have found that sensitivities are greatly enhanced for polycyclic aromatic hydrocarbons containing amino groups. For example, the peroxyoxalate detection limit for aminofluoranthene is lower than that of fluoranthene by a factor of 83 500. For a large number of amino-substituted PAH, we have found that the chemiluminescence method provides detection limits that are improved by at least an order of magnitude over those determined by optimized fluorescence. We are currently investigating the mechanism of this enhancement in sensitivity. Peroxyoxalate chemiluminescence may provide a more sensitive and selective means of determining these compounds in the presence of other fluorophores.
ACKNOWLEDGMENT
5
0
I5
20
TIME
25
30
35
40
(rnin)
Figure 4. Chromatogram of coal tar extract: fluorescence excitation at 305 nm with >389 nm emission filter; chemiluminescence detection as described in the text; PAH assignments the same as in Figure 3.
shows the absorbance arid fluorescence chromatograms obtained a t 254 and 280 nm (UV absorbance and fluorescence excitation). The coal tar extract was examined a t various wavelengths of fluorescence excitation t o search for similar selectivity as observed in the chemiluminescence detection. Chromatograms for cheimiiluminescence and fluorescence excited a t 305 nm, which have similar selectivities, are shown in Figure 4. In our work we have found chemiluminescence very helpful in identifying certain PAH in complex mixtures. Compounds such as benz[a]anthracene, perylene, benzo[a]pyrene, tetracene, and 9,:lO-diphenylanthraceneusually have much higher sensitivities than other compounds having similar retention times. T o further compare the sensitivities of fluorescence and chemiluminescence, we determined optimized fluorescence detection limits for those compounds most sensitively detected in chemiluminescence. As seen in Table 111,two compounds were more sensitively detected by chemiluminescence, one had the same detection limit by both techniques, and the remaining compound was best detected by fluorescence. The chemiluminescent detection system requires significant modifications including an additional solvent pump. Additional improvements are necessary in order for the chemiluminescence system to compete with fluorescence and UV absorbance in a practicad way for these compounds. Commercially available postcoliumn reactors would greatly simplify the detection system and add greater flexibility in manipulating reagent flow rates. At this time, the background emission and resulting mixing noise appear to be the main factors limiting the sensitivity of the method. The detection limits for a few compourilds should be improved by using a cutoff filter to remove some or all of the the background
The authors thank Harold F. Walton and Mitchell S. Gandelman for helpful discussions during the course of this work. We also thank Harold Walton for the loan of several HPLC equipment items provided by a grant from the Environmental Protection Agency, and we thank Waters Associates for the loan of a 6000A pump, Model U6K injector, and Resolve 5 - ~ r nC-18 column. Registry No. Anthracene, 120-12-7;benz[a]anthracene, 5655-3; benzo[b]fluoranthene,205-99-2; 1,2-benzofluorene,238-84-6; benzo[ghi]perylene, 191-24-2;benzo[a]pyrene, 50-32-8;coronene, 191-07-1;dibenz[a,c]anthracene,215-58-7;dibenz[a,h]anthracene, 53-70-3;9,10-diphenylanthracene, 1499-10-1; fluoranthene, 20644-0; fluorene, 86-73-7;indeno[l,2,3-cd]pyrene,193-39-5;perylene, 198-55-0; phenanthrene, 85-01-8; pyrene, 129-00-0; tetracene, 92-24-0;triphenylene, 217-59-4; dansylphenylalanine, 1104-36-5; bis(2,4,6-trichlorophenyl)oxalate, 1165-91-9;naphthalene, 91-20-3; indene, 95-13-6.
LITERATURE CITED Lee, M. L.; Novotny, M. V.; Bartle, K. D. "Analytical Chemistry of Polycyclic Aromatic Compounds"; Academic Press: New York, 1981; Chapter 2. Rauhut, M. M.; Bollyky, L. J.; Roberts, B. G.; Loy, M.; Whitman, R. ii.; Iannotta, A. V. J . Am. Chem. SOC. 1967, 8 9 , 6515. Lechtken, P.;Turro, N. J. Mol. fhotochem. 1974, 6, 95-99. Rauhut, M. M. Acc. Chem. Res. 1969, 2,80-87. Williams, D. C.; Huff, G. F.; Seitz, W. R. Anal. Chem. 1976, 48, 1003-1006. Scott, G.; Seitz, W. R.; Ambrose, G. Anal. Chim. Acta 1980, 175, 221-228. Wllliams, D. C.:Seitz, W. R. Anal. Chem. 1976, 4 8 , 1478. Curtis, T. G.;Seitz, W. R. J . Chromatogr. 1977, 134, 343-350. Kobayashi, S.;Imai, K. Anal. Chem. 1980, 5 2 , 424-427. Kobayashi, S.; Seklno, J.; Honda, K.; Imai, K. Anal. Blochem. 1981, 172, 99-104. Sherman, P. A.; Holzbecher, J.; Ryan, 0.E. Anal. Chlm. Acta 7978, 9 7 , 21-27. Mohan, A. G.; Turro, N. J. J . Chem. Educ. 1974, 5 1 , 528-529. Das, B. S.;Thomas, G. H. Anal. Chem. 1978, 5 0 , 967-973. Christensen, R. G.; May, W. E. J . Llq. Chromatogr. 1978, 7 , 385-399. Rauhut, M. M.; Roberts, B. G.; Maulding, D. R.; Bergmark, W.; Coleman, R. J . Org. Chem. 1975, 4 0 , 330-335.
RECEIVED for review October 25, 1982. Accepted December 2, 1982. The authors gratefully acknowledge the support of the National Science Foundation (Grant No. CHE-79-15801). This work was performed in partial fulfillment of the requirements of the Ph.D. degree (K.W.S) in the Department of Chemistry, University of Colorado, Boulder, CO.
