464
Anal. Chem. 1981, 53, 464-467
Enhancement of Electron Capture Detector Response to Polycyclic Aromatic and Related Hydrocarbons by Addition of Oxygen to Carrier Gas D. A. Miller,' Kristen Skogerboe, and E. P. Grimsrud' Lkpartment of Chemistry, Montana State University, Bozeman, Montana 59717
The normal electron capture responses and the enhancements of response caused by 0.20% oxygen added to the carrier gas of a gas chromatograph have been measured for 30 large aromatic compounds by use of a constant-current electron capture detector (ECD). The compounds studied include three-to-five-ringed polycyclic aromatic hydrocarbons (PAHs), chloro- and methyl-substituted PAHs, and heterocyclic aromatics. The observed magnitudes of the oxygeninduced response enhancements vary greatly throughout this set of compounds. For compounds exhibiting large response enhancements, an obvlous application of oxygen doping is for causing a favorably biased response to these and an improved detection limit. The primary intent of this report, however, is to demonstrate that the oxygen-doped ECD can provide qualitative information concerning structural details of the trace components In a PAH mixture.
Because of the carcinogenic properties of certain polycyclic aromatic hydrocarbons (PAHs), considerable effort is presently being directed toward improving our ability to detect and identify these and closely related hydrocarbons which may be present in the environment. Among the more promising techniques being applied to PAH analyses are those based on prior separation by high-resolution gas chromatography (GC) (1). The GC approach not only offers superior separation of the individual sample components but also is easily mated to any of several sensitive detectors, some of which are capable of providing further qualitative as well as quantitative information concerning the mixture. Of the GC detectors, the mass spectrometer is generally considered to be the most informative. When applied to PAH mixture analysis, however, the mass spectrometer has been shown to be ineffective in distinguishing subtle and even not-so-subtle structural differences. In a report by Lee and co-workers (2),for example, it is shown that even phenanthrene and anthracene are not easily distinguished by either their electron impact or chemical ionization mass spectra, alone. By a more complicated chemical ionization technique involving simultaneous proton-transfer and charge-exchange to the PAH molecule, this group demonstrated that structural differences due to varied arrangements of the fused rings (anthracene vs. phenanthrene) did produce noticeably altered mass spectra. Even with this chemical ionization scheme, however, the more subtle structural variations caused by differing locations of a substituent on a given PAH were not identifiable. Another means by which qualitative information concerning gas chromatographic effluents can be obtained is by the use of two (or more) detectors, each responding by a different fundamental interaction with the sample. This approach would ideally have the response ratios obtained be uniquely traceable to each substance in the sample. A flame ionization Present address: Chemistry and Chemical Engineering Department, Michigan Technological University, Houghton, MI 49931. 0003-2700/81/0353-0464$01 .OO/O
detector (FID) and an electron capture detector (ECD) can provide paired responses of this type to PAHs. Very early in the development of the ECD, this selective detector was noted to provide responses to PAHs which are strong relative to nonaromatic hydrocarbons ( 3 , 4 ) . The combined use of the FID and the ECD for the GC analysis of the PAH content of atmospheric particulates has, in fact, been reported ( 5 , 6 ) . The ECD-to-FID response ratios obtained in these studies varied greatly for the numerous PAH molecules examined. Since the molar response of an FID changes relatively little from one PAH molecule to another of similar size, it is primarily the ECD which causes varied response ratios which can be correlated with individual molecular structures. In a recent report from our laboratory, the enhancement of response of an ECD to five PAH molecules caused by the intentional addition of oxygen to the carrier gas was described (7). The response enhancements observed in that study were in all cases sufficiently large as to be measured reproducibly. The magnitude of the enhancements also appeared to be dependent on structural differences present in the limited set of compounds examined. The measurements reported here extend our earlier study to include a larger set of 30 aromatic compounds. These include 12 three-to-five-ringed PAHs, 11 methyl- and chloro-substituted PAHs, and 7 heterocyclic aromatic hydrocarbons. With this set of measurements it will be shown that a detection scheme which measures oxygeninduced response enhancements will significantly improve the ECD's ability to provide qualitative information concerning a PAH-type mixture. Various aspects of the oxygen-doped ECD technique have been described in previous articles (8-10). Briefly, for many molecules which exhibit weak to moderate ECD responses under normal conditions of the detector, presumably due to a slow electron capture reaction (I),the response is often e-
+ A * products
(1)
increased by the presence of oxygen in the carrier gas, presumably due to the coupled reactions 2 and 3, where 0 2 - acts e- O2* 02(2)
+ 02-+ A
-
products
(3)
as an intermediate by which negative charge is transferred to the analyte molecule, A. Thus, the normal ECD response to a compound usually reflects the rate of electron capture, while the 02-doped response (if a significant response enhancement is observed) reflects the rate of 02-attack on the sample molecule. A measured response enhancement, therefore, is thought to reflect the ratio of the rates of reactions 3 and 1 and may depend strongly on small variations in the structure of the substrate molecule.
EXPERIMENTAL SECTION The gas chromatograph used is a Varian 3700 Aerograph with constant-current, pulse modulated operation of a 63Ni detedor. (It is important that a constant-current ECD be used when measuring oxygen-induced response enhancements, as 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
x'61 /I
a
n I
YI
!I
X I
time
-
Flgure 1. Response of OCECD to 40 ng of benzo[e]pyrene (a) without added oxygen and (b) with 0.10% oxygen added to the carrier gas. Detector temperature was 250 O C . Column temperature was
200
OC.
the magnitude of the enhancement will be very much less if a fixed-frequency, pulsed ECD is used instead (II).) The carrier gas is ultra high purity nitrogen (Matheson) maintained at a flow rate of 40 mL min-'. Since only prepared standards have been studied here, the column is a very simple one, 1.5 ft X '/e in. 0.d. stainless steel tube packed with 3% OV-101 on Chromosorb W. The oven was operated isothermally using temperatures from 140 to 200 "C. Oxygen was mixed into the carrier gas by one of two methods. In the first, oxygen was mixed into a 5-Lstainless reservoir which is part of the carrier gas system preceding the injection port. Oxygen was also added as a makeup gas after the column just prior to the detector by combining the carrier gas flow a t this point with about 4 mL min-' of nitrogen containing 2.0% oxygen. These procedures for introducing oxygen have been described in more detail previously (7,9).The results obtained for each PAH were found to be independent of the choice of method for introducing oxygen. Most of the compounds studied were purchased from commercial suppliers. The methyl-substituted benz[a]anthracenes were obtained courtesy of Melvin S. Newman of The Ohio State University. Standards were prepared by dilution into benzene. Aliquots of 2 pL were syringe-injected into the normal injection port of the instrument. Sample sizes sufficient to produce small, but easily measured, peaks were used. These generally ranged from 1to 100 ng per injection. The retention time of each compound was determined by using an FID prior to its ECD analysis.
RESULTS AND DISCUSSION In Figure 1 is shown the repeated GC-ECD analysis of a prepared standard containing 40 ng per injection of benzo[elpyrene where oxygen-free carrier is used in chromatogram a and 0.10% oxygen is present in the carrier gas of chromatogram b. The peak height and area are increased 200 times by the presence of 0.10% oxygen at 250 "C accounting for the increase in recorder attenuation. Since the noise levels are comparable on the two chromatograms shown, it is seen that an increase in signal to noise of greater than 1 order of magnitude accompanies the 02-doped analysis in this case. Detector temperatures greater than 250 "C were also used, but both the normal and Opdoped responses of the PAHs were considerably weakened with increased temperature. (In some cases the 02-free response was, in fact, inverted a t higher temperatures. This effect and its suspected cause have been discussed previously (7).)Several different oxygen concentrations have been used. As shown previously (7), the measured response enhancement increases continuously as the
465
Table I. Electron Capture Detector Responses and Response Enhancements Caused by 0.20% Oxygen in the Carrier Gas at 250 "C compound RE^ no. RECa EO, 1 phenanthrene
2 anthracene 3 triphenylene 4 chrysene 5 pyrene 6 benz[a]anthracene 7 naph t hacene 8 benzo [elpyrene 9 benzo [alpyrene 10 perylene 11 dibenz [a,c]anthracene 12 dibenz[a,h]anthracene 13 carbazole 14 dibenzofuran 1 5 dibenzothiophene 16 acridine 17 xanthene 18 4-azaphenanthrene 19 1-azaphenanthrene 20 1-chloroanthracene 21 2-chloroanthracene 22 9-chloroanthracene 23 9-chlorophenanthrene 24 2-methylanthracene 25 9-methylanthracene 26 2-methylbenz[alanthracene 27 7-methylbenz[alanthracene 28 12-methylbenz[alanthracene 29 6,8-dimethylbenz[alanthracene 30 7,12-dimethylbenz[alanthracene a
185 165 7.4 400 29 15 190 67 200 175 15 33 130 50 70 4.7 8.9 50 1.7 85 130 140
500
100
14 000
190
150
8 100
230
140
8 700
8500
4.6 62 3.0
1.0 680 0.8 500 1000 5 900
1.0 40 1.5 13 20 130 4000 150 2300 1000 500 640 110 1.6 0.9 1100 14 12 10 2900 3500 2600 4300 80 45 70
140
7 100
16 000 18 000 3 900 26 000 12 000
6 100 77 3.5 9 800 500 160 190 3 000 7 700 34 000 850 1900 1600 6 700
8.9 19 000
Normal ECD molar responses determined from peak
areas all normalized with respect to the case of phenan-
threne. Response enhancement induced by 0.20% oxygen, determined by ratio of peak heights obtained with and without oxygen in the carrier gas. e Relative rate of reaction with 0; normalized to case of phenanthrene, calculated from relationship Roe a REc(RE - l). oxygen concentration is increased. The amount of oxygen which can be added at a given detector temperature is limited by the upper dynamic range of base-line frequency (9) and is also very dependent on the quality (level of bleed) of the column used. In order to survey and compare the ECD behavior of a large number of polycyclic aromatic and related hydrocarbons, we chose a standard condition of 0.20% oxygen and 250 "C. Each analysis, with and w!thout added oxygen, was repeated under this condition a t least three times. The reproducibility of a response enhancement value measured in this way was typically on the order of a few percent and is thought to be limited primarily by our ability to control the oxygen concentration. The results of this survey are shown in Table I. The first 12 compounds listed in Table I are the unsubstituted PAHs. The normal ECD responses (Rm)of these compounds differ by more than 3 orders of magnitude, from phenanthrene, defined to have a relative sensitivity of 1.0, to napthacene with R E C = 4000. For these compounds, the measured response enhancement, RE, caused by oxygen also varies greatly, by about 2 orders of magnitude. Triphenylene has the lowest RE = 3.0 while benzo[e]pyrene with RE = 400 has the largest. I t is clear that the RE values of the PAHs have sufficient magnitude and are sufficiently varied that the measurement of RE values along with the chromatograph retention time could provide additional assistance in the
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
Table 11. Structure and Numbering System for Compounds Listed in Table I 2
3
5
6
>
10
4
7
8
@ /
/
\
4 /
/
/
12
@
13
15
/ \
H
\
16
14
17
l8
identification of the individual PAHs in a complex mixture. The next seven compounds in Table I are heterocyclic molecules in which either nitrogen, oxygen, or sulfur are included into the ring structure. Again, a wide range of RE and RE values are observed. Several interesting features of this group are noted. The three compounds with a five-membered central ring and xanthene also show relatively large response enhancements. The reactivities of acridine and the two isomers of azaphenanthrene are greater than that of the corresponding PAHs without a nitrogen atom, anthracene and phenanthrene. The two isomers of azaphenanthrene yielded response enhancements, 50 and 70, which are distinguishable. The last 11 compounds in Table I beginning with 1chloroanthracene are included to provide an indication of the dependence of the measured RE on differing locations of a substituent. For the case of the three isomers of chloroanthracene, all three have very similar RE values, but have very different 02-enhancement values. The 9-chloro isomer enhances 50 times while the 2- and 1-chloro isomers enhance 8.9 and 4.7, respectively. While 9-chlorophenanthrene has a greater R E C value than the chloroanthracenes, its RE is the smallest (1.7) reported in Table I. For the analysis of chloro-substituted aromatics [which may be produced by chlorination of waste waters, for example (12)] a detector scheme which measures 02-inducedresponse enhancements may offer considerable assistance in the elucidation of the numerous structural isomers which could be present. The effect of structural differences for the methyl-substituted anthracenes are less pronounced than those of the chloroanthracenes. The RE values of 2- and 7-methylanthracene, 85 and 130, are nevertheless easily distinguished. Two of the three monomethylbenz[a]anthraceneswould not be easily distinguished by their RE values, whereas the two dimethylbenz[a]anthracenes have extremely different RE values. The uniquely low RE value of 7J2-dimethylbenz[alanthracene (8.9) appears to be largely due to an unusually large normal response, about 35 times greater than 6,8-dimethylbenz [a]anthracene. For each compound listed in Table I a number under a third row is given. This value, named Ro,, is the contribution of
N?
the oxygen enhancement reaction to the total response observed (again normalized with respect to the case of phenanthrene) and is calculated from the relationships Ro, a RE (RE - 1) (9). The value of Ro, thus determined is thought to reflect the relative reaction rates of 02in reaction 3 with each compound. The Ro, values of the PAHs listed in Table I indicate that the rates of 0; reaction vary more than 4 orders of magnitude from phenanthrene (1.0) to dibenz[a,e]anthracene with Ro, = 26000. A consideration of the Ro, values along with respective RE values provides an improved understanding of the RE measured in each case. For example, the response enhancement of benzo[a]pyrene is lower (29) than that of benzo[e]pyrene (4001, not because their rates of reaction with 02-differ (Ro, values are 18000 and 16000). Rather, the response enhancement of benzo[a]pyrene is lower because its normal ECD response, RE, is significantly greater than that of benzo[e]pyrene. In summary, it appears that large aromatic molecules possess EC sensitivities and exhibit oxygen-induced response enhancements of magnitudes sufficient to allow their measurement in trace analysis schemes involving gas chromatography. The measured RE values are often dependent on subtle structural variations and, in fact, can be used to detect structural differences among isomers that cannot be resolved by existing forms of mass spectrometry. The degree to which measured oxygen-induced response enhancements to these and other compounds will depend on various instrumental factors accompanying differing choices of column technologies, carrier gas supplies, and detector design provide important questions yet to be determined. Assuming the basis of the detector responses reported here is describable in terms of gaseous ion chemistry, however, similar results with other instruments are anticipated. The manner by which RE values can be most reproducibly measured in a single chromatographic analysis is yet to be determined. In addition to using two ECDs and an effluent splitter, several possibilities come to mind, such as tandem ECDs with oxygen added to the carrier, after the first ECD. Alternatively, oxygen-containing and oxygen-free nitrogen might be added as a makeup gas to a single ECD in alternate
Anal. Chem. 1981, 53, 467-471
481
pulses of a few seconds' duration. A simultaneous FID response would also be very desirable. These detector schemes and the inevitable problems associated with each warrent further investigation. Of the 30 compounds examined here, only 11have RES less than 50. For 13 compounds the response enhancements were greater than 100. Thus, in addition to being useful as an indicator of compound identity, an 02-doped ECD is also a means of increasing the sensitivity to normally weakly responding PAH-type molecules.
(3) Lovelock, J. E.; Zlatkis, A.: Becker, R. S Nature (London)1962, 84, 540. (4) Wentworth, W. E.; Becker, R. S. J . Am. Chem. Soc. 1962, 84, 4263. (5) Cantutl, V.; Cartoni, G. P.; Liberti, A,; Torri, A. 0.J . Chromatogr. 1965, 17, 60. (6) Bjorseth, A,; Ekiund, 0. M9C CC,J. Hlgh Resolut. Chromatogr. Chromatogr. Commun. 1979, 2 , 22. Kim, S. H. J . Chroma(7) Grimsrud, E. P.; Miller, D. A.; Stebbins, R. 0.; togr. 1960, 197, 51. (6) Grimsrud, E. P.; Stebbins, R. G. J . Chromatogr. 1976, 155, 19. (9) Grimsrud, E. P.; Miller, D. A. Anal. Chem. 1978, 50, 1141. (10) Miller, D. A.; Grimsrud, E. P. Anal. Chem. 1979, 51, 851. (11) Grimsrud, E. P.; Warden, S. W.; Stebbins, R. G., submbd to Anal.
ACKNOWLEDGMENT We thank Melvin S . Newman, The Ohio State University, for contributing samples of methylated benzanthracene.
(12) Oyler, A. R.; Bodenner, D. L.; Welch, K. J.; Liukkonen, R. J.; Carlson, R. M.; Kopperman. H. L.; Caple, R. Anal. Chem. 1976, 50, 837.
LITERATURE CITED (1) Lee, M. L.: Vassileros. D. L.: White. C. M.: Novotnv. M. Anal. Chem. 1979, 51. 768. (2) Lee, M. L.; Vassilaros. D. L.; Pipkin, W. S.; Sorenson, W. L. NBS Spec. Publ. ( U . S . ) 1976, No. 519, 731.
Chem.
RECEIVED for review September 22,1980. Accepted December 9,1980. This paper is based on work supported by the National Science Foundation under Grant No. CHE-7824515. K.S. was a participant in the Undergraduate Research Program sponsored by the National Science Foundation.
High-Performance Liquid Chromatographic Separation and Fluorescence Detection of Warfarin and Its Metabolites by Postcolumn Acid/Base Manipulation Sun Halng Lee and L. R. Field' Department of Chemistry, 6 0 10, University of Washington, Seattle, Washington 98 195
William N. Howald and Wllllam F. Trager Department of Medicinal Chemistry, 6020, University of Washington, Seattle, Washington 98 195
A high-performance liquid chromatographlc (HPLC) method for the fluorometric determinatlon of warfarin and Its metabolites (dlastereomeric warfarin alcohols, and 4'-, 6-, 7-, and 8-hydroxywarfarin) has been developed. The detection scheme utiilzes a postcolumn acld-base fluorescence enhancement technique that provides high chromatographlc spectficity and sensHivity and is appllcable to both human plasma and urine samples. Detectlon limlts are in the low nanogram range.
Warfarin, 3-(1-phenyl-2-oxobutyl)-4-hydroxycoumarin, has found extensive use both as a rodenticide and as a clinically effective oral anticoagulant in man. Because of extensive clinical use, the quantitation of warfarin and its known metabolites (diastereoisomeric warfarin alcohols; 4-, 6-, 7-, and 8-hydroxywarfarin; benzylic hydroxywarfarin; dehydrowarfarin) as seen in Figure 1 is of importance from a therapeutic standpoint. As a consequence, a variety of assays utilizing spectrophotometric (1,2),fluorometric ( 3 , 4 ) ,thinlayer chromatographic (TLC) (5-71, gas-liquid chromatographic (GLC) (8,9), and high-performance liquid chromatographic (HPLC) (10-16) methods have been developed. However, most of these methods lack either sensitivity, specificity, or both. Most notable of the analytical methods developed are the reversed-phase HPLC assay of Fasco et al. (15, 16) and the excitation-emission matrix analysis of Christian et al. (17). 0003-2700/81/0353-0467%01 .OO/O
The Fasco method utilizes either an isocratic (15)or gradient (16) elution technique combined with UV absorption detection a t 313 nm. While offering high specificity, this assay suffers from the inherent sensitivity limitation of UV photometric detection, often requiring as much 5 mL of plasma to quantitate the metabolites. Christian et al. succeeded in analyzing warfarin and its major metabolite 7-hydroxywarfarin (7-OHwarfarin) by monitoring the fluorescence emission and excitation spectra of the mixture while varying the pH of the solution. This sophisticated methodology, while offering high sensitivity and some specificity, requires expensive equipment and does not attempt to physically resolve each component. Their technique relies on the fact that warfarin fluorescence is quenched under acidic conditions, whereas 7-OH-warfarin is relatively unaffected. Christian's studies suggest that fluorescence detection of warfarin and its metabolites may be enhanced by adjusting the acid-base character of the resolving organic solvent system and consequently may afford a new low level of detection for these compounds. Clearly a more sensitive and selective assay for warfarin and its metabolites is needed, particularly if small animals are to be used in long-term metabolic studies. This paper describes a normal-phase HPLC separation and fluorescence detection method for warfarin and its known metabolites enabling low-volume biological studies.
EXPERIMENTAL SECTION Materials. A packed column (4.6 mm i.d. x 25 cm) of Partisil-10 PAC (10 pm, microparticulate silica with a bonded cyano/amino moiety) was purchased from Whatman (Clifton, NJ). 0 1981 American Chemical Society
