1896
Anal. Chem. 1983. 55. 1896-1901
in deposition parameters (such as time and temperature of sublimation) strongly suggest that the removal of PAHs from SRC I by this procedure is quantitative. The precision of the quantitative results of PAHs in SRC I and the accuracy of the BaP determination in SRM 1580 indicate MIF to be a promising technique for identification and determination of PAHs in unfractionated “real” samples. While frozen-solution Shpol’skii spectrometry has been utilized with considerable success for the determination of PAHs in unfractionated coal liquids (IO), it is less likely (because of solubility requirements) that this technique could be successfully applied to the direct characterization of intractable solid samples such as SRC I. Since quantitative applications of MIF are not constrained by solubility requirements or the efficiencies of solvent-extraction procedures, the greatest success of this technique (as compared with other low-temperature fluorometric methods) should be realized for solid samples containing PAHs in the parts-per-million range. ACKNOWLEDGMENT We thank Peter W. Jones for providing the SRC I sample used in this investigation. Registry No. Benzo[a]pyrene, 50-32-8; perylene, 198-55-0; benz[a]anthracene, 56-55-3;benzo[blfluorene, 30777-19-6; ben63104zo[a]fluorene, 30777-18-5; 7,10-dimethylbenzo[a]pyrene, 33-6; benzo[k]fluoranthene, 207-08-9; pyrene, 129-00-0;phenanthrene, 85-01-8; triphenylene, 217-59-4; chrysene, 218-01-9. LITERATURE CITED (1) Lee, M. L.; Novotny, M. V.; Bartle, K. D. “Analytlcal Chemistry of Polycyclic Aromatic Compounds”; Academic Press: New York, 1981. (2) Futoma, D. J.; Smith, S. R.; Tanaka, J. CRC Crlt. Rev. Anal. Chem. 1982, 73, 117.
Karasek, F. W.; Clement, R. E.;Sweetman, J. A. Anal. Chem. 1981, 5 3 , 1050A. Janardan, K. G.; Schaeffer, D. J. Anal. Chem. 1979, 57,1024. Wehry, E. L.; Mamantov, G. I n “Modern Fluorescence Spectroscopy”; Wehry, E. L., Ed.; Plenum: New York, 1981; Vol. 4, p 193. Shpoi’skil, E. V.; Bolotnikova, T. N. Pure Appl. Chem. 1974, 37, 183. Klrkbrlght, G. F.; de Lima, C. G. Analyst (London) 1974, 99, 338. ColmsJo, A.; Stenberg, U. Chem. Scr. 1978, 9 , 227. Colmsjo, A.; Stenberg, U. Anal. Chem. 1979, 57, 145. Yang, Y.; D’Silva, A. P.; Fassel, V. A,; Iles, M. Anal. Chem. 1980, 5 2 , 1350. Yang, Y.; D’Sllva, A. P.; Fassel, V. A. Anal. Chem. 1981, 53,894. Yang, Y.; D’Sllva, A. P.; Fassel, V. A. Anal. Chem. 1981, 53, 2107. Lai, E. P.; Inman, E. L., Jr.; Wlnefordner, J. D. Talanta 1982, 2 9 , 601. Rima, J.; Lamotte, M.; Joussot-Dubien, J. Anal. Chem. 1982, 54, 1059.
Garrlgues, P.; Ewald, M.; Lamotte, M.; Rlma, J.; Veyres, A,; Lapouyade, R.; Joussot-Dubien, J. Int. J . Envlron. Anal. Chem. 1982, 7 7 , 305.
Garrigues, P.; De Vazelhes, R.; Ewald, M.; Joussot-Dubien, J.; Schmlttar, J.-M.; Gulochon, G. Anal. Chem. 1983, 55, 138. Wehry, E. L.; Mamantov, G. Anal. Chem. 1979, 57,A643. Wehry, E. L. Trends Anal. Chem. 1983, 2 , 143. Maple, J. R.; Wehry, E. L.; Mamantov, G. Anal. Chem. 1980, 5 2 , 920. Maple, J. R.; Wehry, E. L. Anal. Chem. 1981, 53,266. Conrad, V. 6.; Wehry, E. L. Appl. Spectrosc. 1983, 37,46. Hembree, D. M.; Hinton, R. E., Jr.; Kemmerer, R. R.; Mamantov, G.; Wehry, E. L. Appl. Spectrosc. 1979, 33,477. Tokousbalides, P.; Wehry, E. L.; Mamantov, G. J . Phys. Chem. 1977, 87, 1769. Stroupe, R. C.; Tokousballdes, P.; Dlckinson, R . B., Jr.; Wehry, E. L.; Mamantov, G. Anal. Chem. 1977, 4 9 , 701. Farooq, R.; Kirkbright, G. F. Analyst (London) 1976, 707,566. Larsen, I. L.; Hartmann, N. A.; Wagner, J. J. Anal. Chem. 1973, 4 5 , 1511.
RECEIVEDfor review April 25, 1983. Accepted July 15, 1983. This research was supported in part by the National Science Foundation (Grant CHE-8025282) and the Electric Power Research Institute (Contract RP-1307-1).
Spectrofluorimetric Determination of Polycyclic Aromatic Hydrocarbons in Aqueous Effluents from Generator Columns Rance A. Velapoldi,* Patricia A. White, and Willie E. May
Center for Analytical Chemistry, National Bureau of Standards, Washington, D.C. 20234 Keith R. Eberhardt
Center for Applied Mathematics, National Bureau of Standards, Washington, D.C. 20234
An on-stream, standards addltlon spectrofluorlmetrlctechnlque has been used to determine the concentratlons of anthracene, benz[a]anthracene, and benzo[a]pyrene In the effluents of generator columns (which yield saturated solutlons) at temperatures between 10 and 30 OC. Concentratlon values for the standard solutions of PAH’s In water over thls temperature range were as follows: anthracene, 9.9 X IO-’ to 34.4 X mol/L; benz[a]anthracene, 1.5 X lo-’ to 5.7 X IO-’ mol/L; and benzo[a]pyrene, 2.4 X to 8.9 X I O T g mol/L. Confidence llmlts (99 % Worklng-Hotelling) for the values at 25 OC were approxlmately 3-8% of the concentratlon. The method provides data that agree well wlth data from dynamic coupled column hlgh-performance llquld chromatography and, together wlth these values, were used to certlfy the effluent PAH concentratlons of the Standard Reference Material generator columns. The method can be used to determlne PAH concentratlons In aqueous effluents, aqueous solubllities, and octanol-water partltlon coeff lcients In a fast, easy procedure.
The widespread presence in the environment of polycyclic aromatic hydrocarbons (PAH’s), some of which are known carcinogens, has necessitated the development of accurate methods for PAH analyses. The role of standards and more specifically Standard Reference Materials (SRM’s) in method development has been well documented (1,2) and the National Burea of Standards (NBS) has recently certified the concentration of PAH’s in a number of matrices including shale oil (SRM 1580), acetonitrile (SRM 1647), Urban Dust/Organics (SRM 1649), and Water (SRM 1644). Development of the latter was hampered because the usual procedures to produce stable aqueous standard solutions were not successful. Direct gravimetric procedures are hampered by low aqueous solubilities (3,4). Formation of saturated solutions by stirring excess PAH in an aqueous medium leads to the phenomenon of “accommodation” in which microparticles mimic the dissolved PAH (5). Dissolution of the PAH in an organic solvent followed by successive dilutions of the solution is wasteful of organic solvent and sometimes scarce chemicals. Additionally,
This article not subject to US. Copyright. Published 1983 by the Amerlcan Chemical Society
ANALYTICAL CHEMISTRY. VOL. 55, NO. 12. OCTOBER 1983
the final diluted solution retains some organic solvent which can alter the solubility and the aqueous/solvent distribution coefficient. Partition methods where the aqueous solution is formed by equilibration of PAH vapors over water yield good results but are cumbersome and lack general usefulness (6, 7). Even if preparation of standard solutions were possible. adsomtion of the PAH on surfaces in contact with the solution could result in a concentration change. PAH generator columns were developed in response to recommendations from several EPA/NBS workshops for a PAH Standard Reference Material to provide data quality assurance in the monitoring and measurement of PAH containing effluents. The columns provide stable standards of known PAH concentrations in aqueous solutions in a convenient, easy to use manner (8-10)-a first step in the development of accurate analytical techniques. SRM 1644 consists of three generator columns, one for anthracene, the second for benz[a]anthracene (1.2-benzanthracene or BaA), and the third for benzo[a]pyrene (1,2benzopyrene or BaP). A constant flow of water into the column establishes a thermodynamic equilibrium between the PAH in solution and the PAH in the column which is dependent on the temperature and specific PAH solubility. For t h e three specified PAHs, the Concentrations range from approximately 2 x IO4 to 3 x 10.' mol/L (0.5-60 ng/g) over a temperature range from 10 to 30 "C. Certification of the PAH concentrations in the aqueous effluent was dependent upon the development of two independent analytical methods to quantify the three compounds of interest in the effluent from their respective generator columns. T h e first. dvnamic couoled column liauid chromatography (DCCLC), was developed in conjun,&, with the development of the generatorcolumnsand has been previously (8-lo), ~h~ second,a addition technique and "on-stream- spectrofluorimetry to quantify the PAH in the effluents, is reported here. The data Obtained in this study were combined with the DCCLC technique data (&lo) to yield the values given in the certificate for SRM 1644. The experimental system and the PAH concentrations in the effluents as a function of temperature and effluent flow will be discussed. This method should be generally applicable for measuring trace concentrations of fluorescent materials in aqueous effluents and can be used for measuring aqueous s o l u b d h (8-10) and e o l - w a t e r p h i t i o n coefficients (10. I
_
EXPERIMENTAL S E C T I O N Chemicals. All solvents were 'HPLC" grade and were used as received. The PAH's used for the standards were of the highest purity available from commercial suppliers. The assays for the PAH's used as standards were determined by HPLC techniques and the standard solutions were corrected accordindy. Molecular weights used were as follows: anthracene, 178.233;BaA, 228.293; and BaP,252.315 (atomic weights from ref 12). P A R Generator Columns. The columns were produced commercially aceording to NBS specificationsand consist of 0.5% (w/w) of anthracene, BaA, or BaP on fine quintus quartz packed in coiled, stainless steel tubes (nominal dimensions: 50 cm X 0.6 cm internal diameter) equipped with I-um fritted disks and HF'LC fittings ~ ~. ~on each . .end.~ ~ ~ Twenty generator columns were chosen by a random numher selection process from the 240 numbered generator columns of each PAH manufactured for SRM 1644. A minimum of ten columns from this pool of 20 were chosen in random order for the spectrofluorimetric measurements. A blank column was prepared by stripping a generatnr column of deposited PAH using acetonitrile. Measurement Procedure. The basic method used for the determination of the specific PAH Concentration in the effluent was that of standard addition, on-stream, spectrofluorimetric technique using mixed wlvent systems: nominally equal volumes of water/ethanol for anthracene and water/acetonitrile for BaA ~
~~~~
~~~~
1897
W.1.l
R..crrd
U
Rm.
p Wl p Bath
U
W.*,*
U*tioMnd
*.sMllwdmeta
Ana"Z2.1
x-" RCIWECI Digit., "*et*
W a h
Figure 1. Schematic of experimental system used for the on-line determination of PAHs in aqueous effluents.
Table I. Excitation and Emission Wavelengths for the Polycyclic Aromatic Hydrocarbons
PAH anthracene BaA BaP
nm 254 290
he,,
296
kern, nm 384 (or 404) 395 414
and BaP. The total system, depicted schematicallyin Figure 1, consists of an aqueous solvent pump, an organic solvent pump, and provisions for mixing and isolating both sides. Water was pumped from the reservoir through the precolumn (30 em X 0.46 cm diameter, necessary to decrease the effect of pressure fluctuations on the generator column), through a temperature equilibration column (50 cm x 0.2 cm diameter coiled stainless steel) and a blank or generator column (both placed in a stirred hath with temperature control to *0.05 "C), and then mixed with a stream of either organic solvent (ethanol or acetonitrile) or standard solution that was pumped from the organic solvent side. A particle filter (5 r m stainless steel frit) was used to provide additional stream mixing, The resulting mixture was then passed through a fluorescence-free,fused silica flow cell (nominal 20 ,,L volume, samplecavity 1mm x 1mm x 21 mm) of the snectrofluorimeter and the fluorescence signal measured by the several electronic options shown. Three-way control valves Band C were used to isolate either fluid side from the rest of the system while measuring solvent flow or changing columns. Control valve A was used to s e m either the organic solventora standard PAH solution. Changes of standard PAH solutions were made manually by switching valve A to solvent, changing the standard reservoir, and then switching valve A back to standards flow, For data collection, the spectrofluorimeter (Farrand Optical Co., Model Mark 1,Valhalla, NY)was equipped witb 5 or 10-mm slits on both the excitation and emission monochromators yielding band-passes of nominally 10 or 20 nm. Slits yielding band-passes of 1and 2 nm were used in preliminary studies to verify spectra of the PAHs eluting from the generator columns and to check for imwrity interferences. Data were compared to spectra from standard compounds. The excitation and emission wavelengths listed in Table I were selected for each PAH to maximize signal and/or minimize interferences. The output from the spectrofluorimeter was fed into an x-y recorder or to a voltage-to-frequency converter (V-F)-multichannel analyzer (MCA) operated in a multiscaling mode to give digital data. The usual signal integration time was 1s per channel. The electronic system (V-F MCAI was checked for linearitv bv using a calibrated standard voltage &ce and integratpd counts.from 6to luOo00 (rnrrelation coefficient = 0.999997). Sormallg, spectrofluorimetrir data tell between 1500 and 25000 counts s-'. The overall measurement system (standard solutions. flow system, spectwfluorimeter, V-F + MCA) produced straight line responses with typiral rnrrelation coefficients of 0.9999 over the concentration ranges studid. The fluorescencesignal was also monitored with a digital voltmeter to provide stability information during the time that data were collected. The system flow options are listed in Table 11 together with the spectrofluorimetric or solvent flow quantities measured. Although the water and organic solvent flows were set at 3.0 *
+
1898
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
Table 11. Summary of Quantities Measured with Specific Flow Options for the System Represented Schematically in Figure 1 water side column valve C
procedure
a
organic solvent side valve A valve B
P-1 P-2 P-3
blank blank generator
Sa
S S
standards solvent solvent
S S S
P-4
generator
S
standards
S
P-5 P-6
generator generator
waste S
solvent solvent
S waste
measured quantity fluorescence calibration curve fluorescence blank a. column preconditioning b. fluorescence generator column fluorescence generator column t standards flow, organic solvent side flow, water side
S represents spectrofluorimeter.
-
Table 111. Raw Data, Fluorescence Signal in Counts per Second for Blank, Benzo[a]pyrene, Generator Column 215, and Generator Column 215 + Standards at Four Temperatures blank 3714 i. 9 3614 i. 5 15.30 "C Gen Col tStd l a tStd 2 tStd 3 Gen Col water organic solvent total flow
5123 5908 6724 9074 5122 2.88 3.06 5.85
17b,C t 19 i 14 i 24 i. 15 i.
i
20.55 "C 5650 6436 7219 9541 5623
blank 3561 i. 9 3608 i. 6 30.15 "C
25.25 "C
13c i. 1 2 i. 1 6 i. 21 i. 15 i.
6935 7737 10051 6238
flows, mL/min
5.87
i.
16
i. 19 i. f.
24 24
I
7994 i. 24 8766 i. 20 10 999 t 21 7300 i. 35 2.88 3.07 5.85
5.86
a Std 1 = 0.518 ppb (ng/g) BaP. Std 2 = 1.035 ppb ng/g BaP. Std 3 = 2.588 ppb (ng/g) BaP. Estimate of uncertainties for the concentrations of the standards is i.0.276. One standard deviation of the mean. Directional order of measurements.
6 )
0.1 mL/min on each pump (Waters Associates, Inc., Model M-6000A, Medford, MA), the actual flows through the spectrofluorimetric cell were measured by use of a 10-mLvolumetric flask and a stopwatch to provide accurate solvent mixture ratios. These ratios were used to correct the values of the PAH in the effluent that were calculated by fitting concentration vs. relative fluorescence intensity data to a least-squares line using a programmable calculator. The specific procedure for measurements on a single generator column consisted of several steps and were as follows with the procedures in parentheses after each step referring to those in Table II: 1,measure fluorescence blank (P-2); 2, precondition column by purging with specified volume of water (500 mL for anthracene and BaP and 1000 mL for BaA) (P-3a); 3, set bath temperature; 4, measure flows for water and organic solvent sides (P-5,6);5, measure fluorescence signal from generator column effluent (P-3b); 6, measure fluorescence signal from generator column effluent + standard (P-4, minimum of three standards, increasing, decreasing, or mixed order for standard concentrations); 7, measure flows for water and organic solvent side (P-5,6);and 8, measure fluorescence blank (P-2). Measurements for generator column effluents were made at four bath temperatures (nominally 15, 20, 25, and 30 OC); thus steps 3-7 were repeated for each specific temperature. The temperatures were measured to k0.05 O C with a mercury thermometer that had been calibrated against a platinum resistance thermometer.
RESULTS AND DISCUSSION Data for a typical BaP run at 20.55 "C as obtained from the MCA scope are shown in Figure 2a and the digital data for the same run are summarized in Table 111. In Figure 2a, each channel (small dot) represents a fluorescence signal integration time of 1s while each large dot represents a tenth (i.e., tenth, twentieth, thirtieth, ...) MCA channel and thus a 10-s interval. Data from ten channels on the flat portions of the curves were averaged and a standard deviation was determined to give the data in Table 111. These data were then used to produce the graphs in Figure 2b from which the
,y; Column 215
5 il
1.2x104
b,
-5.0
,
0.8
b
0.0 CBenzo(a)pyrenel, ng/g
QC
,30.15
25.25
5.0
(a) Multichannel analyzer CRT display of raw data using standards addition technique. (b) Raw data (X) and fitted least-squares line (-) for the calculation of the benzo[a Ipyrene concentration in the generator column effluent at four temperatures. Flgure 2.
BaP concentrations in the generator column effluents were determined at several temperatures. Imprecision values of approximately 0.2 % were obtained for the measurement of blank, column, or column plus standard during any 10-s period, indicating good, short term equilibrium and instrumental stability conditions. Similar imprecisions were calculated for the fluorescence signals from the generator column at the beginning and end of an analysis at a single temperature (the average difference for the four
ANALYTICAL
CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
18919
Table IV. Solubilities and Lower and Upper 99% Confidence Limits in ng/g for Anthracene, Benz[a ]anthracene, and Benzo[a]pyrene at 5 " C Intervalsa "C
fit
anthracene, ng/g lower
upper
benz [alanthracene, ng/g fit lower upper
benzo[a Ipyrene, ng/g fit lower upper
0.51 0.74 4.55 0.61 (0.56)b 22.02 3.34 (3.42)b 2.46 14.22 10.0 17.70 (17.01)b 0.77 0.88 4.38 5.23 0.82 (0.80) 24.72 4.75 (4.54) 21.07 15.0 22.82 1.08 1.19 7.22 1.13 (1.14) 31.76 6.69 (6.24) 6.19 29.50 20.0 30.61 (31.78) 1.50 1.65 10.09 1.58 (1.62) 9.35 (8.81) 8.66 41.14 44.04 25.0 42.67 (43.66) 2.11 2.37 2.24 (2.29) 14.45 12.97 (12.78) 11.64 56.74 66.05 30.0 61.23 (60.16) a Data calculated from the following equations: anthracene, In K , = -1078.056 t 41884.5(1/T) t 161.175 In T ; BaA, Data in In K, = -93.75982 - 1771.43(1/T) t 13.808 In T ; BaP, In K, = -677.4109 + 23963.0(1/T) + 100.767 In T. parentheses are from ref 16.
temperatures given is approximately 0.3% ). The differences noted for the average of the blank column at the beginning and end of an analysis (-2.2%) are probably due to the lower stability of the measurement system at high sensitivities over a long time frame (-30 min) in addition to flow system disturbances caused by removing the blank column, inserting the generator column, and replacing the generator column with the blank column after measurements were completed on the generator column efflueints at various temperatures. This 2.2% blank difference for these data corresponds to only 1.5% of the average signal for the column plus standards. Additionally, fitting the data and extrapolating to determine the PAH concentration in the effluent reduces the effect of these differences. These differences and measurement imprecisions are taken into account in the calculation of the 99% confidence bands. The data for the BaP columns at different temperatures (aswell as the other columns) were modeled from the equation developed by Clarke and Glew (13) In K,, = A B(1/T) + C In T
a
70T----
581 48
30
1 4
20i -1 I , "x
+
where Kp is the solubility expressed in mole fraction, A , I?, and C are constants, and T i s the temperature in Kelvin. In Figure 3, the experimental data (X), the mathematical fit for the three columns (-), and 99% Working-Hotelling confidence bands (---) (14) are plotted as ng/g of PAH vs. temperature in "C. The extrapolated/interpolated data for the solubilities as a function of temperature along with 99% lower and upper confidence limits (computed as Working-Hotelling confidence bands) are given in Table IV. Values for these columns as determined by DCCLC are given in parentheses after the "fit" values in Table IV. Good agreement was obtained with this independent analytical technique. Detailed statistical analysis (using analysis of covariance) indicated that the small differences obtained for anthracene and BaP by the two methods were not statistically significant. In the case of BaA, the differences between the two analytical techniques were statistically signific,ant which suggests small systematic errors in one or both techniques. No reason for the discrepancy (-6-7% at the 6 ng/g level) could be found (checks were made for impurity contribution, inner filter effect, standards for both methods, etc.). However, the agreement of the values was considered acceptable a t these concentration levels for certification. Accordingly, the assumption was made that the results from the two procedures bracket the true concentration value and the two sets of values were combined for the statistical summary on the SRM certificate (15). A plot of residuals (fitted value - experimental value) vs. temperature from the fitted model for the BaP data is presented in Figure 4. The fact that these residuals show no systematic deviation from the zero reference line, and that the dispersion about the line seems to be fairly uniform, indicates that the data are well-represented by the model, eq 1. The plot shown is typical of the residual plots for the other two PAH's.
5 TEMPERATURE,
, ,
8.5-,
5
10
, , 15
'C
, , 20
,,,,,,,,,,
25
38
TEMPERATURE, *C
Flgure 3. For anthracene (a), benz[a ]anthracene (b), and benz.0[alpyrene (c), the raw (X) and fitted (-) data and the 9 9 % (Working-Hoteiling) confidence bands (- -) for the entire regression curve (eq 1) plotted as PAH concentration units, ng/g, vs. temperature, OC.
-
Thermodynamic Data. Values for the thermodynamic parameters AGO, ASo,AHHo,and ACpo were calculated by using
1900
ANALYTICAL CHEMISTRY, VOL. 55,
NO. 12, OCTOBER 1983
Table V. Summary of Thermodynamic Parameters (AGO, AH",AC", and AS") for Solubilization of Anthracene, BaA, and BaP in Water at 25.0 " C AG"/kJ mol-'
anthracene BaA BaP a Uncertainties
AH"/kJ mol-'
AC,"/kJ mol-'
47.75 * 0.04a (47.69 i 0.05)b 51.3 * 3.7 (47.2 i. 3.5) 1.340 i 1.030 (0.41 * 0.42) 52.1 * 0.1 (52.21 r 0.01) 49.0 i 5.7 (50.0 * 5.9) 0.115 * 1.600 (0.82 i. 0.81) 56.78 * 0.07 (56.71 * 0.041) 50.6 * 3.3 (50.3 i 1.3) 0.838 * 1.000 are reported as 95% confidence intervals. Data in parentheses are from ref 16.
0.2
AS"/
kJ mol-'
12 i 1 2 -11 i 1 9 -20 * 11
I 1
5
I0
20
15
25
30
35
TEMPERATURE, *C EENZO(a)PYRENE DATA
Flgure 4. Restduals of the raw data from the fltted curve uslng the thermodynamic relationship In K, = A B ( l / T ) C In T f o r benzo[a]pyrene, plotted as ng/g vs. OC.
+
+
eq 1 and 2a-d and are summarized in Table V at 298.15 K (25.0 "C) AGO = -RT In Kp = -ART - BR - CRT In T (2a)
AHo = AGO
+ TAS" = -BR + CRT
AC," =
(F)
(2~)
= CR
a m 0
P
The thermodynamic values calculated agree well with those reported elsewhere (16) (see Table IV).The large uncertainties associated with the calculated ASo and ACpo values are consequences of the data set which, to obtain smaller uncertainties, necessitates extremely precise data taken over a large temperature range at very small intervals. Thus, the values for these two parameters are given for information only and really have little meaning. Standards. The standard solutions in organic solvents were prepared reproducibly With dilutions by weight and were stable (three independent preparations gave fluorescence signals that agreed to within 0.2% over a 2-month period). Factors Affecting Analyses. Several factors had to be measured and corrected during the course of the determinations: Flow Rates. The flow rates for both the organic and aqueous solvent sides had to remain constant during a determination since the calculated concentration of the PAH in the effluent was multiplied by a flow correction factor. System and measurement stability were excellent; even though one solvent flow was interrupted, the system could be brought to equilibrium and the run could be continued (see Figure 5a). The presence of an air bubble in the flow cell caused a cyclical signal with an -8-5 period (Figure 5b), which was equated to the double pumping action of the HPLC pumps. Diverting the "aqueous side" flow and allowing only organic solvent to flow through the flow cell eliminated the bubble. Impurities. The materials initially tested for use in the generator columns were found to be better than 99.9% of the
Flgure 5. (a) Graphic illustration of flow interruption in aqueous side (1) and reequilibratlon of flow (2) in total system. (b) Graphic illustration of cyclical fluorescence signal variation due to air bubble in flow cell and the cycle of the HPLC pumps.
desired PAH. However, the materials used by the commercial firm for preparation of the generator columns contained impurities of other PAH's as determined by chromatographic retention times and fluorescence spectra. Corrections were made by subtracting out the contribution of the impurity to the fluorescence signal or by selecting excitation or emission wavelengths at which the impurity was either not appreciably excited or not fluorescent. Anthracene Generator Column. The anthracene generator columns contain very small amounts of phenanthrene; however, due to its very high solubility (1000 pg/kg), the residual amounts of phenanthrene were depleted from the columns at a rapid rate and no correction in the standard addition technique was needed. The high concentrations of anthracene in the effluents resulted in a reduction of the fluorescence signal through an inner filter effect. A correction was made by using eq 3 (17) (3) where Fois the "true" fluorescence signal without inner filter effect, F i s the observed fluorescence signal with inner filter effect, D is the absorbance/cm of the solution, and dl and d2 are the depths of light penetration from which the fluorescence signal is collected. Values d2 and dl were determined from cell dimensions (0.1 and 0.0 mm, respectively). The absorbance D was calculated by determining the molar absorptivity (e) for known concentrations of standards and the usual relationship D/cm = c[C]. Benz[a ]anthracene Column. After equilibration, the BaA column effluent contains as much as 0.3 ng/g of anthracene. Corrections to the relative emission intensities for BaA were not necessary since the excitation and emission wavelengths used reduced any contributions of the anthracene signal to less than 0.5 % . Benzo[a Ipyrene Column: Chrysene (
