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145
where o ( C ) is given by
Nonlinear Model for Ag. T h e graphical representation of the 40 experimental points (C, c) obtained for Ag, Figure 2, shows t h a t a linear fitting is certainly not advisable. We have therefore computed a second-order equation. Discussion. All results are presented in Table V. T h e comparison of the parameters obtained for each element is difficult, when considering the various concentration ranges. T h e slopes ( a ) are always smaller than unity, thus showing t h a t even for t h e elements studied a t relatively high concentration levels (Na, Mg), losses occur during the preconcentration procedure. T h e losses are greater for Ca, Fe, and Cd (slopes of about 0.8),and especially P b (slope 0.6) and Ag. This emphasizes the necessity of a very careful calibration of the preconcentration procedure to compensate for these losses. Some values of the zero intercept ( b ) are significantly different from zero: this could suggest that the choice of a linear fitting is only a first approximation for some of the elements studied in this work. I t will be important in the future to use radioisotope tracers to establish whether the losses observed during the preconcentration are d u e to adsorption processes on the walls of the Teflon bulb because of the extremely low concentrations involved, or to the lack of control of the p H during the final step of recovering the concentrated residues in ultrapure water to obtain the aliquot "B".
a(C) =
m4m "(cAA) for t h e o t h e r elements ml(mp - md
(see Equations 1 and 21, and F in Table L'. As an example, the estimations of precisions for a typical snow sample collected a t South Pole Station, Antarctica ( 1 , 2) are presented in Table VI. By using 95% confidence limits, the relative precision is in the order of 1070,except for P b (23%) and Cd (39%). For this last element, this poor precision is linked to the poor precision of the determination of cAA. No results are given for Ag, as the second-order regression obtained for t h e calibration curve for this element would lead to much more difficult estimations.
ACKNOWLEDGMENT We thank G. Baudin, R. Chesselet, and J. W. Mitchell for helpful discussions.
LITERATURE CITED C. Boutron, Ph.D. Dissertation, University of Grenoble, France, 1978. C. Boutron, S. Martin, and C. Lorius, "Proceedings 9th Int. Conf. Atmos. Aerosols, Condensationand Ice Nuclei, Gilway, Ireland ', Pergamon Press, Elmsford, N.Y., 1978. C. Boutron. Atmos. Environ. in press. C. Boutron and C. Lorius, submitted to Nature (London). C. Boutron, Anal. Chim. Acta, in press, 1978. C. Boutron, Anal. Chim. Acta. 61, 140 (1972). M. Zief and J. W. Mitchell, "Contamination Control in Trace Element Analysis", J. Wiley and Sons, New York, 1976, pp 46-67. C. Patterson and D. Settle, in "Accuracy in Trace Analysis: Sampling, Sample Handling and Analysis", Natl. Birr. Stand. ( U . S . ) ,Spec. Pub/., 422, 321-351 (1976). T. J. Murphy, in "Accuracy in Trace Analysis: Sampling, Sample Handling and Anatysis", Natl. Bur. Stand. (U.S.),Spec. Pub/., 422, 509-539 (1976). A. Hald, "Statistical Theory with Engineering Applications", J . Wiley and Sons, New York, 1967, pp 522-557.
ESTIMATION OF PRECISION For each snow sample, the concentrations C5ample of the initial sample are computed from the parameters of the preconcentration procedure (weights m,, m2,my,and m4;acids contributions g, see Table IV; parameters of the calibration ~ by curves, see Table V), and from the values of c , measured flameless atomic absorption. As the standard deviations on m,, m2, m3, ni4,and g are negligible, t h e precision on the concentrations Csmplrcan be computed from the precision of the calibration curves (given by o(a)and d b ) except for Ag, see Table V) and from the ~ ~ by u(cAA))by precision of t h e determination of c . (given flameless atomic absorption. A11 estimate of the standard deviation on Csample is then:
RECEIVED for review August 4,1978 Accepted October 20, 1978. Work supported in part by the French Ministry of the Environment, the Terres Australes et Antarctiques Francaises, the French Polar Expeditions, and the National Science Foundation's Division of Polar Programs.
Identification of Polynuclear Aromatic Hydrocarbons by Shpol'skii Low Temperature Fluorescence Anders
Colmsjo and Ulf Stenberg*
Department of Analytical Chemistry, University
of Stockholm, Arrhenius Laboratory, S- 106 9 1 Stockholm, Sweden
Polynuclear aromatic hydrocarbons from different sources have been separated by high performance liquid chromatography on reversed phase column. Fractions have been collected and the identification of the PAH was achieved by low temperature fluorescence and the Shpol'skii effect. The spectra obtained were very line-rich, thus giving a high precision in the determination when compared with those of the standard components. Six PAH were identified in automobile exhaust, four in ambient air of Stockholm, and three in the emission from an alumina reduction plant. 0003-2700/79/035 1-0145SO1. O O / O
High performance liquid chromatography has proved to be a \ aluable analytical tool in the separation of' polynuclear aromatic hydrocarbons ( P A H ) . Of the different column systems t h a t have been investigated, the most widely used seems to be bonded phases on which reversed phase chromatography with water and methanol as mobile phase is performed (1 3 ) . Successful attempts have also been made with adsorption chromatography on silica ( 4 ) or aluminum oxide (j), both with cyclohexane as solvent. Because of the low concentrations of air-borne P A H and C
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their suspected carcinogenicity, t h e detection and identification of these atmospheric pollutants require the use of sensitive and selective methods. Conventional UV-absorption (6, 7) or fluorescence spectrometry (8, 9 ) have been applied for this purpose. However, the identifications in cases where purification was not complete, were not unambiguous. T h e specificity of fluorescence emission spectra can be greatly increased by the application of the S.C. Shpol'skii effect, in which the sample is irradiated in the proper n-alkane matrix at a very low temperature. The resulting spectra contain many sharp emission maxima, and the position and the patterns of these maxima are characteristic of the individual PAH. T h e first work in this field was reported in 1952 by the Russian physicist E. V. Shpol'skii ( I O ) . During the following 10-year period, several reports were published concerning this type of fluorescence (11-1 7). However, applications were seldom reported. During t h e past three years further information has been presented on t h e Shpol'skii effect, and t h e authors have pointed out t h e unique selectivity which can be used in analyzing complex mixtures of polyaromatic hydrocarbons (18, 19). T h e conditions known t o affect the quality of these type of spectra, Le., n-alkane solvent matrix, type of cell used (ZO), rate of temperature decrease and temperature in the cell when recording t h e spectra (21), have been discussed previously. There is also another possibility available for low temperature fluorescence determination. T h a t is t o exclude the solvent and instead use a gaseous mixture with nitrogen, argon. or neon which is allowed to condense onto a cold surface (22). In this latter case, a good linear range is reported. which in ordinary Shpol'skii fluorescence is difficult t o achieve (18). In this investigation, we will describe a method for the collection, separation, a n d identification of airborne PAH. P A H s are separated by reversed-phase high performance liquid chromatography (RPHPLC) and individual P A H s identified by low-temperature fluorescence.
EXPERIMENTAL Reagents. Water and methanol were distilled once and cyclohexane and acetone were double-distilled in an all-glass apparatus. The n-alkanes were of p a . quality and showed no fluorescent impurities. Therefore they were used without any further purification. Standard polyaromatic hydrocarbons, obtained from different commercial sources, were used without purification. Sample Collection a n d Preparation. Samples from automobile exhaust gases were taken by a proportional sampling technique that uses a collection device similar to the one reported by Grimmer et al. (23). This method gives a particulate phase (collected on a glass fiber filter) and a condensed water phase. These phases were eventually combined prior to chromatography of the samples. Volatiles were collected from the filter by vacuum sublimation (300 "C, 0.1 Torr, 1 h), and the sublimate was dissolved in acetone:cyclohexane (1:2). The water phase was extracted with cyclohexane and the organic phase allowed to evaporate to almost dryness at room-temperature under a stream of nitrogen. The residue was dissolved in acetone:cyclohexane (1:2) and combined with the filter sublimate solution. Clean-up Procedure. Isolation of the PAH fraction consists of liquid partition extraction with dimethy1formamide:water (9:1) and cyclohexane and subsequent back-extraction into cyclohexane after adding more water to the DMF phase. In some cases it was necessary to remove colored constituents by using column chromatography on silica gel deactivated with 10% water and cyclohexane as solvent. Both of these steps are described in detail by Grimmer et al. ( 2 3 ) . Particulate filter samples of ambient air from central Stockholm and of working air from an alumina refinery (the Soderberg process) were also treated in the above manner. Chromatographic Procedure. A Spectra Physics liquid chromatograph (model 3600 B) equipped with a Schoeffel UV-detector with variable wave-length (model 770) was used. The
Figure 1. (1) Focusing lens, (2) quartz mirror, (3) focusing lens, (4) entrance slit to monochromator, (5) quartz window in removable lid, (6) dry nitrogen gas inlet, (7) hollow copper rod, (8) nitrogen in glass vessel, (9) insulation, (10) evacuation pipe
column was a commercially prepared 26-cm, 2-mm i.d. stainless steel column packed with ODS on 5-gm particles (Spherisorb), and operated at room temperature. A 10-gL Valco injection loop was used. Fractions were collected in 3-mL Pyrex tubes and the solvent was gently evaporated under a stream of dry nitrogen. The mobile phase was in all cases methano1:water. The different gradient,s are listed in Figures 2, 5 , and 7 . Low-Temperature Fluorescence Spectrometry. A sample cell compartment, Figure 1, was constructed which permitted recording of fluorescence spectra from solutions frozen in thin films. Small sample volumes (50 pL) were introduced onto the copper rod which was immersed in liquid nitrogen. Then the nitrogen vessel was closed by attaching the upper part of the compartment and evacuated by a vacuum pump. which permitted a further lowering of the temperature; 63 K was reached at the phase change from liquid nitrogen to solid nitrogen. The temperature of the sample was then stable for about 10 min. A medium-pressure Hg-lamp combined with an Oriel 0.125 m Ebert scanning monochromator was used as excitation source. The luminescence from the sample was analyzed by a Jarrell-Ash 0.5-m Ebert scanning monochromator (series 82-NO). A photomultiplier tube (Hammatsu R 1061, operated at room temperature, was used as detector. and the signal was current amplified and treated by an adjustable low pass active filter.
RESULTS AND DISCUSSION In the PA4Hclean-up procedure reported by Grimmer (23) the automobile exhaust condensate was subjected t o solvent partition followed by adsorption chromatography on silica gel. These two steps were required if gas-liquid chromatography using a flame-ionization detector was used for the analysis. T o decrease the time required for completion of a P A H analysis, we attempted to replace both of these clean-up steps, which are not only time-consuming but also open t o errors, with a single step that uses reversed-phase high performance liquid chromatography (RPHPLC). In preliminary experiments, the RPHPLC-elution behaviors of three different samples were compared. These samples were: a standard mixture of 8 P A H components, Figure 3; an exhaust sample prepared by the method proposed by Grimmer, Figure 3; and an untreated sample, Figure 4. These experiments showed that the aliphatic hydrocarbons and polar components in exhaust condensates which are removed by the solvent partition and silica gel chromatography were eluted from the ODS column before the P A H fraction. Therefore the two-step cleanup of the P A H fraction could be accomplished by RPHPLC. However, t h e column could be used only for a few samples because of irreversible adsorption and/or column deterioration. This column could not be regenerated even by prolonged washing with absolute
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
A
A
3
147
4
-40
30
20
lo
40
(mi
Figure 2. Chromatogram of standard PAH mixture. (1) Anthracene, (2) chrysene/triphenylene, (3) benzo[ a ] pyrene/benzo[ k ] fluoranthene, (4) dibenz[ aclanthracene, (5) dibenzo[ah]pyrenelcoronene. Conditions: 0 % H,O in MeOH; delay, Flow, 0.4 mL min-'; gradient elution, 40 0 min; sweep time, 23 min; abs. X 290 nm, column, ODS 5 p m
-
30
20
lO(rnin1
Figure 4. Uncleaned extract of automobile exhaust. Conditions: See Figure 2 10 9
I
I
A
1\11
25
7
20
TIME
tmin~
i'a
Figure 5. PAH mixture. (1) Phenanthrene, (2) fluoranthene, (3) pyrene, (4) benzo[a]fluorene, (5) benz[a]anthracene, (6) benzo[e]pyrene, (7) benzo[a]pyrene, (8) dibenz[aj]anthracene, (9) dibenz [ahlanthracene, (10) benzo[ghi]perylene, ( 1 1 ) anthanthrene, (12) dibenzo[ai]pyrene, (13) coronene. Conditions: Flow, 0.6 ml. min-'; gradient elution, 40 0 % H,O in MeOH; delay, 0 min; sweep time, 10 min; abs. h 290 nm; column, ODS 5 p m
-
J 40
30
20
lO(min1
Figure 3. DMF-cleaned extract of automobile exhaust. Conditions: See Figure 2
methanol. T h e ODS column deterioration problem was alleviated by including the DMF:H*O-cyclohexane partition step prior to the R P H P L C . T h e chromatographic separation of the PAH was accomplished by passing the sample through the RPHPLC column twice, using two different gradients. The first gradient shown in Figure 5 was used to separate the sample into two fractions. T h e first fraction contained among others, PAH of lower
molecular weight (2 rings and alkylated derivatives), and some polar constituents, and was discarded. T h e second fraction contained the PAH with three or more rings, some of which are considered carcinogenic. The second gradient was used for the chromatographic analysis of' the collected fraction. This two-step separation achieved with a typical auto exhaust sample is shown in Figures 6 and 7. 'The fraction A in Figure 6 which corresponds in retention volume from anthracene to coronene was collected and rechromatographed using a different gradient. For conditions, see Figure 7. Letters in Figure 7 correspond to the fractions which were collected and afterwards determined by low temperature fluorescence. Typical for a chromatogram of an auto exhaust condensate when using X 290 n m as detection wavelength, are the dominant peaks of coronene, benzo[ghi]perylene a n d methylated pyrenes. These peaks are also easily recognized in samples of ambient air from central Stockholm, shown in Figure 8. This may indicate t h a t the main source of polycyclics is automobiles.
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
25 I
25
20
TIME
hin)
10
Figure 6. DMF-cleaned extract of automobile exhaust. Conditions: See Figure 5. Fraction A collected and re-chromatographed. See Figure 7
I P
0
ri
b
30
-
20 TIME
(men)
1
Figure 7. Fraction A. Conditions: Flow, 0.6 mL min-'; gradient elution, 7 . 2 % H,O in MeOH; delay, 8 min, sweep time, 10 min, abs. 15.2 A 290 nm; column, ODS 5 p m , Letters correspond to fractions later determined with low temperature fluorescence, Figures 10-1 5
Figure 8. DMF-cleaned extract of particles in ambient air from Stockholm. Conditions: See Figure 7. Compounds identified with low temperature fluorescence: (1) l-Methylpyrene/4-methylpyrene, (2) benzo[a]pyrene, ( 3 ) benzo[ghi]perylene, (4) coronene
Another considerable source of polycyclics is the alumina refineries, especially when unbaked electrodes of coal and tar are used as in the Soderberg process. The composition of the samples differs considerably from those of ambient air or auto exhaust, especially the amount of lower aromatics, which is higher in samples from an alumina refinery shown in Figure 9. UV spectrometry a t X 290 nm gives only preliminary identification together with the retention time/volume. Most peaks consist of several components which in some cases could be further differentiated by making determinations a t different wavelengths. Fluorescence spectrometry as a detection system has certain advantages over the UV absorbance technique. A better sensitivity can be achieved and the
20 TIME
(mint
10
Figure 9. DMF-cleaned extract of particulate emission from an alumina reduction plant, (the Soderberg process). Conditions: See Figure 7. Compounds identified with low temperature fluorescence: (1) Benzo[a]pyrene, (2) benzo[ghi]perylene, ( 3 ) coronene
I( I
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
149
N
m
m' r*
m
I
1
I
375
400
(nm)
-A
425
Figure 11. Fraction D. low temperature fluorescence spectrum. Conditions: See Figure 10. Compounds identified: 1-methylpyrene and 4-methylpyrene
1 I
I
Figure 13. Fraction J. Low temperature fluorescence spectrum. Conditions: See Figure 12. Compound identified: Anthanthrene. Note: The spectrum is slightly disturbed in the 430-435 n m region due to over-concentration
I
r.
m
a
W
I
400
425
(nm)
-
450
A
Figure 14. Fraction M. Low temperature fluorescence spectrum, Conditions: See Figure 12. Compound identified: Coronene
inition of ca. ten times as many peaks and with a n accuracy better than f0.3 inn. An advantage of this instrument cotnbination also iies in the possibility of isolating compound>,with an expected higher molecular weight than coronene (mol wt 300) or the dibenzpyrenes (mol wt 302). see fraction N in Figure 7 , 'l'his has not yet been found possihle when using gas chroma.
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
by comparing with available standard substances. Therefore, it must be noted t h a t qualitative (and quantitative work) with fluorescence spectrometry is possible only for compounds where an authentic standard is available.
ACKNOWLEDGMENT
m
d
Special thanks are due to Bo Allvin for running the low temperature fluorescence spectra and Bo Runesson for supplying the HPLC fractions. We also thank Beryl Holm for reviewing the manuscript.
I
LITERATURE CITED (1) M. A. Fox and S. W. Staley, Anal. Chem., 48, 992 (1976). (2) A . M. Krstulovic, D. M. Rosie, and P. R. Brown, Anal. Chem., 48, 1383 ~ , i w,.m (3) J. A. Smith, R. A. Henry, R. C. Williams, and J. F. Dieckman, J. Chromat@r. Sci., 9, 645 (1971). (4) H. Boden, J . Chromatogr. Sci., 14, 391 (1976). (5) M. Popl, M. Stejskal, and J. Mostecky, Anal. Chem., 47, 1947 (1975). (6) H. J. Klimisch, J . Chromatogr., 83, 11 (1973). (7) V. MartinCi and J. Janak, J . Chromatogr., 65, 477 (1972). (8) N. G. Vaughan, 8. B. Wheals, and M. J. Whitehouse, J . Chromatogr., 78, 203 (1973). (9) J. F. B. Lloyd, Analyst(London), 100, 1193 (1975). (10) E. V. Shpol'skii, A. A. Ilina, and L. A. Klimova, Dokl. Akad. Nauk SSSR, 87, 955 (1952). (11) B. Moel and G. Lacroix, Bull. SOC. Chim. Fr., 2139 (1960) (12) I . L. Varshavskii, L. M. Shabad, L. Ya. Kheshina, S. S. Khitrovo, V. G. Chalabav, and I . I. Pakholnik, J . Appl. Spectrosc., 2, 105 (1965). (13) E. J. Bowen and B. J. Brocklehurst, J . Chem. SOC., 3875 (1954). (14) E. J. Bowen and B. J. Brocklehurst, J . Chem. Soc., 4320 (1955). (15) E. V . Shpol'skii, Soviet Phys. U s p . , 3, 372 (1960). (16) E. V. Shpol'skii, U s p . Fiz. Nauk., 77, 331 (1962). (17) J. W. Sidman. J . Chem. Phys., 23, 1365 (1955). (18) G. F. Kirkbright and C . G. de Lima, Ana/yst(London), 99, 338 (1974). (19) G. F. Kirkbright and C. G. de Lima, Chem. Phys. Lett., 37, 165 (1976). (20) A . Colmsjo and U. Stenberg, Chem. Scr. 9, 227 (1976). (21) A . Colmsjo and U. Stenberg, Chem. S c r . , 11, 220 (1977). (22) R. C. Stroupe, P. Tokousbalides, R. B. Dickinson Jr., E. L. Wehry, and G. Mamantov, Anal. Chem., 49, 701 (1977). (23) G. Grimmer, A . Hildebrandt, and H. Bohnke, Zentralbl. Bakteriol., Parasitenkd., InfeMionskr. Hyg., Ab?. 1:
[email protected] 8 ,158, 22 (1973). (24) M. L. Lee, K. D. Bartle, and M. Novotny, Anal. Chem., 48, 1566 (1976). I
il I
425
450
(nm)
4+5
-A
Figure 15. Fraction N. Low temperature fluorescence spectrum. Conditions: See Figure 12. Compound NOT identified
tography combined with mass spectrometry, where compounds with higher molecular weights are not eluted from the column ( 2 4 ) . In Figure 15, the fluorescence spectrum of fraction N is shown. It is a line-rich spectrum in the far blue region. This unidentified component also has doublets in the same spectral region as coronene (fraction M, Figure 14), Le., 453.3-453.7 and 459.8-460.2 nm. I t should have a higher molecular weight considering the longer retention time, but no mass spectrum has yet been obtained. Further, three fractions: L, K: and H, respectively, Figure 7, have given spectra with good resolution of compounds which we have been unable to identify
1
.-
RECEILFD for review July 21, 1978. Accepted October 16, 1978. This investigation was supported by the Department of Analytical Chemistry, University of Stockholm. We also thank the National Swedish Environment Protection Board, contract no. 7-49/76 and Swedish Natural Science Research Council, contract no. K 0369-015 for substantial financial support of this work.
