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Method for determination of polycyclic aromatic hydrocarbons by fluorodensitometry
MICROCHEMICAL
JOURNAL
28, 151- 154 (1983)
Method for Determination of Polycyclic Aromatic Hydrocarbons by Fluorodensitometry VIRGINIA L. Corporate
OLSANSKI
AND
Research Drpartment. Wilmingron, Dulall~arr
Received
December
TAMARA
YATZUS
ICI Americas I9897
Inc.,
23, 1981
INTRODUCTION
Thin-layer chromatography has been used extensively for preliminary fractionation of polycyclic aromatic hydrocarbons (PAHs) into groups on a preparative scale prior to quantitative evaluation by GC, HPLC, or fluorescence spectrophotometry (2, 5, 6). Hellman (4) described semiquantitative estimates of PAH by fluorodensitometry in the microgram range. Other workers (8), however, reported the rapid decomposition of nanogram quantities of benzo(a)pyrene (BaP) on silica gel plates and concluded that in situ measurement of PAHs is not feasible. Seifert (9) states that BaP is unstable on normal-phase silica gel but indicated that BaP is relatively stable on reversed-phase silica gel plates (impregnated with paraffin wax). Our own experience confirmed that BaP is relatively stable on reversed-phase silica gel plates, and showed instability on alumina plates as well. The chemically bonded reversed-phase plates used in our work were found to be suitable for fluorodensitometric measurement of nanogram quantities of BaP. The United States Environmental Protection Agency (EPA) has proposed GC and HPLC methods for the analysis of individual PAHs in municipal and industrial wastewater (3). These methods are capable of separating and measuring the 16 PAHs designated by EPA as priority pollutants. The fluorodensitometric method presented in this paper was developed for the estimation of total PAHs in hydroalcoholic solutions (8 and 27% alcohol), corresponding to simulated alcoholic beverages, used for extraction studies on resinous and polymeric coatings (I). The method is equally applicable to the analysis of water samples and could be valuable for rapid screening of wastewater for total PAHs, in order to determine whether a lengthier procedure involving separation of individual PAHs is necessary. MATERIALS
AND METHODS
Reagents. Benzo(u)pyrene (99+%, Aldrich 18,644-9). Standard solutions were prepared to contain 0.1,0.3, 1, and 2 rig/h BaP in cyclohexane. 151 0026-265X183
$1.50
Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
152
OLSANSKI
AND
YATZUS
A mixture of the 16 PAHs designated by the U.S. Environmental Protection Agency (EPA) as priority pollutants (8) was purchased from Supelco, Inc. (PAH mixture 610-M, Catalog No. 4-8743). One milliliter of the concentrated solution was diluted to 100 ml with cyclohexane to give a solution containing 58 ng/pl of total PAHs. Cyclohexane and methanol: distilled in glass solvents. Anhydrous sodium sulfate was reagent grade. Apparatus. Glass Buchner-type funnel: 60-ml capacity, mediumporosity fritted disc, 529/42 outer joint with side arm for vacuum liltration. Evaporation flasks: Pyrex, pear-shaped, 125ml capacity with T29/42 inner joint. Wash bottle stopper, modified: all glass with T29142 outer joint; stopper has inlet and outlet to conduct N, over the cyclohexane surface. Reacti-Vials: 0.3 ml in size, Pierce Chemical Company, Catalog No. 13220. TLC spotting template: Fotodyne Catalog No. 2-6270, universal spotting template or equivalent with three closed sides and marking scales. Glass cutter: carbide tip. TLC tank: Analtech Catalog No. 6020-210 or equivalent (28 x 15.2 x 7.5 cm); tank is lined with 10 x 20-cm saturation pads. Drummond disposable microcaps: 1 ~1 in size. Shimadzu scanning densitometer: Model CS-910, equipped with mercury lamp for fluorometry; peak areas, automatically integrated with a Shimadzu C-RlA recording-integrating data processor, were expressed in integrator units. Procedure. One hundred twenty-five milliliters of aqueous or aqueous alcoholic sample is transferred to a 250-ml separatory funnel. The sample is extracted successively with 50 and 25 ml of cyclohexane, respectively. The combined cyclohexane extracts are filtered through 1% in. of anhydrous Na,SO, on the Buchner funnel into the evaporation flask. The modified wash bottle stopper is inserted into the flask and the cyclehexane filtrate is evaporated in the steam bath while passing a stream of N, over the surface. The concentrated extract is transferred and adjusted to a suitable volume. For very low levels of PAH, the cyclohexane extract may be transferred to a weighted 0.3-ml Reacti-Vial and reduced to about 25 ~1. The final volume of the concentrated extract is determined by reweighing the Reacti-Vial and calculating the volume (sp grav of cyclohexane = 0.778). The recovered extract is retained for thin-layer chromatography . Thin-layer chromatography. The 20 x 20-cm Whatman KC,, plate is placed face down on the template, scored with the carbide glass cutter, and snapped into two 10 x 20-cm plates. EPA priority pollutant
standards.
DETERMINATION
153
OF PAH
A 10 x 20-cm plate is marked into eighteen l-cm channels. Onemicroliter aliquots of sample extracts, standard BaP solutions, and the diluted EPA primary pollutant PAH solution are spotted, leaving three channels vacant for plate blanks. After spotting, a finish line is drawn across the plate 4 cm above the origin. The plate is developed to 4 cm with the solvent system, methanol-H,0 80120and dried in a stream of N,. A second finish line is drawn on the plate 2 cm above the origin, followed by development with methanol. After drying with N,, the plate is scanned with the densitometer in the fluorescence mode (A,, = 365 nm, A,, = 450 nm with L-42 cut off filter, scan speed-20 mmmin, slit width = 0.4 mm, slit height = 8.0 mm). The dimension of the scan (usually about 15 mm in length) is determined by first evaluating the EPA mixture channel. The blanks, standards, and samples are then scanned over the corresponding distance. The BaP response factor is a constant over the range O-2 ng. Total PAHs are calculated as BaP from the area of the sample scan, after correction for the average plate blank. RESULTS
AND DISCUSSION
Since the determination of “total PAHs” is the goal, no attempt was made to separate individual compounds. Instead, the EPA mixture was used as a guide and solvent systems and the stepwise development technique deliberately chosen to push these compounds together as a class. The short length of the PAH zone on the plate served the double purpose of maximizing the total PAH response while minimizing the plate background noise. As little as 0.1 ng of BaP may be detected on the TLC plate. However, the background noise represented by the area corresponding to the average plate blank may equal or even exceed the area corresponding to 0.1 ng of BaP. The lowest level of BaP at which a sufficiently reproducible net area can be obtained is about 0.3 ng BaP. Given a 125-ml sample and a cyclohexane concentrate volume of 25 microliters, the total PAH corresponding to a 0.3-ng BaP response is less than 0.1 ppb. Thus, by increasTABLE
1
FLIJORODENSITOMETRIC DETERMINATIONOF BaP IN SPIKED~ATERAND HYDRO-ALCOHOLICSAMPLESAFTEREXTRACTIONAND THIN-LAYER CHROMATOGRAPHY BaP (ppb)
Sample (125 ml)
Added
Found”
H,O 8% Alcohol 27% Alcohol
0.424 0.424 0.424
0.424 0.434 0.418
n Average of five determinations.
Recovery” (%) 100 102 99
RSD (S,) kO.2 c3.5 i-3.5
154
OLSANSKI AND YATZUS
ing the size of the aqueous sample, PAHs could be detected at the low parts per trillion level, providing there were no interferences. For validation purposes, replicate samples of water and hydroalcoholic solutions were spiked with BaP and carried through the procedure. The results of five independent determinations of BaP in water and in 8 and 27% alcohol solutions, respectively, are summarized in Table 1. These results show BaP recoveries ranging from 99 to 102% with an average percentage relative standard deviation of 22.4. SUMMARY A TLC fluorodensitometric method for the determination of total PAHs in aqueous and hydroalcoholic solutions at the parts per billion level has been developed. The PAHs are extracted into cyclohexane and separated as a class on a chemically bonded reversed-phase TLC plate using stepwise development. In the fluorescence scanning of the plate the PAHs are located by reference to a mixture of 16 PAHs designated by EPA as primary pollutants and measured as benzo(a)pyrene.
REFERENCES 1. “Code of Federal Regulations, Title 21,” Chap. 1, Food and Drug Administration, 175.300. Revised as of April 1980. 2. Daisey, J. M., and Leyko, M. A., Thin-layer-gas chromatographic method for the determination of polycyclic aromatic and aliphatic hydrocarbons in airbourne particulate matter. Anal. Chem. 51, 24-26 (1979). 3. EPA Method 610-Polynuclear aromatic hydrocarbons. Fed. Reg. 44, No. 233, Dec. 3, 1979. 4. Hellman, H., Vereinfachte routinemassige Bestimmung von polycyclishen Aromaten. Z. Anal Chem. 275, 109-113 (1975). 5. Howard, J. W., Turicchi, E. W., White, Richard H., and Fazio, T., Extraction and estimation of polycyclic aromatic hydrocarbons in vegetable oils. J. Assoc. Off. Anal. Chem. 49, 1236-1242 (1966). 6. Kadar, R., Nagy, K., and Fremstad, D., Determination of polycyclic aromatic hydrocarbons in industrial wastewater at the t&ml level. Talanta 27, 227-230 (1979). 7. Ogan, Kenneth, Katz, Elena, and Slavin, Walter, Determination of polycyclic aromatic hydrocarbons in aqueous samples by reversed-phase liquid chromatography. And. Chem. 51, 1315-1320 (1979). 8. Rao, A. M. M., and Vohra, K. G., Concentration of benzo(a)pyrene in Bombay. Armos. Environ., 403 (1975). 9. Seifert, B., Stability of benzo(a)pyrene on silica gel plates for high-performance thinlayer chromatography. J. Chromatog. 131, 417-421 (1977).
JOURNAL
28, 151- 154 (1983)
Method for Determination of Polycyclic Aromatic Hydrocarbons by Fluorodensitometry VIRGINIA L. Corporate
OLSANSKI
AND
Research Drpartment. Wilmingron, Dulall~arr
Received
December
TAMARA
YATZUS
ICI Americas I9897
Inc.,
23, 1981
INTRODUCTION
Thin-layer chromatography has been used extensively for preliminary fractionation of polycyclic aromatic hydrocarbons (PAHs) into groups on a preparative scale prior to quantitative evaluation by GC, HPLC, or fluorescence spectrophotometry (2, 5, 6). Hellman (4) described semiquantitative estimates of PAH by fluorodensitometry in the microgram range. Other workers (8), however, reported the rapid decomposition of nanogram quantities of benzo(a)pyrene (BaP) on silica gel plates and concluded that in situ measurement of PAHs is not feasible. Seifert (9) states that BaP is unstable on normal-phase silica gel but indicated that BaP is relatively stable on reversed-phase silica gel plates (impregnated with paraffin wax). Our own experience confirmed that BaP is relatively stable on reversed-phase silica gel plates, and showed instability on alumina plates as well. The chemically bonded reversed-phase plates used in our work were found to be suitable for fluorodensitometric measurement of nanogram quantities of BaP. The United States Environmental Protection Agency (EPA) has proposed GC and HPLC methods for the analysis of individual PAHs in municipal and industrial wastewater (3). These methods are capable of separating and measuring the 16 PAHs designated by EPA as priority pollutants. The fluorodensitometric method presented in this paper was developed for the estimation of total PAHs in hydroalcoholic solutions (8 and 27% alcohol), corresponding to simulated alcoholic beverages, used for extraction studies on resinous and polymeric coatings (I). The method is equally applicable to the analysis of water samples and could be valuable for rapid screening of wastewater for total PAHs, in order to determine whether a lengthier procedure involving separation of individual PAHs is necessary. MATERIALS
AND METHODS
Reagents. Benzo(u)pyrene (99+%, Aldrich 18,644-9). Standard solutions were prepared to contain 0.1,0.3, 1, and 2 rig/h BaP in cyclohexane. 151 0026-265X183
$1.50
Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
152
OLSANSKI
AND
YATZUS
A mixture of the 16 PAHs designated by the U.S. Environmental Protection Agency (EPA) as priority pollutants (8) was purchased from Supelco, Inc. (PAH mixture 610-M, Catalog No. 4-8743). One milliliter of the concentrated solution was diluted to 100 ml with cyclohexane to give a solution containing 58 ng/pl of total PAHs. Cyclohexane and methanol: distilled in glass solvents. Anhydrous sodium sulfate was reagent grade. Apparatus. Glass Buchner-type funnel: 60-ml capacity, mediumporosity fritted disc, 529/42 outer joint with side arm for vacuum liltration. Evaporation flasks: Pyrex, pear-shaped, 125ml capacity with T29/42 inner joint. Wash bottle stopper, modified: all glass with T29142 outer joint; stopper has inlet and outlet to conduct N, over the cyclohexane surface. Reacti-Vials: 0.3 ml in size, Pierce Chemical Company, Catalog No. 13220. TLC spotting template: Fotodyne Catalog No. 2-6270, universal spotting template or equivalent with three closed sides and marking scales. Glass cutter: carbide tip. TLC tank: Analtech Catalog No. 6020-210 or equivalent (28 x 15.2 x 7.5 cm); tank is lined with 10 x 20-cm saturation pads. Drummond disposable microcaps: 1 ~1 in size. Shimadzu scanning densitometer: Model CS-910, equipped with mercury lamp for fluorometry; peak areas, automatically integrated with a Shimadzu C-RlA recording-integrating data processor, were expressed in integrator units. Procedure. One hundred twenty-five milliliters of aqueous or aqueous alcoholic sample is transferred to a 250-ml separatory funnel. The sample is extracted successively with 50 and 25 ml of cyclohexane, respectively. The combined cyclohexane extracts are filtered through 1% in. of anhydrous Na,SO, on the Buchner funnel into the evaporation flask. The modified wash bottle stopper is inserted into the flask and the cyclehexane filtrate is evaporated in the steam bath while passing a stream of N, over the surface. The concentrated extract is transferred and adjusted to a suitable volume. For very low levels of PAH, the cyclohexane extract may be transferred to a weighted 0.3-ml Reacti-Vial and reduced to about 25 ~1. The final volume of the concentrated extract is determined by reweighing the Reacti-Vial and calculating the volume (sp grav of cyclohexane = 0.778). The recovered extract is retained for thin-layer chromatography . Thin-layer chromatography. The 20 x 20-cm Whatman KC,, plate is placed face down on the template, scored with the carbide glass cutter, and snapped into two 10 x 20-cm plates. EPA priority pollutant
standards.
DETERMINATION
153
OF PAH
A 10 x 20-cm plate is marked into eighteen l-cm channels. Onemicroliter aliquots of sample extracts, standard BaP solutions, and the diluted EPA primary pollutant PAH solution are spotted, leaving three channels vacant for plate blanks. After spotting, a finish line is drawn across the plate 4 cm above the origin. The plate is developed to 4 cm with the solvent system, methanol-H,0 80120and dried in a stream of N,. A second finish line is drawn on the plate 2 cm above the origin, followed by development with methanol. After drying with N,, the plate is scanned with the densitometer in the fluorescence mode (A,, = 365 nm, A,, = 450 nm with L-42 cut off filter, scan speed-20 mmmin, slit width = 0.4 mm, slit height = 8.0 mm). The dimension of the scan (usually about 15 mm in length) is determined by first evaluating the EPA mixture channel. The blanks, standards, and samples are then scanned over the corresponding distance. The BaP response factor is a constant over the range O-2 ng. Total PAHs are calculated as BaP from the area of the sample scan, after correction for the average plate blank. RESULTS
AND DISCUSSION
Since the determination of “total PAHs” is the goal, no attempt was made to separate individual compounds. Instead, the EPA mixture was used as a guide and solvent systems and the stepwise development technique deliberately chosen to push these compounds together as a class. The short length of the PAH zone on the plate served the double purpose of maximizing the total PAH response while minimizing the plate background noise. As little as 0.1 ng of BaP may be detected on the TLC plate. However, the background noise represented by the area corresponding to the average plate blank may equal or even exceed the area corresponding to 0.1 ng of BaP. The lowest level of BaP at which a sufficiently reproducible net area can be obtained is about 0.3 ng BaP. Given a 125-ml sample and a cyclohexane concentrate volume of 25 microliters, the total PAH corresponding to a 0.3-ng BaP response is less than 0.1 ppb. Thus, by increasTABLE
1
FLIJORODENSITOMETRIC DETERMINATIONOF BaP IN SPIKED~ATERAND HYDRO-ALCOHOLICSAMPLESAFTEREXTRACTIONAND THIN-LAYER CHROMATOGRAPHY BaP (ppb)
Sample (125 ml)
Added
Found”
H,O 8% Alcohol 27% Alcohol
0.424 0.424 0.424
0.424 0.434 0.418
n Average of five determinations.
Recovery” (%) 100 102 99
RSD (S,) kO.2 c3.5 i-3.5
154
OLSANSKI AND YATZUS
ing the size of the aqueous sample, PAHs could be detected at the low parts per trillion level, providing there were no interferences. For validation purposes, replicate samples of water and hydroalcoholic solutions were spiked with BaP and carried through the procedure. The results of five independent determinations of BaP in water and in 8 and 27% alcohol solutions, respectively, are summarized in Table 1. These results show BaP recoveries ranging from 99 to 102% with an average percentage relative standard deviation of 22.4. SUMMARY A TLC fluorodensitometric method for the determination of total PAHs in aqueous and hydroalcoholic solutions at the parts per billion level has been developed. The PAHs are extracted into cyclohexane and separated as a class on a chemically bonded reversed-phase TLC plate using stepwise development. In the fluorescence scanning of the plate the PAHs are located by reference to a mixture of 16 PAHs designated by EPA as primary pollutants and measured as benzo(a)pyrene.
REFERENCES 1. “Code of Federal Regulations, Title 21,” Chap. 1, Food and Drug Administration, 175.300. Revised as of April 1980. 2. Daisey, J. M., and Leyko, M. A., Thin-layer-gas chromatographic method for the determination of polycyclic aromatic and aliphatic hydrocarbons in airbourne particulate matter. Anal. Chem. 51, 24-26 (1979). 3. EPA Method 610-Polynuclear aromatic hydrocarbons. Fed. Reg. 44, No. 233, Dec. 3, 1979. 4. Hellman, H., Vereinfachte routinemassige Bestimmung von polycyclishen Aromaten. Z. Anal Chem. 275, 109-113 (1975). 5. Howard, J. W., Turicchi, E. W., White, Richard H., and Fazio, T., Extraction and estimation of polycyclic aromatic hydrocarbons in vegetable oils. J. Assoc. Off. Anal. Chem. 49, 1236-1242 (1966). 6. Kadar, R., Nagy, K., and Fremstad, D., Determination of polycyclic aromatic hydrocarbons in industrial wastewater at the t&ml level. Talanta 27, 227-230 (1979). 7. Ogan, Kenneth, Katz, Elena, and Slavin, Walter, Determination of polycyclic aromatic hydrocarbons in aqueous samples by reversed-phase liquid chromatography. And. Chem. 51, 1315-1320 (1979). 8. Rao, A. M. M., and Vohra, K. G., Concentration of benzo(a)pyrene in Bombay. Armos. Environ., 403 (1975). 9. Seifert, B., Stability of benzo(a)pyrene on silica gel plates for high-performance thinlayer chromatography. J. Chromatog. 131, 417-421 (1977).