DISCUSSION AND CONCLUSIONS The Monte Carlo program previously developed for discrete energy photon excitation (1) has been adapted to account for the continuous energy photon excitation required when treating a conventional X-ray tube. The results of the Monte Carlo calculations agree quite well with values calculated numerically ( 4 , 5 ) that have been experimentally verified. We have obtained an estimate through private communication (11) that the computer time now required for the analytical method for the case when it can be used is probably a factor of from four to 40 times less than that for the present Monte Carlo model. However, it should be reiterated that it is not suggested that the Monte Carlo model compete directly with the analytical method. Rather it should be used in those situations where the analytical method either cannot be applied or for those applications where the analytical method requires more calculation time. This should include applications for thin samples, for cases in which diverging incidence or exit beams exist, or for layered samples. In general, it is applicable when the geometry of the system is complex. The Monte Carlo program requires only about 45 seconds on an IBM 370/165 computer to calculate sufficient histories (7000) to yield a standard deviation of 1.5% or less for ternary samples. This indicates that the model will be very useful to the analyst. We have already begun investigations of the use of the model directly in the inverse calculation of elemental amounts from experimental X-ray intensities on radioisotope-source, energy-dispersive systems. We estimate that optimization of the program by choosing
more efficient random number generation methods (12) could lead to a further reduction in the required computer time of 50% or so.
APPENDIX The flow chart of the Monte Carlo program, Figure 4, outlines the basic steps involved in the calculation of the primary, secondary, and tertiary intensities of a ternary alloy. The pertinent equations and derivations may be found in the appendix of Reference 1. LITERATURE CITED (1) Robin P. Gardner and Alan R. Hawthorne, X-Ray Spectrom., 4, 138 (1975). (2) J. Sherman, Spectrochim.Acta., 7 , 263 (1955). ( 3 ) J. Sherman, Spectrochim. Acta., 15, 466 (1959). (4) T. Shiraiwa and N. Fujino, Jpn. J. Appl. Phys., 5 , 866 (1966). (5) T. Shiraiwa and N. Fujino, Bull. Chem. SOC.Jpn., 40, 2289 (1967). (6) The International Union of Crystallography, "International Tables for X-Ray Crystallography". Vol. 111, Kynoch Press, Birmingham, 1962. (7) W. Bambynek et al., Rev. Mod. Phys., 44, 716 (1972). (8) W. J. Veigele, Atomic Data Tables, 5 , 51-1 11 (1973). (9) J. V. Gilfrich and L. S. Birks, Anal. Chem., 40, 1077 (1968). (IO) W. E. Selph and C. W. Garrett, "Monte Carlo Methods for Radiation Transport". Reactor Shielding for Nuclear Engineers. N. M. Schaeffer, Ed., TID-25951, US. Atomic Energy Commission, 1973, pp 207-257. (1 1) J. W. Criss, personal communication, 1975. (12) E. J. McGrath et al.. "Techniques for Efficient Monte Carlo Simulation", ORNL-RSIC-38 (1975).
RECEIVEDfor review April 25, 1975. Accepted August 8, 1975. The authors acknowledge partial financial assistance from the Environmental Protection Agency under Grant NO. R-802759.
Sensitive Method for Determination of Phthalate Ester Plasticizers in Open-Ocean Biota Samples C. S. Giam,' H. S. Chan, and G. S. Neff Department of Chemistry, Texas A&M University, College Station, Texas 77843
The use of gas chromatography (GC) with an electron capture detector (ECD) provldes the basis for a very sensitive method for the detection of phthalate ester plasticizers in open-ocean biota samples. The chlorinated hydrocarbons, as the DDT family and the polychlorinated biphenyls which are present in almost all marine biota samples, interfere with the ECD-GC analysis of the phthalates. Thus, a separation of these classes of compounds is required prior to GC; this separation was achieved with column chromatography on deactivated ( 3 % water) Florisil. The very low levels of phthalates anticipated in these samples required the reduction of background contamination to levels much lower than had previously been reported. With careful decontamination of all reagents and equipment and precautions to avoid recontamination during the procedure, background levels as low as 25 ng of dlbutyl phthalate and 50 ng of di-2-ethylhexyl phthlate (DEHP) were attained. Typical results of biota analyses are given; an average of approximately 5 ppb of DEHP was found.
Phthalic acid ester plasticizers are in wide use with production approximating 1 billion pounds per year. There are Author t o whom all correspondence should be addressed.
indications that several of these esters are toxic to aquatic organisms (1-3) and that they are widely dispersed in the environment (4-8). However, no detailed studies of the fate of phthalates in the marine environment have been reported. As part of an investigation into the distribution of the phthalates in the open-ocean environment, this study was undertaken to establish methodology necessary for the determination of phthalate levels in open-ocean biota. A major problem in the analysis of open-ocean samples is the reduction of background contamination to levels less than the very low (parts per billion or ppb) levels generally present in the samples. This problem of background contamination has been more serious in the trace analysis of phthalates than in the studies of many other pollutants (including the chlorinated hydrocarbons) because phthalates are present in almost all equipment and reagents used in the laboratory. Non-plastic materials like cork, glass wool, Teflon sheets, and aluminum foil have been found to contain the most prevalent of the phthlates, di-2-ethylhexyl phthalate (DEHP). Although there are procedures for determining phthalate esters in biota samples (6-9), procedure blank levels, when reported, have tended to be very high ( 7 , 9). For example, 1500 ng of DEHP and 1000 ng of dibutyl phthalate (DBP) were the background levels reported by one investigator in spite of rigorous purification (7).
ANALYTICAL CHEMISTRY, VOL. 47,
NO. 13,
NOVEMBER 1975
2225
SAMPLE C H 3 C N , 200 ml 40
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Flgure 1. Analytical scheme
Also, many of the previously reported procedures have been based on the use of the flame ionization detector (FID) for gas chromatographic (GC) analyses. However, the more sensitive and specific electron capture (EC) detector is preferred for open-ocean samples because of its ability to detect the very low levels of phthalates expected. While this detector provides increased sensitivity, it also demands a much lower level of laboratory contamination than has ever been reported. In addition, the high sensitivity of the EC detector to chlorinated hydrocarbons requires a prior separation of the phthalates from the chlorinated hydrocarbons to avoid interference during GC analyses. This paper reports a very low background procedure for the determination of phthalates in the presence of chlorinated hydrocarbons using an EC detector for GC analyses.
EXPERIMENTAL Apparatus. A Tracor Model MT-220 GC with a Nickel-63 (10 mCi) EC detector in a dc mode, equipped with a 6 ft X '14 in. 0.d. borosilicate column packed with 3% SE-30 on Gas Chrom Q (100200 mesh) was used for analyses. The injector, detector, and column temperatures were 250, 275, and 220 "C, respectively. Nitrogen was used as the carrier gas a t a flow rate of 60 cm3/min. Second-column confirmation was done on a Barber-Colman Model 5360 GC with a Tritium (150 mCi) EC detector in a pulse mode, equipped with a 6 ft X $4 in. 0.d. borosilicate column packed with 1.5% SP-2250 and 1.95% SP-2401 on Supelcon AW-DMCS (100-200 mesh). The injector, detector, and column temperatures were 210, 210, and 195 "C, respectively. The operating sensitivity for both of the detectors was about 5 ng DEHP giving 50% full scale deflection (fsd). Reagents. Organic solvents of high purity were used in the extraction, partition, and cleanup processes. Most of the solvents were purchased from Mallinckrodt Chemical Company. Except for diethyl ether, normally these solvents were sufficiently pure for use without further purification. Only freshly distilled diethyl ether was used because contamination occurs during storage. Water was purified by liquid-liquid extraction in a 15-liter extractor with petroleum ether as the extracting solvent. The petroleum ether was changed a t 24-hour intervals until a 50-fold concentrate demonstrated no impurities by EC-GC. Sodium sulfate, sodium chloride, and Florisil were heated and stored in a 320 "C oven before use. Deactivated Florisil (3%) was obtained by mixing the heated Florisil with water (3% w/w) in a rotating mixer. All solvents and reagents were checked for contamination prior to use, either by concentrating solvents 200-fold or by concentrating solvent rinses of solid reagents. Materials used in these analyses demonstrated no EC detectable impurities under these conditions. Procedure. Cleaning. The equipment for analysis included glassware, porcelain ware, filter paper, blender, spatula, forceps, dissecting knife, and Teflon stopcocks. Contamination from glassware was one of the major causes of high background levels be2226
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Elution pattern of DEHP on undeactivated Florisil
cause the large amount of glassware being used provided a large surface area for adsorption of contaminants. The following procedure describes the decontamination of the equipment used in our analytical procedure. All equipment was first washed with cleaning solution (Micro, International Products Corporation) and laboratory distilled water. (Tap water should be checked for its DEHP level before it is used to wash equipment.) After rinsing the equipment with water, it was rinsed with 3 X 50 ml distilled acetone (certified ACS grade) and once with 50 ml of Nanograde acetone. The acetone for rinsing was distilled over potassium permanganate through a 5-ft column. Teflon stopcocks which cannot be heated were wrapped with heattreated aluminum foil after the final rinse. All equipment was heated in a 320 "C oven for at least 10 hours before use. The equipment from the oven was covered with purified aluminum foil while cooling; this prevented the possible contamination from dust particles in the air. When the equipment had cooled to room temperature, it was rinsed with 2 X 100 ml of Nanograde petroleum ether. The petroleum ether washing was concentrated to about 0.5 ml and checked with the gas chromatograph for contaminants. If the background level of DEHP was greater than 50 ng, the rinsing process was repeated. The number of rinses needed depends on the previous history of the equipment and the length of time in the oven. Aluminum foil and Mason jars were used for storing samples. Aluminum foil was used for the cap liners for the Mason jars and also for wrapping whole fish samples; it was cleaned by heating in a 320 OC oven. Mason jars were used to store tissue and plankton samples; they were cleaned as other glassware. For homogenization or maceration, a Virtis blender was preferred because it is more convenient to clean than Waring blenders. The various parts of the Virtis blender-the container, the shaft, and the blades-can be cleaned by heating in a 320 "C oven. Also cross-contamination between samples can be avoided since Mason jars can be used as blender jars. Contamination from filter paper has been a major problem (IO). The complete removal of these contaminants has been difficult, even by Soxhlet extraction. Whatman GF/A glass fiber filter paper is preferred since it can be cleaned just by heating in the oven. In order to avoid contamination from rubber stoppers, a Teflon sheet ( 2 X 2 in.) with a hole (3/4-in.diam.) in the center was used to support the Buchner funnel in the filtration process. The Teflon sheet was cleaned by washing with acetone and heating in a 200 "C oven. Analysis. The scheme for the analysis of biota samples is shown in Figure 1. Samples were kept at or below 0 "C until they were ready for analysis. If possible, the skin or shells of biota samples were dissected off and only the inner or sub-dermal tissues were used (about 50 to 100 g for muscle and whole animal samples and less than 30 g for liver samples). The sample was weighed into a tared blender or Mason jar. Nanograde acetonitrile (100 ml) was added to the container and the tissue was macerated for at least 2 min. This acetonitrile extract was filtered and another 100 ml of acetonitrile was added to the tissue. It was again blended and filtered and then combined with the previous extract in a l-liter separatory funnel. One hundred ml of methylene chloride-petroleum ether (1:5) was added to the separatory funnel followed by 650 ml of purified salt water (5% NaC1). After partition, the organic layer was washed with 50 ml of purified water and dried by sodium sulfate in a 100-ml stoppered cylinder. Then the extract was transferred to a Kuderna-Danish evaporative concentrator. About 10 ml of isooctane was used to rinse the sodium sulfate and combined with the extract. Then the solution was evaporated to less than 10
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
Table I. D E W Content of Some Common Laboratory Items PPb
2 x 10: Tygon tubing’ Neoprene stopper‘ 1.6 x l o 6 Black rubber tubing‘ 250000 Black rubber stopper‘ 30000 Cork‘ 6000 Amber latex tubing’ 4000 Glasswool 1000 Polyethylene tubing‘ 800 Teflon sheet 400 Aluminum foil 300 Sodium sulfate 2 Sodium chloride 1.5 Tap water 1.5 Florisil 0.05 Blank 500-1000 ng a Hexane content of 1-2 mm of “minced” material
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ml for the Florisil cleanup. For the cleanup procedure, a 2.2-cm i.d. glass column was charged with 35 g of deactivated Florisil (3% water w/w). About 1 inch of sodium sulfate was added at the top of the Florisil column. The concentrate was introduced onto the column and three 200-ml eluate fractions were collected: (1) 6%, (2) 15%and (3) 20% diethyl ether in petroleum ether. These fractions contained DDT and PCBs, DEHP and DBP, and DBP respectively. Each fraction was concentrated to about 5 ml and analyzed by GC. For samples with low lipid content (less than 0.5 g), a 10-g (1.0 X 17.0 cm) Florisil column was found to be sufficient for the cleanup. This column was eluted with 40 ml of petroleum ether t o remove the chlorinated hydrocarbons and 40 ml of 20% ether for
DEHP and DBP. R E S U L T S AND DISCUSSION Florisil Separation of Phthalates f r o m DDT and PCBs. The most common of the phthalate esters are dimethyl-(DMP), diethyl-(DEP), dibutyl-(DBP), and di-2ethylhexyl-(DEHP). The discussion in this paper will be focused on DEHP and DBP because they are among the most abundantly produced of the esters and also because there were indications from scattered studies that the other phthalates were not present in environmental samples. Our previous studies in the Gulf of Mexico have shown that chlorinated hydrocarbons (DDTs and polychlorinated biphenyls (PCBs)) were present in virtually all biota samples in the parts per billion (ppb) range (11-14). Thus, any procedure to analyze the phthalate esters by EC should include a separation scheme for the ubiquitous chlorinated hydrocarbons. This study included the development of separation procedures as well as of analytical and background reduction techniques. Since our laboratory has used Florisil in the analyses of DDT and PCBs in biota samples, and since the elution of D E H P from Florisil had been reported ( 6 ) ,the Florisil separation of chlojinated hydrocarbons and phthalates was attempted. This separation is needed prior to GC analysis because some of the peaks from the PCBs overlap with the phthalate esters. An overlap of the PCB, Aroclor 1260, and D E H P can interfere with the identification of DEHP. The higher detector response of the interfering PCB component can also produce an erroneous quantitation. A similar overlap of DBP and Aroclor 1254 was also observed. Initial attempts, using the procedure of Mayer e t al. ( 6 ) , to recover the D E H P added to an activated Florisil column with 15% ethyl ether in petroleum ether were not reproducible. Elution was then performed using higher percentages of ethyl ether. With a 30-g (2.2 X 12.0 cm) column, a 5.3-pg sample of D E H P and 200-ml fractions, the elution pattern
DEHP from
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