ods require that the mercury to be determined is present in a rather high concentration in a small sample volume and hence cannot be applied to natural water samples. The risk of losing mercury on preconcentration by evaporation has Sample t r e a t m e n t Hg f o u n d , pgl1.a been stressed ( 4 ) . Therefore, the method proposed by .-_____ Omang (7) for the destruction of dissolved mercury comUnirradiated 0.31 pounds by treatment with potassium permanganate was Irradiated 1 0 min 1.01 1.00 combined with the method reported by Omang and Paus (ZnCdHg lamp) 30 min 1.15 1.11 (8) for the destruction of geological materials by digestion 30 min 1.05 1.12 Stored with KMnO, 2% KMnO, 0.98 (0.06) 1.02 (0.03) with hydrofluoric, hydrochloric, and nitric acid in a Teflon 4% KMnO, 1.04 (0.08) 0.97 bomb. The results are shown in Table I. 4%KMnO, 1.07 1.01 It is seen that the irradiation time of 10 min, judged as Stored with KMnO,, 2% KMnO, 1.00 (0.24) 1.06 (0.23) sufficient from Figure 5, is too short for the complete departly evaporated 4% KMnO, 1.07 (0.27) 1.06 (0.31) struction of all mercury compounds in the sample. Howand redi1ute.d Stored with KMnO,, 2% KMnO, 1 . 1 2 (0.38) 0.99 (0.39) ever, the results of prolonged irradiation compare nicely partly evaporated, 4% KMnO, 1.08 (0.42) 1.09 (0.43) with the results obtained after complete wet-chemical dedigested in bomb struction. The latter method is considerably more compliand rediluted cated and yields substantially higher blank values than the a Blank values were obtained from deionized water. When photolytic method. they exceeded the detection limit (0.03 pg/l.) they were Since the photochemical method requires only the use of subtracted from the results. Subtracted blanks are given in HC1, SnC12, and uv light, it can easily be adapted for unatparentheses. tended automated operation. Table I. Comparison of Photochemical and Wet Chemical Decomposition of Mercury Compounds in an Acidified Natural Water Sample (River Waal)
LITERATURE CITED that for deionized water or for inorganic mercury in natural water (not drawn) pointing to a slight incompleteness of the decomposition of the added organomercurials. However, even if the added compounds are completely decomposed, this may not be the case for the compounds originally present in the sample, especially if the latter has not been filtered. The organomercurials may be strongly adsorbed on or even absorbed in solid particles in the sample. Therefore, we compared the photochemical destruction method with a wet chemical method. Many wet-chemical digestion meth-
(1) (2) (3) (4) (5) (6)
(7) (8) (9)
W. R. Hatch and W. L. Ott, Anal. Cbem., 40, 2085 (1968). L. Magos, Analyst (London),96, 847 (1971). Y. Kimura and V. L. Miller, Anal. Cbim. Acta, 27, 325 (1962). T. C. Rains and 0. Menis, J. Assoc. Off. Anal. Cbem., 5 5 , 1339 (1972). P. 13.Goulden and B. K. Afghan, Tech. Bull. 27 (1970), Inland Waters Branch, Dept. of Energy, Mines, and Resources, Ottawa, Canada. F. A. J. Armstrong, P. M. Williams, and J. D. Strickland, Nature (London), 211, 481 (1966). S. H. Omang, Anal. Chim. Acta, 5 3 , 415 (1970). S. H. Omang and P. E. Paus, Anal. Cbim. Acta, 5 6 , 393 (1971). B. G. Gowenlock and J. Trotman, J. Cbem. Soc., 1454 (1955).
RECEIVEDfor review July 7, 1975. Accepted October 23, 1975.
Determination of Aliphatic and Aromatic Hydrocarbons in Marine Organisms J. S. Warner Battelle Columbus Laboratories, 505 King Avenue, Columbus, Ohio 4320 1
A simple and sensitive procedure is described that is suitable for determining aliphatic and aromatic hydrocarbons in large numbers of samples of marine organisms. The procedure involves aqueous caustic digestion, ether extraction, silica gel chromatography, and gas chromatography. Recoveries greater than 70% were obtained from organisms that contained petroleum components at levels of 0.1 to 10 bg/g. Many of the aromatic hydrocarbons were Identifled by chemical ionization mass spectrometry. The method is applicable to a wide variety of organisms.
Assessment of the extent and effects of contamination of the marine environment by petroleum fractions or crude oils requires a knowledge of the petroleum hydrocarbon uptake of marine organisms. Because of factors such as seasonal variations, discontinuous feeding habits, differences in life stage, specimen variability, and varying environmental stresses, numerous samples need to be analyzed in order 578
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
to obtain reliable and meaningful results. The analytical methodology involved needs to be diagnostic and reliable, but also needs to be as simple as possible to permit the analysis of relatively large numbers of samples. Previously reported methods have been used primarily to obtain a gas chromatographic fingerprint and a quantitative determination of saturated hydrocarbons, especially pristane, phytane, and normal paraffins. In much of our work, we were more interested in aromatic hydrocarbons which are generally more toxic, more water soluble, and more readily concentrated by many marine organisms than saturated hydrocarbons (1, 2 ) . We also studied the olefinic hydrocarbons associated with the aromatic fraction. The procedures used by Clark and Blumer (3-5) involve a prolonged Soxhlet extraction of moist tissues using methanol or benzene-methanol followed by a series of separatory funnel extractions using pentane. Blaylock et al. (6) used an alcoholic KOH digestion procedure which facilitated the complete release of hydrocarbons from cellular particles; however, six or more separatory funnel extractions
w e r e r e q u i r e d . A l t h o u g h both of the a b o v e a p p r o a c h e s usually give adequate recovery of hydrocarbons from tissues, we developed the p r o c e d u r e described below t o facilitate the analysis of m a n y samples. B y using aqueous caustic for digestion, t h e r e w a s no need for e x t r a c t i o n steps to r e m o v e alcohol p r i o r to the silica gel c h r o m a t o g r a p h y . Ethyl ether w a s used as the e x t r a c t i o n s o l v e n t w h i c h readily gives c o m p l e t e e x t r a c t i o n of h y d r o c a r b o n s . The e n t i r e digestion and e x t r a c t i o n steps w e r e conveniently c a r r i e d out i n a single s c r e w - c a p centrifuge tube. Three f r a c t i o n s w e r e o b t a i n e d : a s a t u r a t e d fraction, a m o n o - and d i a r o m a t i c fraction, and a t r i a r o m a t i c fraction. Olefinic h y d r o c a r b o n s appeared in the a r o m a t i c fractions. V a l i d a t i o n e x p e r i m e n t s w e r e carried out to d e t e r m i n e the recoveries o b t a i n a b l e at various h y d r o c a r b o n c o n c e n t r a t i o n s . GC-MS w a s used t o identify the aromatic components.
EXPERIMENTAL Materials. Because of the widespread use and occurrence of hydrocarbon oils and waxes, special attention was given to the cleanliness of glassware, the purity of solvents, and the avoidance of contamination of the samples. Only glass, Teflon, or metal were permitted to come in contact with the samples. All glassware items were given a final cleaning in high-purity toluene or carbon tetrachloride in an ultrasonic bath followed by rinses with distilled-inglass grade solvent and drying in an oven a t 150 "C. Thoroughly cleaned Teflon or foil liners were used on all bottle or vial closures. Distilled-in-glass grade solvents (from Burdick and Jackson Laboratories, Inc.) were used throughout this work. Blank runs were made from time to time to check for contamination. Ethyl ether was obtained without preservative, stored a t 5 "C, and passed through an alumina column to remove peroxides prior to use. Preservation of Samples. The tissue samples used were frozen in wide-mouth glass jars with foil-lined screw caps, shipped in dry ice, and stored at -75 "C until used. Tissue Extraction. Ten grams of tissue which had been homogenized by a Tekmar Tissumizer, was mixed with 4 g of 4 N NaOH in a 50-ml centrifuge tube having a Teflon-lined screw cap. The sample was placed in an oven at 90 "C for 2 h and shaken thoroughly after the first hour. I t was then cooled to room temperature, and shaken vigorously for 1 min with 15 ml of ethyl ether. After centrifuging a t 2000 rpm for 10 min, the ether layer was withdrawn with a 30-ml syringe having a long 15-gauge needle and added to a 1 - 0 2 narrow-mouth bottle having a Teflon-lined screw cap. The aqueous layer remaining in the centrifuge tube was reextracted with a 10-ml portion of ether in a similar manner. The extracts were combined and dried over 1 g of anhydrous magnesium sulfate. T h e ether extract was decanted into a 25-ml evaporator tube containing an ebullator which had been fitted with a modified Snyder column. The contents were concentrated to 1 ml using a Kontes Tube Heater a t 75 "C. The ether was replaced with hexane by adding 2 ml of hexane and reconcentrating to 1 ml in a tube heater at 110 "C. After adding a small stainless steel pellet to promote boiling, the tip of the tube was placed in a 3-mm recess in an aluminum block insert in a tube heater a t 120 "C to cause refluxing. This was continued for at least a minute to rinse down the walls of the concentrator tube. Silica Gel Chromatographic Separation. The column used was a 0.9 X 25 cm Fischer and Porter glass column fitted with a fritted glass disc, a Teflon stopcock, a 100-ml reservoir, and Teflon seals. The column was packed by filling it with petroleum ether and slowly adding 10.0 g of silica gel (MCB No. SX-144-7, activated a t 150 "C overnight), while vibrating the column gently with an electric vibrator to remove bubbles. The stopcock was opened and 2-3 psi of nitrogen pressure was applied (using Teflon tubing and oil-free fittings) until the solvent level was about 1 mm above the silica gel. The column was never permitted to run dry; a thin layer of solvent was left on top of the silica gel at all times during its use. Any traces of hydrocarbon in the silica gel were removed by washing the column with 25 ml of methylene chloride followed by two 2-ml petroleum ether rinses and a 40-ml petroleum ether rinse prior to the addition of a tissue extract. The elution rate was 1-2 ml/min. T h e concentrated tissue extract was transferred to the column and allowed to move down the column using nitrogen pressure until the solvent level was about 1 mm above the silica gel. The
walls of the column were rinsed with petroleum ether and nitrogen pressure was applied until the solvent level was again about 1 mm above the silica gel. The column was then eluted with 25 ml of petroleum ether. The eluate was collected in a 25-ml concentrator tube. The total eluate a t this point, Fraction 1, contained all of the saturated hydrocarbons. After adding 50 ml of 20% methylene chloride in petroleum ether, v/v, to the reservoir, two 25-ml eluates were collected, Fraction 2 and Fraction 3. Fraction 2 contained most of the mono- and diaromatic hydrocarbons.as well as most of the biogenic olefinic hydrocarbons. Fraction 3 contained most of the triaromatic hydrocarbons. An internal standard (100 pl of heptane containing 100 pg of nctotriacontane, equivalent t o 10 pug per gram of tissue) was added to each fraction prior to concentration to 1-2 ml in a tube heater. Each sample was further concentrated to 100-200 pl by adding a small stainless steel pellet and placing the tip of the tube in a 3-mm recess in an aluminum block insert in a tube heater operated a t 130 'C. A disposable transfer pipet attached by a Teflon sleeve to a 1-ml tuberculin syringe was used to transfer each concentrate to a 200p1 sample vial. Each vial was capped with an aluminum crimp cap and a Teflon-lined septum that had been preextracted by a 16-h Soxhlet extraction with hexane. The samples were refrigerated until assayed by gas chromatography. Gas Chromatographic Analysis. A 3-pl sample of the concentrates obtained by silica gel chromatography was injected into a Varian Model 1840 Gas Chromatograph using a Hewlett-Packard Automatic Sampler. The gas chromatograph was equipped with dual flame ionization detectors and dual 10-ft X 2-mm i.d. glass columns packed with 10% SE-30, 3% OV-17, or 3% OV-1 on 100120 mesh Gas Chrom Q. The helium carrier gas flow rate was maintained a t 30 ml/min; the injector and detector temperatures were kept a t 250 and 325 "C, respectively; and the column temperature was programmed from 60 to 300 "C at 8"/min, then maintained a t 300 "C for 20 min. The detector signal was fed into an Infotronics CRS-204 electronic integrator connected to a teletype having a punched paper tape output. For determining individual gas 'chromatographic peaks, the integretor was operated in the integrate mode. The punched paper tape which contains peak area and retention time information was fed into a CDC 6400 computer. The computer was programmed to print out for each gas chromatographic peak its retention time (in minutes), the area relative to the n-dotriacontane internal standard, and the normalized area. T h e total of the areas of all of the peaks relative to the internal standard was also obtained. I t is recognized that there are some differences in flame ionization response factors for different hydrocarbons; however, the differences were not considered great enough to be of concern in this work. For determining the total hydrocarbon content represented by both the gas chromatographic peaks and the underlying envelope typical of weathered oils, the integrator was operated in the digitized mode with integration every 20 s. The computer in this case was programmed t o give the total area for each four-carbon range relative to the internal standard. A heptane blank was used as the base line that was subtracted from each sample chromatogram by the computer. Mass Spectrometric Analysis. A Finnigan Model 1015 quadupole mass spectrometer equipped with a chemical ionization source and interfaced with a Varian Model 1740 gas chromatograph was used to help identify some of the gas chromatographic peaks obtained. The gas chromatographic conditions were similar to those described above. Methane was used as the carrier and ionizing gas. Control and data acquisition were performed by a Finnigan System 150 interactive computer system. Unit mass scans were made over an m / e range of 100 to 350. A reconstructed gas chromatogram was obtained based on the total ion intensity of each individual spectrum. In printing out a mass spectrum from an individual spectrum number, the background represented by the base line or valley preceding the peak was usually subtracted out.
RESULTS AND DISCUSSION The recovery and the reproducibility of recovery achieva b l e b y the a b o v e p r o c e d u r e w a s d e t e r m i n e d b y s p i k i n g commercially frozen oyster tissue w i t h k n o w n amounts of a s t a n d a r d m i x t u r e c o n t a i n i n g 0.1% of e a c h of 1 2 h y d r o c a r b o n s i n heptane at levels of 0.1 to 10.0 wg/g of tissue. T h e results are given i n T a b l e I. All of the saturated h y d r o c a r ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
579
~~
Table I. Recovery of Hydrocarbons from Spiked Oyster Tissue Amount added, icg/g
10.0 10.0 10.0 Av Av dev Av % recovery
A m o u n t of given hydrocarbon0 f o u n d , y g l g b c14
Cl9
c24
TM B
T
N
MN
DMN
T MN
€3
F
P
11.60
11.70 10.00 7.32 7.45 7.38 8.09 8.52 9.70 8.23 11.11 9.63 8.00 9.72 10.02 4.45 4.74 5.40 6.65 8.76 9.70 8.15 9.61 10.72 7.82 9.61 10.20 7.60 7.82 8.23 9.43 11.17 11.73 9.60 10.27 11.15 9.14 9.78 10.64 6.46 6.67 7.00 8.06 9.48 10.38 8.66 10.99 9.83 0.71 1.63 0.15 1.34 1.29 1.07 0.94 1.12 0.90 0.18 0.63 0.32 91 106 81 98 65 67 70 95 104 87 98 110 1.11 2.0 1.66 2.06 1.03 1.12 1.35 2.06 1.40 1.63 1.53 1.78 2.09 1.63 2.0 1.51 2.01 1.36 1.55 1.40 1.99 1.67 1.90 1.80 1.96 2.12 1.63 2.0 2.02 1.81 2.00 2.09 1.90 1.95 2.16 2.27 1.75 1.37 1.98 Av 1.64 2.03 1.54 1.40 1.47 2.00 1.74 1.93 1.66 1.69 1.70 2.06 Av dev 0.01 0.02 0.27 0.28 0.04 0.30 0.28 0.22 0.28 0.11 0.22 0.06 Av % recovery 8 2 102 100 70 74 77 83 87 97 85 85 103 0.4 0.39 0.49 0.45 0.33 0.34 0.32 0.38 0.34 0.42 0.18 0.22 0.30 0.4 0.21 0.31 0.39 0.24 0.26 0.27 0.32 0.30 0.38 0.24 0.34 0.35 0.4 0.20 0.29 0.39 0.16 0.18 0.19 0.24 0.24 0.30 0.20 0.29 0.32 Av 0.27 0.36 0.24 0.41 0.31 0.26 0.26 0.29 0.37 0.21 0.28 0.32 Av dev 0.08 0.08 0.03 0.06 0.05 0.05 0.05 0.04 0.04 0.04 0.04 0.02 Av % recovery 68 102 90 60 65 78 73 93 65 53 70 80 0.1 0.05 0.09 0.04 0.09 0.05 0.05 0.05 0.04 0.08 0.04 0.09 0.10 0.1 0.08 0.10 0.10 0.03 0.03 0.04 0.05 0.04 0.06 0.09 0.03 0.08 0 ..1 0.05 0.09 0.09 0.04 0.05 0.08 0.05 0.05 0.08 0.07 0.05 0.07 Av 0.06 0.09 0.09 0.04 0.04 0.05 0.04 0.06 0.07 0.04 0.08 0.08 Av dev 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Av % recovery 60 90 90 40 50 40 60 40 70 40 80 80 Q C , , , C,,, and C,, = normal paraffins of given chain length; TMB = 1,2,3,5-tetramethylbenzene; T = tetralin; N = naphB = biphenyl; thalene; MN = 2-methylnaphthalene; DMN = 2,3-dimethylnaphthalene;TMN = 2,3,6-trimethylnaphthalene; F = fluorene; P = phenanthrene. b Tissue weight is wet basis. -.
-
Table 11. Recovery of Hydrocarbons from Contaminated Clam Tissue Tissue D Fa
2 2 2
A m o u n t of given hydrocarbonb f o u n d , pg/gC Cl,
c14
0.52
0.95
0.66
1.06
C16
‘19
N
2-MN
DMN
TMN
B
F
P
MP
1.01 0.63 4.61 5.90 3.26 1.32 0.22d 0.46d 0.88d 0.56 0.01 0.56 6.52d 8.26d 4.21d 1.54d 0.18 0.28 0.85 0.58d 0.70d 1.21d 1.21d 0.65d 2.83 3.65 2.32 1.00 0.13 0.32 0.80 0.54 Av 0.63 1.07 1.08 0.61 4.65 5.94 3.26 1.29 0.18 0.35 0.84 0.56 Av dev 0.07 0.09 0.09 0.04 1.24 1.55 0.63 0.19 0.03 0.07 0.03 0.01 Av % recovery 90 89 89 94 71 72 78 84 82 76 95 97 10 0.11 0.27 0.26 0.14 2.05 1.82 0.73 0.24 0.03 0.09 0.12 0.15 10 0.10 0.27 0.27 0.13 0.75 0.91 0.45 0.17 0.01 0.04 0.08 0.12 10 0.09 0.25 0.27 0.17 2.18 1.95 0.85 0.26 0.04 0.12 0.16 0.14 Av 0.10 0.26 0.27 0.15 1.66 1.56 0.68 0.22 0.03 0.08 0.12 0.14 Av dev 0.01 0.01 0.01 0.02 0.61 0.43 0.15 0.03 0.01 0.03 0.03 0.01 Av % recovery 71 107 111 115 127 94 81 71 68 88 68 121 25 0.01 0.08 0.10 0.07 0.34 0.45 0.23 0.05 ... ... ... ... 25 0.02 0.06 0.11 0.06 0.23 0.31 0.18 0.07 25 0.01 0.06 0.08 0.06 0.41 0.59 0:29 0.11 ... ... Av 0.01 0.07 0.10 0.06 0.33 0.45 0.23 0.06 ... ... ... ... Av dev ... 0.01 0.01 0.01 0.06 0.09 0.04 ... ... ... ... ... Av % recovery 18 72 103 115 63 68 68 49 ... ... ... 100 ... ... ... ... 0.03 0.04 0.02 0.01 ... ... ... ... 100 ... ... ... ... 0.05 0.09 0.05 0.01 ... ... ... ... 100 0.06 0.08 0.04 0.01 Av ... ... ... ... 0.05 0.07 0.04 0.01 ... ... ... ... Av dev ... ... ... ... 0.01 0.02 0.01 0.00 ... ... ... ... Av % recovery ... ... ... ... 38 42 48 32 ... ... ... ... a The contaminated clam tissue, from clams exposed t o fuel oil in water, was diluted with commercial frozen oyster tissue to give the dilution factor ( D F ) indicated. b C,,, C,,, C , , , and C , , = normal paraffins of given chain length; N = naphthalene; 2-MN = 2-methylnaphthalene; DMN = a dimethylnaphthalene; TMN = a trimethylnaphthalene; B = biphenyl; F = fluorene; P = phenanthrene; MP = a methylphenanthrene. C Tissue weight is wet basis. d This value was used as the basis for calculating percent recovery.
bons appeared in Fraction 1. Most of the phenanthrene and fluorene, about half of the biphenyl, and traces of the naphthalene compounds appeared in Fraction 3. T h e rest of the aromatic compounds appeared in Fraction 2. T h e 580
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
oyster tissue blanks contained traces of a mineral oil b u t not enough to interfere with the analyses. T h e recoveries averaged at least 80% in the majority of cases with a n average deviation of less than 20%. Even at the 0.1 pg/g level,
a = Biphenyl = 0 22 p g / g 5 = Methylbiphenyl = 0 2 5 p g / g c : Fluorene = 0 4 6 p g / g d = Methylfluorene = 0 54 p g / g e Phenanthrene = 0 80 p g / g f = Methylphenanthrene = 0 56 p g / g g = C2- Phenanthrene = 0 I 3 p g / g
e
I 5 16 I 7 l 8 1
I
5
4
1
1
1
1
I I IO 15 Retention Time, minutes
I
20
Figure 1. Gas chromatogram of Fraction 1 from clams found to contain normal paraffins at the levels indicated
a= D= c = d= e =
Naphthalene = 0 54 p g / g 2-Methylnaphthalene = 0 8 4 p g / g I-Methylnaphthalene = 0 49 pq/g C2-Naphtnalene = 0 5 5 p g / g C3-Nophtnolena = 0 17 ,+q/g
1
1
I
IO 15 Retention Time,minutes
5
25
1
1
25
20
Figure 3. Gas chromatogram of Fraction 3 from clams found to contain biphenyls, fluorenes, and phenanthrenes at the levels indicated
I
: 2:
45
6:
8i
CC
20
43 B,:
193Tc
201
26C
32C
35:
151:
'43
483
S p e c - r u n Ndnoer
Figure 4. Reconstructed gas chromatogram from a GC-MS run of a Fraction 2 obtained from clams exposed to a No. 2 fuel oil
Table 111. Mass Spectral Identification of Gas Chromatographic Peaks from a Fraction 2
b
Major c o m p o n e n t Spectrum NO.^
0
5
IO 15 Retention Time, minutes
20
25
Figure 2. Gas chromatogram of Fraction 2 from clams found to contain naphthalenes at the levels indicated
the recoveries were usually above 50%. T h e recoveries and reproducibilities were considerably better for the saturated hydrocarbons than for the aromatics. Although the above spiking procedure is commonly used, it does not involve the efficiency of 'extracting hydrocarbons out of cellular and subcellular particles. In an effort to study the latter problem, a second validation study was carried out using clams t h a t had been exposed to No. 2 fuel oil. T h e fuel oil was taken up by the clams and incorporated into the tissue. T h e homogenized contaminated clam tissue was diluted with various amounts of homogenized oyster tissue. The highest value obtained for each hydrocarbon in the least diluted sample was used as the basis of calculating the percent recovery for the other samples. The results are given in Table 11. T h e objective of this validation study was to determine whether the extraction is as efficient a t low levels as it is a t high levels when oil is actually incorporated into tissue.
46 60 72 88 95 122 133 141 153 159 169 184 188 201 237 246 257 271 283 313 329 347 362 373 385 402 420 457 467 0 See Figure 4.
M o l wt
134 134 132 128 146 146 146 145 146 142 142 160 160 160 156 156 156 156 156 168 170 170 170 170
168 184 184 182 182
Tentative identification
C,-Benzene C,-Benzene Tetralin Naphthalene Methyltetralin M 2 thy 1t etr ali n Methyltetralin Methyltetralin Methyltetralin Methylnaphthalene Methylnaphthalene C,-Tetralin C,-Tetralin C,-Tetralin C, -Naphthalene C,-Naphthalene C,-Naphthalene C, -Naphthalene C,-Naphthalene Methylbiphenyl C,-Naphthalene C;Naphthalene C;Naphthalene C,-Naphthalene Methylbiphenyl C;Naphthalene C,-Naphthalene C,-Biphenyl C,-Biphenyl
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
581
Table IV. Mass Spectral Identification of Gas Chromatographic Peaks from a Fraction 3 Major c o m p o n e n t
Spectrum N0.a
a
Mol w t
92 138 148 175 184 192 198 222 231 24 7 265 273 280 288 29 5 327 333 See Figure 5.
Tentative identification
Biphenyl Methylbiphenyl Methylbiphenyl Fluorene Meth ylfluorene C,-Biphenyl (&-Biphenyl Methylfluorene Dibenzothiophene Phenanthrene C,-Fluorene Methyldibenzothiophene Methyldibenzothiophene Methylphenanthrene Methy lphenanthrene C,-Phenanthrene C,-Phenanthrene
154 168 168 166 180 182 182 180 184 178 194 198 198 192 192 206 206
", 60
Z
t
M/E
Flgure 6. Chemical ionization (CH4)mass spectrum of a methylphenanthrene present in an extract of clam tissue. (This is spectrum No. 295 from Figure 5)
'4 N = . 2 0 4
C2,
iEXSEUE
LM.]+ i 90
I10
130
150
I70
150
210
230
253
270
250
310
330
M/ E
Figure 7. Chemical ionization (CHI) mass spectrum of a &-hexaene present in an extract of abalone viscera Spectruw Nunber
Figure 5. Reconstructed gas chromatogram from a GC-MS run of a Fraction 3 obtained from clams exposed to a No. 2 fuel oil
The data show t h a t the average recoveries were usually greater than 70% and that the extraction efficiency did not drop off appreciably until the hydrocarbon levels were below 0.1 wg/g. The completeness of the extraction procedure was also checked by reprocessing the aqueous phase. Additional heating or additional ether extractions increased the recoveries by less than one percent. Figures 1-3 show examples of gas chromatograms, using an OV-17 GC column, of the three different fractions obined by the silica gel chromatography. These fractions were isolated from clams that had been exposed to No. 2 fuel oil and had retained low levels of identifiable hydrocarbons. T h e OV-17 column gave good separation of C ~ and T pristane but unlike the OV-1 column did not resolve CIS and pristane. OV-17 was selected primarily because it gave somewhat better resolution of aromatic hydrocarbons than did OV-1. The saturated hydrocarbons (in Fraction 1)were identified by retention times. The aromatic hydrocarbons (in Fractions 2 and 3) were identified by retention times and by combined gas chromatography-chemical ionization mass spectrometry (GC-CIMS) using methane as the carrier-ionizing gas. Aromatic hydrocarbons show intense [M H ] + ions, weak [M CzHj]+ adduct ions, and very little fragmentation (7). Reconstructed gas chromatograms of the two aromatic fractions from an extract of clams that had been exposed to No. 2 fuel oil are shown in Figure 4 and 5 . The identification of the major peaks, based on their mass spectra and expected retention times, are given in Tables I11 and IV. A representative CI mass spectrum of an aromatic hydrocarbon, that of a methylphenanthrene found in a tissue extract, is shown in Figure 6. Various biogenic olefinic hydrocarbons are frequently present in marine samples (8, 9). In our analysis scheme, olefinic hydrocarbons showed up with the aromatic hydro-
+
582
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
carbons in Fractions 2 and/or 3 depending upon the amount of unsaturation present. Abalone, which consume considerable quantities of algae, were found to contain relatively large amounts of olefinic hydrocarbons in their gut. Olefinic hydrocarbons yield weak [M H]+ ions and a preponderance of fragmentation when studied by CI mass spectrometry(l0). A representative mass spectrum of an olefinic hydrocarbon, that of a C21-hexaene found in abalone, is shown in Figure 1. Although the procedure described here can detect individual hydrocarbons in marine organisms a t levels down to 0.01 Fg/g in many cases, the presence of interfering biogenic hydrocarbons often makes it impossible to detect petroleum contamination a t total oil levels as low as a few wg/g. The presence of petroleum is indicated by the nature of total gas chromatographic fingerprint of a given fraction. Fraction 1 from a crude oil typically shows a series of dominant n-paraffin peaks with no odd or even preference, a series of smaller isoparaffin peaks, and an underlying envelope of numerous cycloparaffin components. Unfortunately, Fraction 1 from biogenic material may also contain a series of n-paraffins. Although many of the odd-carbon nparaffins, especially CIS, C17, and those in the C25 to C33 range, usually predominate in biogenic material, it is not possible to determine whether a small portion of the paraffins represents petroleum contamination. The detection of petroleum n-paraffins is also complicated by the fact that in some oils, especially in highly weathered oils, the n-paraffins are present in such small amounts that they are obscured by the underlying envelope. Fraction 2 from a crude oil typically shows peaks of some individual components, mainly alkylbenzenes and alkylnaphthalenes, and an underlying envelope of numerous alkyl fused ring compounds in which only one or two of the rings is aromatic. Unlike the n-paraffins, none of these aromatic hydrocarbons of petroleum are formed biogenically to any significant extent. The aromatic hydrocarbons are, therefore, good indicators of petroleum. Unfortunately, biogenic olefins also appear in Fraction 2 and, in some cases, are so numerous that trace amounts of aromatic hydrocarbons can
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not be determined in their presence. This problem may be largely avoided by hydrogenation of the olefins as described by Giger and Blumer (11).The interference by biogenic olefins is much less severe for Fraction 3 which contains the phenanthrenes, fluorenes, biphenyls, and dibenzothiophenes of petroleum. Although these latter compounds are good indicators of petroleum, they are present only a t relatively low levels. Further insight into the complexity of petroleum is provided by the work of Coleman and co-workers (12). Because of the above limitations, the procedure described here, like previously reported methods, is not suitable for determining ambient levels of petroleum components in base-line samples from relatively pristine areas. However, the procedure is very useful in determining levels of petroleum in animals exposed to oil a t toxic ievels in the laboratory or exposed to petroleum in the field a t levels significantly above the levels in relatively pristine areas. For example, mussels collected from Coal Oil Point, a natural oil seep area near Santa Barbara, Calif., contain very significant amounts of weathered petroleum detectable by this procedure. We have used the overall extraction-gas chromatographic procedure on several hundred tissue samples including mussels, lobsters, clams, oysters, fish, shrimp, abalone, crabs, starfish, sea urchins, and bloodworms and found it to be quite convenient. Mass spectrometry was useful for the further characterization of representative samples.
ACKNOWLEDGMENT Mass spectrometric analyses were performed by R. Foltz and P. Clarke.
LITERATURE CITED (1) D. B. Boylan and E. W. Tripp, Nature (London),230, 44 (1971). (2) J. W. Anderson, J. M. Neff, E. A. Cox, H. E. Tatem. and G. M. Hightower, Mar. Biol., 27, 75 (1974). (3) R. C. Clark, Jr., and M . Blumer, Limnol. Oceanogr., 12, 79 (1967). (4) M. Blumer, G. Souza, and J. Sass, Mar. Biol., 5 , 195 (1970). (5) R. C. Clark, Jr., and J. S.Finley, Proceedings of Joint Conference on Prevention and Control of Oil Spills, March 13-15, 1973, p 161. (6) J. W. Blaylock, P. W. O'Keefe, J. N. Roehm, and R . E. Wilding, Ref. 5, p 173. (7) M. S.E. Munson and F. H. Field, J. Am. Chem. SOC.,89, 1047 (1967). (8) M. Blumer and D. W. Thomas, Science, 148, 370 (1965). (9) M. Blumer, R. R . L. Guillard, and T. Chase, Mar. Biol., 8, 183 (1971). (10) F. H. Field, J. Am. Chem. SOC.,90, 5649 (1968). (11) W. Giger and M. Blumer, Anal. Chem., 46, 1663 (1974). (12) H. J. Coleman, J. E. Dooley, D. E. Hirsch, and C. J. Thompson, Anal. Chem., 45, 1724 (1973).
RECEIVEDfor review January 15, 1975. Accepted December 1, 1975. This work was supported by the American Petroleum Institute under Contract No. OS-20-G. Tissue samples were submitted by Jack Anderson of Texas A&M University under API Contract No. OS-20-C and Dale Straughan of the University of Southern California under API Contract No. OS-20-D.
Pyridine Catalyzed Reaction of N-Nitrosodi methylamine with Heptaf Iuorobutyr ic Anhydride Terry A. Gough," Martin A. Pringuer, Keith Sugden, and Kenneth S. Webb Laboratory of the Government Chemist, Cornwall House, Stamford Street, London SE 1 9N0, England
Colin F. Simpson University of Sussex, Brighton BN 1 9QJ, Sussex, England
The pyridine catalyzed reaction of N-nitrosodimethylamine with heptafluorobutyric anhydride has been studied and the products have been separated by gas chromatography. The constituents of the reaction mixture were identified by combined gas chromatography and mass spectrometry, and a volatile N-nitrosodimethylamine derivative was detected. Electron impact mass spectra indicated a molecular weight of 268 and high resolution measurements gave an empirical formula of C6H3N202F7.Supporting evidence was obtained from field desorption spectra and a microwave plasma detector. The structure of this compound was deduced from the mass spectral evidence, supported by nuclear magnetic resonance spectrometry. The compound has a retention index of 790 at 135 O C on a silicone (OV1) stationary phase in contrast to a value of 1335 for the derivative previously reported.
T h e preparation of derivatives which can be separated by gas chromatography and detected by electron capture is frequently employed for detecting trace constituents. The use of heptafluorobutyric anhydride (HFBA) to form elec-
tron capturing derivatives of volatile N-nitrosamines has been described previously ( 1 ) . The nature of a series of dialkyl and heterocyclic nitrosamine derivatives has been studied by gas-liquid chromatography and mass spectrometry (2). In this earlier work, it was reported that two HFBA-nitrosodimethylamine derivatives were formed, with Kovats retention indices of 790 and 1335, on a silicone (OV1) stationary phase. Only the derivative having the longer retention time was examined and a proposed structure presented. The aim of this further work is to study the nature of the other reaction product and confirm t h a t it is also derived from the reaction between N-nitrosodimethylamine and HFBA.
EXPERIMENTAL Apparatus. A Pye 104 chromatograph was fitted with a 6 m X 4 mm i.d. glass column containing 5% Carbowax 20M on 80-100
mesh Chromosorb G acid washed and DCMS treated. Detection was by flame ionization and microwave plasma (Applied Research Laboratories Ltd, Model 850).Electron impact spectra and accurate mass measurements were obtained using an AEI Model MS 902 double focusing mass spectrometer interfaced to the chromatograph via a silicone membrane separator. Field desorption spectra were obtained on a Varian MAT CH5 mass spectrometer using ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
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