(39) Lloyd, A. C., h t . J . Chem. Kinet., VI, 169 (1974). (40) Stedman, D. H., Wu, C. H., Niki, H., J . Phys. Chem., 77, 2511 (1973). (41) McAfee, J . M , Pitts, J. N., Jr., Winer, A. M., “In-Situ Long-Path Infrared Spectroscopy of Photochemical Air Pollutants in an Environmental Chamber,” presented Pacific Conference on Chemistry and Spectroscopy, San Francisco, Calif., October 16-18. 1974. (42) Calvert, .J.’G., Kerr, J . A,, Demerjian, K . L., McQuigg, R. D., Science, 175,751 (1972). (43) Greiner, N. R , J . Chem. Phys., 53,1070 (1970). (44) Gorse, R. A., Volman, D. H . J . Photochem., 3,115 (1974). (45) Stuhl. F., Z. Naturforsch, 28A, 1383 (1974). (46) Grovenstein, E., JI., Mosher, A. J., J Amer. Chem. SOC.,92, 3810 (1970). (47) Boocock, G., Cvetanovic, R. J., Can. J Chem., 39, 2436 (1961). (48) Jones, G. R. H., Cvetanovic, R. J., ibid., p 2444. (49) Mani, I., Sauer, M . C., Jr., Aduan. Chem. Ser., 82, 142 (1968).
(50) Bonanno, R. A., Kim, P., Lee, J. H., Timmons, R. B., J. Chem. Phys., 57, 1377 (1972). (51) Stuhl. F.. Ber. Bunseges. Phvsilz. Chem.. 77,674 (1973). . , (52) Bradley,’J. N., Ha&, W., Hoyermann’, K.., Wagner, H. G., J.. Chem. Soc., Farad. Trans. I., 69,1889 (1973). (53) Johndon, H., in “Project Clean Air Task Force Assessments,” Vol. 4, Task Force 7, University of California, 1970. (54) Smith, I. W. M., Zellner, R., J . Chem. SOC.,Farad. Trans. II, 69, 1617 (1973).
Received for revieu: September 9, 1974. Accepted October 31, 1974. This work was supported in part by the California Air Resources Board (Contract No. 3-017),.the Environmental Protection Agency (Grant No. R-800649) and the National Science Foundation-RANN (Grant No. GI-41051). The contents d o not necessarily reflect the views and policies of the Environmental Protection Agency, the California Air Resources Board or the National Science Foundation-RANN, nor does mention o f trade names or commercial products constitute endorsement or recommendation for use.
Quantitation of Environmental Hydrocarbonsby Thin-Layer Chromatography Gravimetry/Densitometry Comparison Leon Hunter Naval Biomedical Research Laboratory, School of Public Health, University of California at Berkeley, Naval Supply Center, Oakland, Calif. 94625
The application of thin-layer chromatography to the detection and analysis of trace amounts of hydrocarbons has been investigated. Samples can be separated into saturates (alkanes) and unsaturates (mainly aromatics) at sensitivities to 0.5 pg. Quantitations by a densitometric method and by gravimetric recovery of the spots were compared. Comparable accuracy is possible, provided authentic calibration samples are available. In the absence of the latter, quantitation by densitometry is considerably less accurate than gravimetry due to variable hydrocarbon response to the visualization method.
Monitoring and control of environmental pollutants require their detection, identification, and quantitation. Frequently difficulties can‘ arise, for example, from the low levels being measured (especially in the presence of interfering compounds), from the need to identify the more hazardous individual components in a complex pollutant mixture, and from the losses or inadvertent contamination that is difficult to avoid when recovering small amounts of pollutants from various environmental media. These problems are most evident in the area of hydrocarbon pollution. Such pollution, while resulting largely from acute or chronic oil spills, may also come from other sources, such as road oil, engine lubricants, engine exhausts, machine lubricants, and cutting fluids. The wide variety of methods proposed to meet the problems of hydrocarbon pollution monitoring is perhaps indicative of the absence of any widely accepted general technique. Most of the analytical procedures reported in the literature involve some form of chromatography and/or spectrometry. Gas chromatography has been widely used, especially in the analysis of volatile air pollutant hydro-
carbons but also in the analysis of hydrocarbons in marine organisms (1-3) and in seawater ( 4 ) . The method is limited by the difficulty of handling high-boiling, high-molecularweight hydrocarbons that are a major component in many spills. Liquid chromatography has frequently been used in preliminary recovery and cleanup procedures ( 5 ) . Its use in the final stages of hydrocarbon separation has been limited by poor resolution compared with other chromatographic techniques. Thin-layer chromatography, while lacking the very high resolution of gas chromatography, is not hampered by low-volatility considerations and has the additional merit of requiring inexpensive equipment. Many of the prior applications of this technique have been qualitative, as in the analysis of gasoline/lube oil mixtures in carp (6), or have involved the separation and recovery of specific hydrocarbons (7), generally polyaromatics, and often for quantitative determination by other procedures (8). Spectrometric methods have found wide qualitative and quantitative application alone or as a supplement to chromatographic separations. Fluorescence spectra have proved useful in the analysis of various types of crude and refined oils (9, IO), but in general are limited to aromatics as the only fluorescing species. Infrared absorption has been used to fingerprint and differentiate oil types (11) and to provide an estimate of total oil levels on the basis of certain common absorption bands (12). Mass spectrometry is useful in the identification of specific hydrocarbons as in the analysis for aromatics in mullet (2, 3 ) . A major requirement in oil-pollution monitoring is the determination of total hydrocarbon levels, preferably with breakdown by type (alkanes, aromatics, olefins). In any individual spill this requirement may be met by the selection of some marker compound in a reference sample of the oil. However, this approach must be applied with increasing caution as the postspill period is extended and as inevitable weathering changes take place. Furthermore, Volume 9, Number 3, March 1975
241
the technique is completely inapplicable where the effects of multisource chronic oil pollution are under investigation, since reference samples are generally not available, Such conditions exist in many areas of coastal waters subject to repeated or continuing contact with commercial and recreational activities. A similar lack of reference samples is also probable where baseline studies are being made in threatened areas with low but increasing pollution levels. The Naval Biomedical Research Laboratory recently undertook a project to investigate the levels of chronic oil pollution and its effects on the marine food chain in San Francisco Bay. After reviewing the current methods we concluded that thin-layer chromatography came closest to meeting the requirements for a rapid, inexpensive procedure for the quick comparison of polluted and unpolluted samples. The possibility of adapting TLC for quantitative measurements was sufficiently promising to warrant investigation. The results are reported in the present paper. Experimental
Silica gel was used as an adsorbent in all the work to be described. Both commercially and laboratory-prepared plates were used. Most commercial plates were unsuitable for sulfuric acid visualization owing to the presence of small amounts of organic binder material that caused an overall darkening during the relatively high-temperature visualization procedure. An exception was the silica gel G Uniplate (Analchem, Inc.) which contained no organic binder and gave very uniform results. Plates (20 x . 2 0 cm and 5 x 20 cm) were prepared in the laboratory a t a nominal 250 p thickness from silica gel G (Brinkmann Instruments, Inc.) using a Desaga/Brinkmann Adjustable Applicator. Plates were dried and activated a t 110°C. Immediately prior to use, the plates were cleaned by migration with chloroform or, in the case of plates, intended for gravimetric estimates, with 60/40 hexanes/acetone. Chromatograms were developed with hexanes solvent (Mallinckrodt AR), used without further purification. The major solvent contaminants were traces of polar compounds retained tightly at the bottom of the plate and, if necessary, could be readily removed by scraping off a 1.5cm band of silica gel prior to visualization. For the gravimetric procedure the silica gel was scored to provide strips, 1 cm wide, for each sample, run in duplicate. The hydrocarbons (0-100 pg) were recovered from the appropriate areas (delineated by charring identical samples on a separate plate) by scraping off the silica gel, packing it in a miniature column made from a Pasteur pipet plugged with glass wool, and eluting with a mixture (60/40v/v) of distilled hexanes (Mallinckrodt AR) and distilled reagent grade acetone. The eluent, collected in two 0.2-ml portions, was placed in a small (1-cm diam) preweighed aluminum cup and allowed to evaporate a t room temperature. The hydrocarbon residue was weighed on a microbalance (Cahn Gram Electrobalance) with a precision of 1 pg or better. All equipment used in this procedure was prerinsed with the hexane/acetone elution solvent to minimize contamination. As an additional check, blank determinations were run on identical silica gel strips and the hydrocarbon values corrected appropriately (blanks generally in the 0-2-pg range). In general the second 0.2-ml eluent contained 10% of the amount of nonvolatiles in the first, indicating essentially complete recovery of all recoverable material in 0.4-ml eluent. For the densitometric determination, charring was conducted a t a number of temperatures. To minimize problems of spot migration and diffusion, due largely to temperature variations during the charring process, the plates 242
Environmental Science & Technology
were heated in a disposable box placed on a large (21 x 21 cm) hotplate. The box (22 X 22 X 10 cm) was constructed from heavy-gage aluminum foil and was open on one side (for insertion of the clamped TLC plate). The hydrocarbon spots were scanned with a densitometer (Photovolt Corp.) along the long axis of the plate. The labor involved in scanning was greatly minimized by recording the densitometer output, suitably modified by a logarithmic amplifier to give a linear logarithmic response. The areas of the peaks of the resulting optical density vs. distance plots were measured by planimeter. Hydrocarbons used for test purposes were generally a t least 95% pure. Many were obtained from hydrocarbon sample kits purchased from Chem Service, Inc. (West Chester, Pa.). Results and Discussion
The basis of a TLC procedure for determining hydrocarbons is the ability to (a) separate all hydrocarbons from all other compounds and (b) obtain some separation by type among the hydrocarbons themselves. It is evident from the references previously quoted and from our own observations that hydrocarbons may be readily separated from other materials by taking advantage of their generally low polarity and by using nonpolar 1. (
ALKANES
OLEF INS
0.E
0.6 Rf
0.4
0. 2
Figure 1. Thin-layer
chromatography of hydrocarbons ( 7 3 )
Loci of specific compounds marked by X
1 om n-Octadecane 0 Phytane
5-
4VI
L
40
80
1M
160
SAMPLE SIZE, ug
Figure 2.
Effect of temperature on hydrocarbon response curve
migrating solvents such as hexane. Since essentially all other compounds are more polar than hydrocarbons, they are retained under these conditions a t the starting point on a TLC plate while the hydrocarbons are spread out along the plate. The fact that hydrocarbons can be spread out on a TLC plate raises the possibility for separation by type. Kirchner and Miller (13) examined the behavior of a variety of hydrocarbons on silica gel using hexane as the developing solvent. When these compounds are classified by type and their ‘I’LC positions plotted by Rf value, they fall into well-defined groups as shown in Figure 1. Clearly, it would be possible to identify in an unknown hydrocarbon mixture the presence of aromatics, olefins and, presumably, alkanes (which would be expected to migrate close to the hexane solvent front). As a further check on hydrocarbon behavior under our TLC conditions, a brief survey of a variety of aromatic and alkane hydrocarbons was carried out with the results shown in Table I. While there appears to be a general shift to lower Rf values relative to Kirchner, there is a clear-cut separation into two groups with arornatics ranging from Rf 0.33-0.49 and saturates from Rf 0.78-0.86. These results confirm that TLC can readily establish the presence or absence of these two major types of hydrocarbons in a sample.
~
Table I. Approximate Rf Values for Hydrocarbons
Phenathrene Anthracene 2,3-Dimethylnaphthalene 1,2,3-Trimethylbenzene Naphthalene 1-Methylnaphthalene 2-Methyl n a p h t ha le ne n-Dodecane
0.33 0.33 0.39 0.44 0.45 0.47 0.49 0.78
Phytane n-Tetradecane n-Octadecane n-Docosane n-Triacontane n-Hexacosane mHexadecane
0.79 0.82 0.82 0.84 0.84 0.86 0.86
Table II. Gravimetric Recovery of TLC Hydrocarbons Spotted, W9
Aromatic Mix I* Aromatic M i x (le n-Docosa n e Alkane Mix I d Hydrocarbon Mixe a
40.4 40.4 20.1 44.5 44.5 26.2 26.2 43.6 43.6
Recovereda
-
!4
%
33.4 30.2
83‘L 79 751 95
19.1 40.9 38.5
23.1 23.7 39.6 42.7
\;
89
):
89
91’1 95 981
From c o m b i n e d e l u e n t (0.4 ml). * A o o r o x i m a t e l v e a u a l a m o u n t s of
Gravimetric Analysis
The determination of hydrocarbon levels by recovery from a silica gel separation and actual weighing is the nearest approach to an absolute method, provided recovery approaches :loo%. In a series of tests designed to investigate this point, samples containing known amounts of alkanes or aromatics were chromatographed and recovered. The results are shown in Table 11. It will be seen that recoveries of 90% or better can generally be achieved for both aromatics and alkanes. The reproducibility of duplicate determinations is also very satisfactory (within 3-5% of the averaged values). It is concluded that the quantitative determination of trace hydrocarbons by gravimetry/TLC is capable of giving results correct to within this reproducibility. The utility of the method was demonstrated by the analysis of hydrocarbons extracted from polluted mussels taken from San Francisco Bay. The hydrocarbons were readily separated into an alkane spot (recovery range Rf 0.65-0.9) and an aromatic streak (recovery range Rf 0.10.5) in the following amounts (corrected on basis of above recoveries): Alkanes: kg recovered, 20.1; ppm on wet tissue, 52 Aromatics: pg recovered, 13.3; ppm on wet tissue, 34 Total: kg recovered, 33.4; ppm on wet tissue, 86 Densitometric Ari alysis
Upon first consideration the densitometric determination of hydrocarbon spots would appear to be a much more convenient, considerably less tedious and exacting procedure than gravimetry. For example, some of the operations such as spot visualization can be carried out simultaneously on all the spots on one plate. Scanning is a much quicker procedure than the careful repeated weighings necessary ,at microgram levels to ensure reliable gravimetric results. For these reasons it was hoped to establish the accuracy of the method in determining single hydrocarbons and then perhaps extend it to the much more useful measurement of mixed hydrocarbons. T o establish minimum conditions necessary for satisfactory quantification, a number of variables were examined, including visualization reagent and temperature, carbon number, and hyldrocarbon type (e.g., branched or linear
alkanes, linear or nonlinear fused aromatic rings). Effect of Temperature. One of the requirements established early in the investigation was the need for a controlled temperature environment during the charring procedure. The effect of temperature on the area vs. sample weight curve for n-octadecane is shown in Figure 2 . Subsequent work suggested that the reduced response a t the higher temperature is largely due to increased volatilization prior to charring. Close temperature control is difficult to maintain in a system where considerable air movement is necessary to vent the sulfuric acid fumes. While the aluminum foil “oven” described earlier was of considerable help in stabilizing the temperature, it was still desirable in quantitative work to minimjze the effect of temperature and other variables by simultaneously running all the samples required for one determination on a single TLC plate. Quantitative Determination of Single Hydrocarbons
With only the few conditions and controls mentioned above, it is readily possible to measure single hydrocarbons in the range 1-100 gg. If we use n-octadecane and phenanthrene as aliphatic and aromatic examples, respectively, the method involves running duplicate spots with known amounts of hydrocarbon a t two levels in the 1-100 pg range, visualizing with H*S04/Se a t 200”C, and using the resulting calibration curve (drawn through spots and origin) to calculate the amount of hydrocarbon in the unknown samples, run preferably in duplicate, on the same plate. The results of typical test runs (Table 111) show that accurate measurements can be made on individual hydrocarbons in the 1-50 ,ug range. Even in the case of phenanthrene which has a poor charring response curve (see later section) average values were only in error by 1wo. Quantitative Determination of Hydrocarbon Types
TLC for the analysis of single hydrocarbons is mainly of interest for high molecular weight, essentially nonvolatile Volume 9, Number 3, March 1975
243
Table 111. TLC Analysis of Single Hydrocarbons by Densitometry Found
Sample
Actual,
n-Octadecane
Single determination, pg
Average, pg
Error, %
15
0 15.5 43.0)
45
}
45.3
0.7
’O’g\ 12.0)
11.5
11.5
47.5)
Phenanthrene 13
point (195°C) close to the 200°C visualization temperature. The problem is aggravated by the relatively high resistance to charring shown by aliphatic hydrocarbons. Any attempt to reduce volatility by lowering the temperature had a proportionally greater effect on slowing the charring reaction. Effect of Visualization Agent. A variety of charring agents was tried including HzSO4, H&04/Se, and oxidizing systems such as H2S04/HN03, H ~ S 0 4 / K M n 0 4 ,and chromic acid. The oxidizing mixtures, used indiscriminately, were generally unsuitable as they tended to oxidize the hydrocarbon spot completely, especially when the latter was present in small amounts. The effect of adding limited amounts of an oxidizing agent such as potassium dichromate to the sulfuric acid was examined. By oxidizing a limited fraction of the hydrocarbon a t a lower temperature (140°C),it was hoped to eliminate or at least reduce the carbon number effect by “freezing” the samples on the TLC plate as carboxylic compounds with greatly reduced volatility. The latter could thus be exposed to the charring action of the sulfuric acid for a much longer time. Approximate calculations indicated that a spray containing 0.25% w/v potassium
2
Ln
!= z 3
5 z1
M
40
BO SAMPLE SIZE,119
i0
Figure 3. Response curve for C I 4 ,C Z 2 ,and C30 alkanes Visualization agent, H2S04/Se *Measured on different scale frcm Figure 2
compounds difficult to detect by gas chromatography. Of much greater usefulness is its ability to analyze mixed hydrocarbons and give an integrated measure by type of the total amount, including high boilers, without the need for authentic comparison samples. The gravimetric procedure described above is one example of this approach. The feasibility of a similarly general densitometric method is predicated on eliciting approximately equal spot responses from all the members of each hydrocarbon t: ?e group. Some of the factors, pro and con, involved are ;cussed below. Effect of Chain Branching. Comparison was made of the linear n-octadecane with the branched C20 hydrocarbon, phytane (2,6,10,14-tetramethylhexadecane).Figure 2 shows essentially similar response to the visualization procedure. This is a significant observation in view of the likelihood of mixed linear/branched populations in alkane mixtures typically found in oil spills. Effect of Carbon Number. The response curves for three representative hydrocarbons, covering the range C14 to (230, are shown in Figure 3. The substantial variation with carbon number appears to be largely due to the different volatilities of the test compounds. This is supported by the converging plots in Figure 4.These predict zero response (charring) from undecane (Cll) which has a boiling
io
10 $0 CARBON NUMBER Figure 4. Effect of carbon number on TLC response at different
sample levels Visualization agent, HzS04/Se
1
t
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Environmental Science & Technology
2 0 4 0 60 SAMPLE SIZE,ccg
80
Figure 5. Response curves for C14,Cz2, and C30alkanes Visualization agent, H2S04/K2Cr207
Table
IV. TLC Analysis of a
Hydrocarbon Mixture
Total hydrocarbon Actual, pg
8.7
Found, pg
''7
Error,
Average, pg
8.0
%
8
8.3 38.3
44
34\ 54 1
Table
15
V. Determination of Mixed Aromatics Amount determined
TLC method
Densitometric
Single deterAmount mination, applied, pg pg Average, fig
2.5
2.51
Error,
%
2.8
12
31.9
21
3.1
Gravimetric
40.4
33.41 } 30.21
considerably reduced and is obviously dependent on the proper selection of a calibration hydrocarbon corresponding most closely to the averaged response of the particular hydrocarbon mixture being analyzed. (Whether the aromatic responses of various pollutant hydrocarbon mixtures are sufficiently variable to cause significant differences is not known a t this time.) If the above test mixture of four hydrocarbons is considered representative, then it would appear that the higher charring response of the pyrene, anthracene, methylnaphthalene group is more typical and therefore selection of, for example, anthracene as a calibration standard rather than phenanthrene would be more likely to give hydrocarbon levels approximating to the true values. The results of anthracene-calibrated analyses of an aromatic mixture comprising the above four compounds are compared in Table V with a gravimetric analysis of the same mixture. While the results are of comparable accuracy, this may not necessarily be true when an unknown hydrocarbon mixture is analyzed. In the latter case the gravimetric error which appears to be due largely to mechanical losses would not be expected to change (and in fact can be corrected for on that basis), while the densitometric error would be an unknown quantity. Comparison of Densitometric and Gravimetric Analyses
To obtain some idea of the variation in hydrocarbon levels as determined by the two procedures, a sample of dichromate would produce the limited oxidation reaction the San Francisco Bay mussel (Mytilus edulis) extract that was desired. previously mentioned was analyzed. The results as preThe results are shown in Figure 5 . Most significantly, sented in Table VI show that the charring/densitometric the C22 and C30 curves are now virtually superimposable technique tends to significantly exaggerate the amounts of (cf. Figure 3). Furthermore the difference between the both aromatics and alkanes. They also show that anthramore volatile n-tetradecane (C14) and the higher carbon cene is a somewhat better model for the aromatics than compounds has been significantly reduced (50% of C22 ren-CC22 is for the alkanes, despite the more favorable response compared with 25% without dichromate). In view sults reported earlier with the latter. Presumably this is of the fact that the environmental impact of the more voldue to the presence of alkanes, linear and branched, of atile hydrocarbons typified by tetradecane is likely to be much higher molecular weight than n-Cnz, that are minimal and short-lived (due to weathering), the reduced charred more efficiently. The densitometer values are all response to the lower end of the alkane range is not exwithin a factor of two of the gravimetric values assumed pected to cause a significant error in the total alkane to approximate the true values. Such accuracy may be value. An additional dividend of the dichromate visualizaperfectly adequate for many environmental monitoring tion is the increased sensitivity, down to 0.1-0.5 kg. applications, especially since the relative ratings are corTo test the method, analyses were run on solutions of a rect and provided the method possesses other favorable synthetic hydrocarbon mixture containing approximately aspects, such as speed and convenience. However, the apequal amounts of n-tetradecane ( C I ~ ) ,n-heptadecane parent differences in favor of the densitometric method (C17), n-docosame ( C Z ~ )and , 2,6,10,15,16,23-hexamethyl- are not as great as might be expected when one takes into tetracosane (C30). The developed hydrocarbon spot was account the necessity for repeated calibration checks. visualized by spraying with the H2S04/K2Cr207 mixture, heating for 30-60 min at approximately 140°C and finally at 200-250°C to complete the charring and drive off the sulfuric acid. The results based on standard n-docosane spots on the same plate, are tabulated in Table IV. The values, not unexpectedly, are less accurate than those obtained for a single hydrocarbon. Nevertheless, this TLC procedure is capable of detecting microgram quantities of ali2.0 phatic hydrocarbon and providing a reasonable estimate, .ENE in this case within &20% of the actual amount. Effect of Aromatic Type. Application of the improved (H2S04/K2Cr207)procedure to a series of four aromatic compounds of varied structures, namely a two-dimensionally fused ring system (pyrene), a linear fused ring system (anthracene), an angular fused system (phenanthrene), and an alkylated aromatic (1-methylnaphthalene) gave the curves shown in Figure 6 that includes for comparison a n-C22 curve run on the same plate. It will be seen that 10 20 substantial differences exist not only between the aromatSAMPLE SIZE, w g ics and the n-docosane but also among the aromatics Figure 6. Response curves for selected aromatic hydrocarbons themselves. As a result, the precision of the densitometric Visualization agent, HZSO4/K2Cr207 procedure as a general method for aromatic mixtures is
I
1
Volume 9 , Number 3, March 1975
245
Table VI. Hydrocarbons in San Francisco Bay Mussel TLC Method
Alkanes
Aromatics
Total
Gravimetric, ppm“ 52 Densitometric, ppm (% error)b 99
34 86 57 (+68)d 156(+81) a Corrected for 90% recovery. Based on gravimetric values. Based on W C ? :calibration. Based on anthracene calibration.
Moreover if correction of the data to agree more closely with the true, i.e. gravimetric, values is desired, it cannot be assumed without the accumulation of more extensive comparative data that the correction factors applicable to the above mussel analysis (0.53 for alkanes; 0.6 for aromatics) will be the same for a wide range of hydrocarbon samples. Conclusions
The analysis by type and amount of low levels of hydrocarbons by gravimetric and densitometric thin-layer chromatographic techniques has been investigated. Separation into saturates (alkanes) and unsaturates (aromatics) is readily achieved. The separation of olefins, while appearing to be feasible, was not pursued at this time, since their presence in oil spills is likely to be minimal. The gravimetric recovery of hydrocarbon spots gives directly values which are consistently within 10-20% of the actual amount and which therefore can be corrected to yield values close to the true values. Charring of the spots followed by densitometric measurement lacks accuracy due to variable hydrocarbon response; the resultant need for repeated checks and controls (calibration) makes the method only marginally more convenient than the more accurate gravimetric procedure. In combination with the appropriate extraction procedures (14, the TLC methods described in this paper can readily be used to detect and measure microgram levels of hydrocarbons. At the present time they are being success-
246
Environmental Science & Technology
fully applied to investigate hydrocarbon levels in marine organisms in the chronically polluted environment of San Francisco Bay (14, 15) and have shown similar promise for the investigation of marine sediments. Acknowledgment
The author would like to express his appreciation to Harold E. Guard and Louis H. DiSalvo for many helpful discussions.
Literature Cited (I) Blumer, M., Souza, G., Saas, S., Marine Biol. 5 , 195 (1970). (2) Sidhu, G. S., Valc, G. L., Shipton, J., Murray, K. E., F A 0 Symposium on the Detection and Monitoring of Pollutants, Rome 1970. (3) Shipton, J., Last, J . H., Murray, K. E., Valc, G. L., J . Sei. Food Agr., 21,433-6 (1970). (4) Bovlan. D. B.. T r i m . B. W.. Nature, 230 44-7 (1971). ( 5 ) American Public ‘Health Association, “Standard Methods for the Examination of Water and Wastewater,” 13th ed., 413, 1971. (6) Pflaum, W., Kempf, T., Luedemann, D., Motortech., 29, 84 (1968). (7) Stanley, T. W., Morgan, M . J., Grisby, E. M., Enuiron. Sci. Technol., 2,699-702 (1968). ( 8 ) Bender, D. F., ibid., pp 204-6. (9) Zitke, V., Bull. Environ. Contam. Toxicol., 5,559-64 (1971). (10) Thurston, A . D., Knight, R. W., Enuiron. Sei. Technol., 5, 64-9 (1971). (11) Kawahara, F. K., ibid., 3,150-3 (1969). (12) Mark, Jr., H. B., Ta-ching Yu, Mattson, J . S., Kolpack, R. L., ibid., 6,833-4 (1972). (13) Kirchner, J . G., Miller, J. M., Znd. Eng. Chem., 44, 318 (1952). (14) DiSalvo, L. H., Guard, H . E., Hunter, L., Cobet, A . B., Center for Wetland Resources, Louisiana State University Publication No. LSU-SG-73-01, 205-20 (1973). (15)‘DiSalvo, L. H., Guard, H. E., Hunter, L., Enuiron. Sei. Technol., in press.
Received for review March 29, 1974. Accepted November 11, 1974. Mention of cpmmercial products is for identification only and does not constitute endorsement by the Environmental Protection Agency of the L!S. Government.
