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Comparison of Thin-Layer and Column Chromatography for Separation of Sedimentary Hydrocarbons Juanita N. Gearing* and Patrick J. Gearing Marine Ecosystems Research Laboratory, Graduate School of Oceanography, University of Rhode Island, Kingston, Rhode Island
0288 1
Thomas F. Lytle and Julia S. Lytle Gulf Coast Research Laboratory, P.O. Drawer AG, Ocean Springs, Mississippi 39564
Several thin-layer (TLC) and column chromatographic (CC) methods were compared for efficiency and precision of separation of standard mixtures into hydrocarbon classes. TLC with activated silica gel G and CC with 2:l (v/v) activity grade I silica gel-alumina gave similar and excellent separation of aliphatic from aromatic hydrocarbons. Extracts of 12 replicate sediment samples from Narragansett Bay, R.I., separated by these two methods gave x f s values (pg hydrocarbons per g dry weight of sediment) of 111.5 f 26.2 (TLC) and 116.9 f 14.4 (CC) for the aliphatic hydrocarbon fraction, and 29.9 f 9.7 (TLC) and 31.3 f 8.6 (CC) for the aromatic hydrocarbon fraction. The results suggest that the two techniques may be used interchangeably for purification of sedimentary hydrocarbon extracts. Interlaboratory precision for sediment samples from the same area is equal to that reported for artificial calibration samples.
With the great increase in worldwide transportation of petroleum and petroleum products (refined oil, solvent, and plastics to name only a few), concern has grown over the extent and effect of accidental spills and other inputs to the marine environment. I t has become necessary to increase not only the number but also the accuracy and replicability of measurements of hydrocarbons in the environment. I t is now critical t h a t many laboratories be able to carry out these complex measurements on an almost routine basis. Because of the chemical variety of extracts from environmental samples and because of the experimental difficulties involved in analyzing a number of different sample types, it may never be possible or advisable to set a standard method for hydrocarbon analyses. Thus, the characteristics and replicability of the different methods used must be firmly established so t h a t results from different laboratories may be compared. Much of this work is now being done. It has been centered upon the extraction techniques since incomplete extraction can affect not only the quantitative but also the qualitative results. Relative efficiencies have been determined for extraction of hardshell clams (I) and for surface sediments ( 2 ) . Examination of results on intercalibration samples (tuna meal and cod liver oil) analyzed in several laboratories ( 3 ) points out the importance of the extraction procedure. Relative standard deviations less than 20% have been reported for homogenized sediments analyzed by three laboratories ( 4 ) . However, such close agreement is not always the case. Hilpert et al. ( 5 ) gave the precision of total sedimentary hydrocarbon determinations between eight laboratories. Relative standard deviations were 114 and 122% for two different samples, while intralaboratory results on the same samples had relative standard deviations of 25% (12 samples) and 30% (9 samples). It should be noted that these results were obtained on actual homogenized sediments with very low hydrocarbon concentrations. 0003-2700/78/0350-1833$01.00/0
One aspect of the methods currently in use which has not received much attention is the purification and separation into “aliphatic” and “aromatic” hydrocarbon fractions by solidliquid chromatography. Many different chromatographic columns, plates, and elution schemes have been used to separate the vast number of compounds in petroleum into general classes (5,6). In 1961, Synder (7) demonstrated the applicability of both silica gel and alumina to petroleum hydrocarbons. Since then, columns of alumina overlying silica gel have been favored. Variability occurs in the proportions of the two materials, in their mesh size, in their state of activation, and in the eluents. Column chromatography (CC) with ratios of silica gel to alumina (v/v) of 4:l ( 3 ) ,3:l( 5 ) ,2:l (8), 3:2 (9),and 1:l (3) has been done. Elution with varying proportions of pentane, hexane, heptane, petroleum ether, benzene, toluene, methylene chloride, and carbon tetrachloride has been used. Work with thin-layer chromatography (TLC) has often been carried out on plates coated with silica gel and developed with hexane or petroleum ether (3, 20-12). It has been routinely assumed that these many schemes can be used interchangeably without affecting the precision or accuracy of the measurement. Such assumptions are dangerous when not supported by published experimental evidence. This paper reports results of separation of standard hydrocarbon mixtures by TLC and by CC using several of the more widely applied chromatographic conditions. We have also examined the relative efficiency of the two techniques in separating aliphatic and aromatic fractions from each other and from more polar compounds in extracts of an actual marine sediment.
EXPERIMENTAL All solvents used were distilled in glass to remove any hydrocarbon contaminants. Standards included a mixture of nalkanes (with 16, 17, 18, 24, 28 and 32 carbon atoms), the isoprenoid pristane (2,6,10,14-tetramethylpentadecane), two monounsaturated paraffins (n-CI6 and n-CL8 3-methylnonadecane, and a number of aromatics (1,2,4,5-tetramethylbenzene, naphthalene, 2,3,6-trimethylnaphthalene, phenanthrene, and anthracene). The standards were purchased from Aldrich Chemical Company, Analabs, and Applied Science Laboratories. Column chromatography used silica gel (Woelm) and neutral alumina (Woelm). Both reagents were activated by heating overnight at 200 “C. Deactivation was by thorough mixing with the appropriate volume of extracted tap water. All columns had an adsorbent to sample weight ratio greater than 100 and were prewashed with two column volumes of hexane before the sample was charged. Thin-layer chromatography was carried out using the technique described by Quinn and Wade (11). Plates were coated with a 0.37-mm layer of silica gel G Type 60 (Analabs) containing no organic binder, activated at 110 “C for 12 h and then precleaned with CH2C12:CH30H(80:20, v/v). After spotting, samples were added to aid in visualization eluted by hexane with 1%“,OH of bands. A mixture of 3-methylnonadecane and phenanthrene standards was eluted on the same plate to act as a guide in removing the hydrocarbon fractions of the sample. Bands were C 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
Table I. Fractionation of Standards by Column Chromatography u g recovered from column (silica ge1:alumina) 3:2, 5% deactivated 4:1, activated 2:1, activated ug hexane benzene hexane benzene hexane hexane hexane charged ( 3 vols) ( 3 vols) (2 vols) (2 vols) 1st vol. 2nd vol. 3rd vol. n-alkanes 5075 2535 175 2630 20 3640 55 0 isoprenoids 460 710 39 495 0 605 0 0 unsaturates 1050 380 53 1030 5.5 660 145 0 aromatics 2900 945 74 0 24 1010 90 0
benzene (1vol.) 0 0 0
2365
Table 11. Fractionation of Standards by TLC and CC (percent recovery, two runs) CC, 2 : l ( v / v ) silica ge1:alumina (activated) TLC, activated silica gel aliphatic band aromatic band hexane (2 vols) benzene (2 vols) 3-methylnonadecane 1,2,4,5-tetramethylbenzene naphthalene 2,3,6strimethylnaphthalene phenanthrene
46 0 0 0 0
53 0 0 0 0
visualized by spraying the plate with bromothymol blue and viewing under UV light. A sediment extraction procedure similar to that of Boehm and Quinn (12) was followed. Twelve aliquots were taken from a wet, homogenized silty clay from central Narragansett Bay (i1°22'N, 41'35'W) which had been put through a 0.82-mm sieve to remove large organisms and shells. After addition of 3-methylnonadecane plus phenanthrene internal standards, each aliquot was refluxed for 4 h in equal portions of methanolic KOH (1N) and benzene, with 5% pre-extracted water added. The nonsaponifiable fraction was extracted with three separate portions of benzene. taken to 5 mL on a rotary evaporator, and treated with activated copper to remove elemental sulfur. Six aliquot extracts were then separated on columns consisting of 2:l (v/v) silica gel under alumina (both activity grade I) and eluted with two column volumes of hexane (aliphatic fraction) followed by two column volumes of benzene (aromatic fraction). The other six extracts were separated into two fractions by TLC in the procedure described above. The same procedure, using TLC, was carried out on 25 samples of sediment from the tanks at the Marine Ecosystems Research Laboratory, Rhode Island. These tanks were originally filled with sediment from the same location as the homogenate described in the preceding paragraph. Analysis of hydrocarbons was by gas chromatography with a Perkin-Elmer model 3920B or 990 chromatograph equipped with dual flame ionization detectors and dual 2 m X 3.2 mm stainless steel columns packed with 4% FFAP on 80/100 mesh Gaschrom 2. Temperature programming was from 90 to 230 "C at 8 "C m i d with a 4-min initial hold and final hold until Kovats Index 3200 was reached. RESULTS AND DISCUSSION Comparison of S t a n d a r d s on Various Columns. Table I shows the separations achieved with a known hydrocarbon mixture put through three columns containing differing proportions of silica gel to alumina. The mixture contained all the standards listed previously except 3-methylnonadecane and phenanthrene. In comparing the composition of the eluates from the three columns, it can be seen that the 3:2 (v/v), 5% deactivated column and the 4:l (v/v), activity grade I columns with the elution schemes described were ineffective in separating the hydrocarbon classes. All of the standards eluted predominantly with the hexane in both columns. The column made up of 2:l (v/v) silica gel to alumina (both activity grade I) was most effective of those examined. Two column volumes of hexane removed virtually all of the aliphatic hydrocarbons while leaving the aromatics on the column. Aromatics began to be eluted by the third column volume of hexane and were removed completely when the elution solvent
1
6 3 50 54
2 31
32 0
17
0 0 0
67 69
20 0 0 0 0
0 24 26 57
80
58
84
0
27 19
was changed to benzene for the fourth column volume. It was concluded that excellent separation of aliphatic from aromatic hydrocarbons could be obtained by use of such a column and elution with two volumes of hexane (aliphatic fraction) followed by two volumes of benzene (aromatic fraction). This comparison of the three column chromatography schemes was designed primarily to test the efficiency of the procedure routinely used in our laboratory. T h e results do not imply that the many schemes used in other laboratories may not be just as efficient, but that any procedure used should be experimentally validated. In particular, it should be noted that vastly different separations may be obtained by the use of silica gel and alumina of different mesh ranges. Comparison of S t a n d a r d s Separation by T L C a n d CC. A mixture of one aliphatic and four aromatic hydrocarbons (see Table 11) was prepared. Two aliquots were separated by the column conditions shown to be effective in the last section, and two by TLC. All four runs gave nearly identical separations. Because of evaporative losses of the lower molecular weight aromatics, the final proportions in the aromatic fractions differed slightly from the standard mixture. There was, however, virtually no overlap of aliphatics with aromatics in either method. This indicates that TLC and CC may be used interchangeably, resulting in increased flexibility and thus efficiency in hydrocarbon analysis, for each method has certain definite advantages. With TLC, standards may be run routinely on the same plate as the samples, providing a check on each separation. It is particularly good with small samples, several of which may be developed on the same plate. However, samples must be reduced to a very small volume for spotting on a TLC plate, increasing the risk of loss of the more volatile compounds by evaporation (see Table 11, where some loss of tetramethylbenzene and naphthalene was observed during TLC). Moreover TLC involves greater exposure of the hydrocarbons to sunlight, increasing the risk of photo-oxidation. Column chromatography may be faster than TLC but, as previously noted, great care must be taken to standardize a chromatography scheme and to periodically test its efficiency since standards cannot be run with each sample. The greater ratio of solvent to hydrocarbons in CC lessens the danger of evaporative loss. Comparison of Sediment Replicates by T L C a n d by CC. The separation of standard hydrocarbon mixtures tested the precision of the methods on a simple sample. These mixtures contained ten or fewer compounds. Environmental
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
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Table 111. Fractionation of Twelve Replicate Sediment Extracts by TLC and CC (concentrations, clg/g) aromatic fraction total hydrocarbons aliphatic fraction
TLC
cc
TLC
44.7 11-2 106 25.1 114 118 20.7 90.1 91.4 20.8 130 79.3 37.5 131 127 30.5 119 152 29.9 116.9 111.5 mean 9.7 26.2 14.4 std. dev. 32.4% 12.3% 23.5% rel. std. dev. t statistic (tabulated value at 99% confidence) variance ratio, F (tabulated value at 99% confidence)
cc 17.2 24.4 38.9 34.5 34.6 38.0 31.3 8.6 27.5%
TLC 151 139 111
100 165 183 141.4 31.6 22.3%
cc 130
142 130 164 165 157 148.2 16.4 11.1%
3 . 7 (10.97) 0.49 (4.03)
Table IV. Total Hydrocarbon Concentrations in Sediments from Adjacent Areas of Narragansett Bay' no. of rel. std. date samples ref. laboratoryb range, ug/g mean, pg/g std. dev. dev., % 3 130-1 60 147 15.3 10.4 1973 3 ( 4) 141-185 157 16.8 10.7 1976 6 (16) 1, 2 144-210 185 27.6 14.9 Aug. 1976 6 (16) 2, 3 Aug. 1976 12 this w o r k 1 100-183 145 24.3 16.8 Sept. 1976 25 this work 1 45.1-186 140 34.2 24.4 1977 8 (1 7 ) 2 130-168 144 13.9 9.7 a All sediments except those in this work are from 71" 22" and 41"35'W. Laboratories are (1)this laboratory, ( 2 ) T. Wade a n d J. Quinn, University of Rhode Island, and ( 3 ) J. Farrington, Woods Hole Oceanographic Institute. samples may contain hundreds or even thousands of compounds, including not only n-alkanes, isoprenoids, and fused-ring aromatics such as in the standard mixtures, but a vast array of less readily identifiable compounds such as alicyclic hydrocarbons. Such complex samples are needed to test the separation efficiencies of the methods under actual conditions. Table I11 gives hydrocarbon concentrations and standard deviations, based on gas chromatography, of 12 aliquots (six extracts separated by TLC and six by CC) of the homogenized sediment. The variance ratio (F)was calculated and when compared with the tabulated values at the 99% level ( a = 0.01) showed that the standard deviations of values from the two different methods are statistically indistinguishable. Thus, although the values suggest that column chromatography may be slightly more reproducible, the greater precision is not statistically significant. The equality of variances allows a simple t test to be performed, which showed that the two means are also equal a t the 99% level. The hydrocarbons in central Narragansett Bay surface sediments give complex chromatograms with compounds eluting from Kovats Index 1400 to 3200+. Peaks of C25 cycloalkenes and odd carbon number n-alkanes from n-C25 to n-CB1are superimposed on a large, unresolved complex mixture (UCM). The source of the C25alkenes, also found in Buzzard's Bay and Gulf of Maine (13),Scotian Shelf ( 1 4 ) , and Gulf of Mexico shelf sediments (B), is presently unknown. The heavy n-alkanes are thought to originate from terrestrial plant waxes ( 1 4 , 15). The UCM contains naphthenic, naphthenoaromatic, and aromatic hydrocarbons, which strongly suggest an anthropogenic source for that component (13, 1 4 ) . This assemblage of hydrocarbons from disparate sources provides a good test for the separating efficiency of thin-layer and column chromatography. Examination of the gas chromatograms of fractions separated by the two methods showed no discernible qualitative differences in the patterns. P r e c i s i o n a n d A c c u r a c y of Sediment Analyses. The accuracy of the results of the 12 sediment analyses was tested relative to published data by other investigators on sediments from the same area of Narragansett Bay, as well as to additional determinations in our own laboratory. These data
are summarized in Table IV. A one-sided F test of variance coupled with a t test on the means indicated that neither the means nor the standard deviations were statistically different when comparing the 12 determinations reported in Table I11 with either the 21 determinations by other laboratories (F1,31 = 1.80, t31= 1.335)or with all the other observations (21 from other laboratories and 25 from our laboratory) = 0.184, tjs= 0.095). The one-sided F test of variance was also applied to the data as grouped by laboratories. The variance between laboratories was not statistically greater than the variance within laboratories ( F = 1.234; the tabulated value for F2,34 is 5.29 at the 99% confidence level). Thus the results obtained by both column chromatography and thin-layer chromatography are accurate (assuming that the mean of 48 analyses is accurate) as well as precise within normal experimental levels. Moreover, the results indicate the interlaboratory reproducibility which can be obtained. The methods of analysis used by the three laboratories were very similar, but the sediments were collected and analyzed by a least 5 people over 5 years in 3 laboratories. The relative standard deviations ( 1 2 5 % ) compare favorably with intralaboratory variances. Farrington and Tripp (18) reported relative standard errors of 137% for western North Atlantic surface sediment with hydrocarbon concentrations from 113 to 150 ug/g. Hilpert et al. (5)reported intralaboratory relative standard deviations of 25 and 30% on sediments in the 0.5 to 1 pg/g range, but interlaboratory ranges (8 laboratories) of up to two orders of magnitude for the same homogenized sediments. The low concentrations of these Alaskan sediments make the analysis for total hydrocarbons much more difficult. However, where hydrocarbon concentrations are greater and similar methods are used, much better interlaboratory precision is achievable. ACKNOWLEDGMENT The authors thank Harry McCarty for laboratory assistance. LITERATURE C I T E D (1) J. W. Farrington and G. C. Medeiros, "Proceedings, 1975 Conference on Prevention and Control of Oil Pollution", American Petroleum Institute, Washington, D.C., 1975, p 115. (2) J. W. Farrington and €3. W. Tripp, M a r . Chern. CoastalEnviron., 18 (ACS Symp. Sew.), 267 (1975).
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(3) J. W. Farrington, J. M. Teal, G. C. Medeiros, K. A. Burns, E. A. Robinson, J. G. Quinn, and T. L. Wade, Anal. Chem., 48, 1711 (1976). (4) T. L. Wade and J. G. Quinn, submitted for publication in Environ. Pollut. (5) L. R. Hilpert, W. E. May, S. A. Wise, S. N. Chesier, and H. S. Hertz, Anal. Chem., 50, 458 (1978). (6) R. C. Clark, Jr. and D. W. Brown, in "Effects of Petroleum on Arctic and Subarctic Marine Environments and Organisms", D. C. Malins, Ed., Academic Press, New York, N.Y., 1977, pp 1-90, (7) L. R . Snyder, Anal. Chem., 33, 1527 (1961). (8) P. Gearing, J. N. Gearing, T. F. L e , and J. S.Lytle, Geochim. Cosmochim. Acta, 40, 1005 (1976). (9) R. S.Clark and J. S. Finley, "Proceedings of the 1973 Joint Conference on Prevention and Control of Oil Spills", American Petroleum Institute, Washington, D.C., 1973, p 161. (10) L. Hunter, Environ. Sci. Techno/., 9, 241 (1975). (1 1) J. G. Quinn and T. L. Wade, Marine Memorandum Series No. 33, University of Rhode Island, Kingston, R.I., 1974, 8 pp. (12) P. D. Boehm and J. G. Quinn, Estuarine Coastalnlbr. Sci., 6, 471 (1978).
(13) J. W. Farrington, N. M. Frew, P. M.Gschwend, and 6. W. Tripp, Esfuarine Coastal Mar. Sci., in press. (14) P. D. Keizer, J. Dale, and D. C. Gordon. Jr., Geochim. Cosmochim. Acta, 42, 165 (1978). (15) J. W. Farrington and P. A. Meyers, in "Environmental Chemistry", Vol. 1, G. Eglinton, Ed., The Chemical Society, Burlington House, London, 1975. (16) J. W. Farrington and J. G. Quinn, Estuarine Coastalnlbr. Sci., 1, 71 (1973). (17) T. L. Wade, University of Rhode Island, Kingston, R I . , personal communication, 1978. (18) J. W. Farrington and B. W. Tripp, Geochim. Cosmocbim. Acta, 41, 1627 (1977).
RECEIVED for review June 23,1978. Accepted August 14,1978. Work supported by Grant R803902020 of the Environmental Protection Agency.
Determination of Volatile Halogenated Hydrocarbons in Water with XAD-4 Resin Lars Renberg Special Analytical Laboratory. National Swedish Environment Protection Board, Wallenberg Laboratory, University of Stockholm, S- 106 9 1 Stockholm
The determination of trihalomethanes, chloroethenes, and dichloroethane in water was carried out by adsorption on an XAD-4 polystyrene resin, followed by elution with ethanol. The method results in an extract concentrated enough for both chemical determination and small scale biological tests. By using two series-connected columns, the degree of adsorption was studied and the chloroethenes were found lo be more strongly adsorbed than the haloalkanes. The recovery was found to be 60-95% of the substances studied.
the determination of total organic halogen content in water has been described (15). The advantages of using the XAD-4 technique are several. The method is simple and allows sampling in situ, thus avoiding changes of water sample composition between time of sampling and time of analysis. The procedure involves a concentration step which makes it possible to reach desired detection limits, as these limits may vary with applications. It also allows the collection of samples large enough for both chemical determination and small scale biological tests such as the Ames' bacterial system for mutagenicity determination (16).
The presence of volatile halogenated hydrocarbons such as trihalomethanes and chloroethenes in water has been established in water samples from several sources. The trihalomethanes have been shown to be formed during the chlorination process, used in water treatment plants, and the average levels of chloroform, bromodichloromethane, and chlorodibromomethane in drinking water samples from 80 cities in the U.S.A. were found to be 21, 6, and 1.2 Fg/L, respectively (I). Also the incoming water to a water treatment plant has been shown to contain several halogenated compounds such as the commonly used solvents 1,2-dichloroethane, trichloroethene, and tetrachloroethene (2). The main interest in these types of compounds is focused on possible toxic effects, particularly carcinogenicity. Using bacterial systems, bromodichloromethane, chlorodibromomethane, trichloroethene, and 1,2-dichloroethane have been shown to possess mutagenic properties (3, 4 ) . T h e need for determination of volatile hydrocarbons in water samples has resulted in several methods such as solvent extraction ( 5 ,6), gas stripping ( 7 ) ,head space (8),and direct injection techniques (9). In this paper, a method is described which involves the adsorption of volatile halogenated hydrocarbons on an Amberlite XAD-4 resin followed by elution with ethanol. The use of XAD-2 or XAD-4 resins for the determination of less volatile substances has been described earlier (10-1.2), and adsorption of haloforms on acetylated XAD-2, followed by elution with pyridine ( I 3 ) ,and on non-acetylated XAD-'2 (14) has also been reported. Recently, the use of XAD-resins for 0003-2700/78/0350-1836$01.OO/O
EXPERIMENTAL Materials. Amberlite XAD-4 (Rohm and Haas Company), acetone (pesticide grade), methanol and ethanol (spectroscopic grade) were used. The purity of the methanol and ethanol was checked by the gas chromatographic system described below. Polymeric Resin Cleanup. Amberlite XAD-4 was placed in a column with a glass filter. Five bed volumes of acetone were passed through the column with a flow rate about 0.1 bed volume/min. Then water was passed upflow through the column at a rate sufficient to expand the bed by about 50% to remove the smallest particles. The resin was then extracted by means of a Soxhlet apparatus with methanol for at least 6 h, then replaced into the column and eluted with 20 bed volumes of purified water and finally stored under purified water. The purity of the resin was checked in a blank procedure. Purified Water. Deionized water was distilled through an all glass apparatus. The first 1070 of the distillate was discarded and the 1G-5070portion was passed through a column containing 10 mL XAD-4 at a flow rate of about 30 mL/min. If the resin was taken directly from the Soxhlet extraction and thus containing methanol, the first 200 mL of the eluate was discarded. Adsorption and Desorption Steps. Two glass columns, 15 X 1.1(id.) cm, with Teflon stopcocks, were each filled with 5 mL of XAD-4. Purified water was added to the top of the columns and the beds were stirred t o release air bubbles. The columns were connected in a series and the water sample was allowed to pass through the columns at a flow rate of 30 mL/min. The columns were allowed to run dry and disconnected. To each column, ethanol (3 mL) was added, the beds were stirred, and the resins were allowed to swell for at least 20 min. The beds were stirred once more t o release air bubbles and the columns C 1978 American Chemical Society
