Sample extraction and purification for determination of polycyclic

J. Jacob , J. J. Belliardo , W. Karcher , A. S. Lindsey , P. J. Wagstaffe. Separation ... Determination of mono- to tetracyclic aromatic hydrocarbons ...
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Anal. Chem. 1980, 52, 2027-2031

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Sample Extraction and Purification for Determination of Polycyclic Aromatic Hydrocarbons by Reversed-Phase Chromatography Bruce P. Dunn' and Robert J. Armour Environmental Carcinogenesis Unit, British Columbia Cancer Research Centre, 60 7 West loth Avenue, Vancouver, British Columbia. Canada V5Z 1L3

Hydrocarbons are extracted from samples by alkaline digestlon, and Interfering materlals are removed by adsorption onto Fkrlsy, partttknlng ot the sample between isooctane and dimethyl sutfoxlde, and colwnn chromatography on Sephadex LH-20. A radioactive Internal standard Is used to correct for losses during procedures. Twenty polycyclic aromatic hydrocarbons are determlned by reversed-phase hlgh-pressure ilquid chromatography, utlliring simultaneous detectlon by ultraviolet absorbance and fluorescence monitors.

There is increasing concern about the contamination of air, water, and food by polycyclic aromatic hydrocarbons (PAHs) with carcinogenic and mutagenic properties. The problem of assessing the degree of environmental contamination by PAHs has given rise to a large number of procedures for separating and measuring various PAH isomers. Recent trends have been toward the separation of compounds by high-resolution gas chromatography (on capillary or long packed columns) or by high-pressure liquid chromatography (HPLC). We have previously described procedures for measuring the PAH isomer benu>[a]pyrene in marine samples by a combination of thin-layer chromatography and fluorimetry (I , 2). In an extension of this work, we have now developed routine procedures for measuring 20 PAH isomers by high-pressure liquid chromatography, employing reversed phase columns and a combination of UV absorption and fluorescence detection. Extracts of some types of samples such as air pollution particulates may be chromatographed with a minimum of sample purification. However, in a majority of samples of interest, PAHs are accompanied by a complex array of other organic compounds which either physically prevent successful chromatography or cause interfering peaks or excessive base-line shifts. Such samples require more or less extensive purification procedures to isolate PAHs as a purified fraction before resolution of individual PAH isomers is attempted. We have previously published a sample extraction and purification scheme which gives a PAH fraction suitable for thin-layer chromatography (I). Radioactively labeled benzo[a]pyrene was used as an internal standard to monitor and correct for losses of carcinogen during sample purification and handling procedures. Here we describe a modification of this procedure and demonstrate its use in the analysis of PAHs in three types of samples by HPLC. EXPERIMENTAL SECTION Materials. Sample extraction solvents were practical or technical grade for economy and were redistilled before use in an all-glass still with a Vigreaux reflux column. Solvents were stored in glass-stoppered containers and were protected from contact with rubber or plastic at all times (3). Dimethyl sulfoxide (Me2SO) was spectral grade and was generally used without further purification. Polycyclic aromatic hydrocarbon reference compounds were from Eastman (Rochester, NY), K and K Laboratories (Plainview, 0003-2700/80/0352-2027901 .OO/O

NY), Analabs Inc. (New Haven, CT), Sigma (St Louis, MO), and from the Community Bureau of Reference, Brussels, Belgium. The latter organization has available for purchase certified standards of benzo[blchrysene, benzo[b] fluoranthene, benzo[klfluoranthene, benzoljlfluoranthene, benzo[e]pyrene, and indeno[1,2,3-cd]ppene. Radioactively labeled benzo(a1pyrene ([3H]benzo[a]pyrene, specific activity 25 Ci/mmol) was purchased from the Amersham Corp. and routinely purifed before use by extracting the material dissolved in hexane with 0.25 M NaOH in 40% ethanol to remove degradation products formed in storage (4). Stock solutions of radioactive benzo[a]pyrene were stored at room temperature in ethanol in the dark. Sodium sulfate and KOH were reagent grade and were used without further purification. Florisil (60-100 mesh) was from Matheson Coleman and Bell and was prepared as previously described, except that it was deactivated with 5% rather than 2% water (I). Sample Extraction. A 20-100-g sample of mussel tissue or 5-10 mg of finely divided creosoted wood was placed in a 300-mL round-bottomed flask, and 150 mL of ethanol, 7 g of KOH, two or three boiling chips, and an aliquot of radioactive benzo[a]pyrene (25 OOO dpm 3HB(a)P, ca. 0.1 ng) were added. The tissue was digested by refluxing gently for 1.5 h with occasional swirling to prevent sticking on the bottom of the flask. The digest was added while hot to 120-150 mL of water, the amount of water being adjusted to yield a final ethanol/water mixture of 5 0 4 5 % ethanol (ethanol total volume = 200 mL, water total volume including that in the wet sample). The digestion flask was rinsed with an additional 50 mL of ethanol which was added to the separatory funnel. The mixture was extracted three times with 200 mL of isooctane, and the isooctane extracts were combined and washed with 4 X 200 mL of warm (60 " C ) water. Wet sediment samples weighing 10-100 g were refluxed in 100 mL of ethanol with 5 g of KOH, boiling chips, and radioactive tracer. The contents of the flask were then swirled to suspend the particulate material and poured into 100-mL centrifuge tubes. The suspension was centrifuged for 5 min at 500 g and the supernatant decanted. The particulate material was washed twice with 50 mL of ethanol by stirring and resuspension followed by centrifugation and decantation. The original supernatant and ethanol washes were combined with 150 mL of water in a separatory funnel. The mixture was then extracted with isooctane as previously described for tissues. Column Chromatography on Florisil. The isooctane from the extraction step was rotary evaporated to a volume of approximately 10 mL and then 100 mL of toluene added and the volume again reduced to 10 mL to remove the isooctane. The extract was then made up to 100 mL in toluene. A column of 30 g of Florisil (deactivated with 5% water) covered with 60 g of coarse Na2S04was prepared in a glass column (40 X 400 mm) with a coarse fritted glass disk. The column was prewashed with 100 mL of toluene, which was discarded. The sample in toluene was then applied to the column, followed by two washes each of 100 mL of toluene. Me2S0 Partition. Five milliliters of M e a 0 was added to the combined toluene eluate from the column, and the mixture was rotary evaporated to remove the toluene, leaving the sample in MezSO (which is nonvolatile at a rotary evaporator water bath temperature of 60 OC and normal aspirator vacuum). The M e 3 0 was transferred to a separatory funnel containing 10 mL of isooctane, and the evaporating flask washed with an additional 5 0 1980 American Chemlcal Society

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mL of Me2S0which was also added to the separatory funnel. The contents of the separatory funnel were shaken, and the MezSO layer was transferred to a second separatory funnel containing 40 mL of water and 20 mL of isooctane. An additional 10 mL of fresh Me2S0 was added to the first separatory funnel, the contents were shaken, and this Me2S0 was also transferred into the second separatory funnel. The second funnel (containing 20 mL of isooctane, 40 mL of water and 20 mL of Me2SO) was shaken, and the Me2SO/water phase transferred into a third separatory funnel containing 20 mL of isooctane. The contents of the third funnel were shaken and allowed to separate, and the Me#O/water layer was discarded. The isooctane layers from the second and third separatory funnels were then combined and washed twice with 40 mL of water. The isooctane was rotary evaporated to a volume of approximately 5 mL containing a few drops of separated water from the undried solvent. Ten milliliters of 10% ethanol-QO% toluene was added, and the mixture rotary evaporated to a volume of approximately 1mL. This removes water by azeotropic distillation and changes the solvent to toluene. The extract was then dried down into Me2S0 for chromatography (see below) or subjected to further purification by chromatography on Sephadex LH-20. Chromatography on Sephadex LH-20 (Optional). The toluene extract from the Me2S0 partition step was transferred with washes of toluene into a graduated 15-mL centrifuge tube and then dried to a volume of 0.5 mL with gentle warming and a stream of dry nitrogen. An equal volume of ethanol was added, and the sample applied to a column of Sephadex LH-20 (bed size 10 mm X 200 mm) packed in toluene/ethanol (1:l).The material was drawn into the column bed utilizing gentle suction on the column outlet and then the column was eluted by using gentle suction with 18 mL of toluene/ethanol (1:l)which was discarded. The PAH fraction was then recovered from the column by elution with a second 18 mL of solvent and the column regenerated for subsequent reuse by washing with 36 mL of solvent. The PAH fraction was reduced in volume to approximately 1mL by rotary evaporation and dried down into M e 8 0 for chromatography (next section). Elution volumes on the Sephadex LH-20 column were determined for an individual column utilizing either reference compounds, as described by Giger and Blumer (5) or a mixture of PAHs from a diluted sample of commercial creosote. Elution volumes were checked from time to time, as they changed as the chromatography adsorbant settled in the column. High-pressure Liquid Chromatography. Purified samples (approximately 1mL volume in toluene) from the M e a 0 partition or Sephadex LH-20 purification steps were transferred with toluene washes into a 15-mL centrifuge tube. A 100-pL sample of Me2S0 was added and the toluene evaporated by gentle warming and a stream of dry nitrogen, leaving the purified PAH fraction dissolved in the nonvolatile Me8O. Samples were stored at room temperature in the dark in Me2S0 in small cone vials (Alltech Associates, internal volume 0.3 mL with a Teflon-lined screw cap). Ten-microliter aliquots of the samples were taken with a microsyringe to determine the recovery of radioactively labeled benzo[a] pyrene by scintillation counting. Chromatography was carried out with a Perkin-Elmer Series 2/2 dual pump gradient chromatograph, coupled to a PerkinElmer LC-55 variable-wavelength UV absorption detector in series with a Varian Fluorichrom filter fluorescence detector. Reversed-phase chromatography columns were slurry packed with Perkin-Elmer HC-ODS 10-pm packing in carbon tetrachloride, utilizing 2.1 mm i.d. X 250 mm columns (Alltech Associates). The packing reservoir (Separations Group) was pressurized a t 5000-6000 psi by the liquid-chromatograph pumps. Such laboratory-packed columns yielded approximately 3000 theoretical plates for benzo[a]pyrene at a flow rate of 0.5 mL/min and a k' of 5. Aliquots of 2-10 p L were injected onto the column, utilizing a Rheodyne 7105 variable volume syringe loading injector. Chromatography was carried out at 30 "C, with the column submerged in a stirred, thermostatically controlled water bath. Eluting solvent at 0.5 mL/min consisted of 60% acetonitrile-40% water for 6 min, followed by a linear gradient of 3% acetonitrile/min to 99% acetonitrile. The composition of the eluting solvent was then held at 99% until all UV absorbing and fluor-

escent material had eluted from the columns. PAHs were detected by UV absorption at 296 nm (absorption maximum for benzo[alpyrene), followed by fluorescence utilizing a broad excitation band (340-380 nm, selected by a Corning 7-54 filter in series with a 7-60 fiiter) and measuring emission at wavelengths greater than 400 nm (selected by a Coming 3-73 W cutoff fiiter in series with a 4-76 red blocking filter to eliminate red leakage from the 754/7-60 excitation filter combination). Solvents were air saturated, and a variable back-pressure restrictor (Varian Associates) was used on the output line of the second detector to maintain an internal pressure of approximately 200 psi inside both detectors to suppress bubble formation. Each detector output was fed to a separate dual pen recorder, with one pen set at 10 times the sensitivity of the other pen to increase the dynamic range of chart recorder traces which could be easily measured. Chromatographic peaks were identified by retention times, by cochromatography of reference compounds, and by the ratio in response between W and fluorescence detectors. Peak areas were measured by triangulation,and the amount of injected component was determined with the use of response factors prepared with reference compounds. The degree of contamination of the originally processed sample was calculated utilizing the amount of a component calculated from a chromatogram,the injected volume of extract, the recovery of material per unit volume of the injected extract (determined by measurement of the [3H]benzo[a]pyrene internal standard), and the weight of the sample before extraction. The [3H]benzo[a]pyrene internal standard totally corrects for losses of benzo[a]pyrene during sample purification and analysis. The same recovery figure is also applied to other PAHs-in this case the recovery figure corrects for any mechanical losses or dilution changes which affect the sample as a whole but does not correct for any differential losses which occur more to one compound than to another.

RESULTS Sample Purification. We have previously described the use of Florisil deactivated with 2% water and eluted sequentially with isooctane and benzene as a purification procedure for PAH from marine samples (I). Recoveries of benzo[a]pyrene using this procedure were somewhat variable. Florisil deactivated with greater than 2% water gave greater recoveries of benzo[a]pyrene but also entailed substantial losses of lower molecular weight PAHs such as fluoranthene which were only partially retained on the column during elution with isooctane. In the present procedure, the sample in toluene is passed through a column of Florisil deactivated with 5 % water. Pigments and other polar material are retained during this rapid one-step elution. Aliphatic hydrocarbons and other nonpolar interfering materials pass through the column with the PAHs. These are largely or completely removed during the subsequent dimethyl sulfoxide/isooctane partition step. This latter procedure, which effectively separates PAHs from a variety of interfering materials (6),cannot easily be performed on the raw extract because of emulsion-forming materials in the extract which are removed by adsorption onto Florisil. During the course of methods development, samples from various stages of the extraction and purification procedures were chromatographed. Prior to Florisil chromatography, samples were generally impossible to chromatograph except at large dilutions. Removal of polar compounds by adsorption onto Florisil removed a substantial fraction of the weight of the extracts and generally allowed successful chromatography. At this stage of purification, chromatograms using the fluorescence detector were often but not always interpretable, but those from the U V absorption detedor generally contained numerous peaks which could not be attributed to known PAHs. Further purification of extracts by partitioning between MezSO and isooctane was usually sufficient to give a sample yielding interpretable UV and fluorescence chromatograms.

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Flgve 1. Chromatogaphy of polycyclicaromatic w o c a r b c nreference compounds. Chromatography conditbns are desaibed h the text. Peak identification is as follows: 1, phenanthrene; 2, anthracene; 3, flue ranthene; 4, pyrene; 5, triphenylene; 6, benz[a]anthracene; 7, chrysene; 8, benzo[e]pyrene; 9, benzo[j]fluoranthene; 10, perylene; 11, benzo[b]fluoranthene; 12, dibenz[ac]anthracene (shoulder); 13, benzcb [ k]fluoranthene; 14, benzo [ a ] pyrene; 15, dibenz [ahlanthracene; 16, benzo[gh/]perylene; 17, lndeno[l,2,bcd]pyrene; 18, benzo[b]chrysene; 19, coronene; 20, dibenz[ ai] pyrene.

Chromatography on Sephadex LH-20 was generally not as useful as dimethyl sulfoxide/isooctane partitioning as a purification step after removal of polar sample components by adsorption onto Florisil. Extracts of marine organisms purified by Florisil adsorption and then chromatography on Sephadex LH-20 often contained relatively large amounts of nonvolatile organic residues, were difficult to concentrate for chromatography, and had UV absorbing compounds which complicated the detection of PAH during HPLC. In contrast, samples purified by Florisil adsorption and MezSO partitioning were generally easily dried down into a small volume of M e a 0 and chromatographed with little difficulty. Sephadex chromatography, however, was sometimes useful as an additional purification step in cases where samples purified by Florisil adsorption and dimethyl sulfoxide/isooctane partitioning did not give interpretable chromatograms. For example, extrach of commercial coated glass fiber air conditioning filters, purified only by Florisil adsorption and dimethyl sulfoxide/ isooctane partitioning, contained very large amounts of UV absorbing and fluorescent material which obscured the PAHs present. These interfering materials were effectively removed by chromatography on Sephadex LH-20. Some types of samples, however, consistently contain non-PAH material which is not removed by any of the purification procedures described. For example, a majority of lobster samples examined contained a series of four to six prominent W absorbing but nonfluorescent peaks eluting with a retention time approximately that of coronene. High-pressure Liquid Chromatography. Four types of reversed-phase chromatography packings were evaluated for the separation of higher molecular weight PAHs. Merck RP-8 and Merck RP-18 packings gave columns with good efficiencies for the chromatography of individual PAH standards but with a relatively poor ability to separate PAH isomers with four and five fused rings. Vydac TP and Perkin-Elmer HC-ODS reversed-phase columns gave similar separations and were able to resolve a number of isomers not separable by Merck columns. Perkin-Elmer material was chosen for further use. Initial chromatography studies were carried out at a column temperature of 60 "C. A t this temperature and a solvent program of 40% acetonitrile to 99% acetonitrile, benz[a]anthracenelchrysene and dibenz[a,h]anthracene/benzo[ghilperylene were not separated. Lowering the column temperature to 30 "C and changing the solvent program to a linear gradient starting a t 60% acetonitrile resulted in the complete separation of the first pair of isomers and the partial separation of the other pair. Thermostatic control of column temperature was found to be necessary to ensure adequate reproducibility of retention times.

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Flgwo 2. Chromatography of extracts of marine samples. Chromatography conditions are described In the text: A, creosoted wood from a piling; B, sediments from base of piling; C, mussels (Mytilus edulis) from concrete brklge abutment 3 m from pilings.

Figure 1 reproduces UV absorption and fluorescence chromatograms of a mixture of 20 PAH standards. Compounds 1-7 are present at 100 ng/injection, while compounds 8-20 are present at 25 ng/injection. Of the compounds chromatographed, there is complete or partial resolution between all,with the exception of the pairs benzo[j]fluoranthene/benzo[e]pyrene and perylene/benzo[b]fluoranthene. The pairs benzo[k]fluoranthene/dibenz[a,c]anthracene and benzo[ghi]perylene/dibenz[a,h]anthracene show partial resolution-these pairs of compounds chromatographed as partially separated peaks or as single peaks with shoulders, depending on the relative concentrations of the isomers. Use of more efficient columns than the laboratory-packed columns used in this study would improve the ability to separate these isomer pairs. We have used the extraction and analysis procedures described for a large number of different samples and sample types. As a demonstration of the usefulness of the procedures, Figure 2 shows chromatograms of extracts of three types of marine environmental samples taken in the vicinity of a group of creosoted pilings in tidal waters. We have previously shown that such groups of pilings represent point sources of PAH pollution in coastal waters (7,8). Figure 2a shows the UV absorption and fluorescence chromatograms of the purified PAHs extracted from a small sample of the surface wood of one of the pilings. Most major peaks could be identified as resulting from parent, nonalkylated PAHs on the basis of cochromatography of reference compounds and the ratio in response between UV and fluorescence detectors. In re-

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versed-phase chromatography, alkylated Compounds elute with greater retention volumes than the parent PAH. In samples containing extensive amounts of alkylated PAHs, peak identification may be expected to be less easy because of cochromatography of alkylated PAHs with nonalkylated PAHs with the next greater number of fused rings. Figure 2b shows the PAHs isolated from the edible tissue of mussels (Mytilus edulis) growing on a concrete bridge abutment at a distance of 3 m from the pilings, while Figure 2c shows PAHs isolated from surface sediments at the base of the pilings. These samples contained the same range of PAHs, although the shape of the chromatographic base line due to unresolved compounds was of a different shape. A complete quantitative interpretation of these chromatograms will not be given here; however, it may be noted for comparison purposes that the level of benzo[a]pyrene in mussel tissue was approximately 50 ng/g wet weight, the level in sediments approximately lo00 ng/g wet weight, and the level in surface samples of creosoted wooden pilings 5OOOOO ng/g wet weight. The chromatograms of PAHs from mussels and sediments presented in Figure 2b,c are typical of chromatograms of organisms and sediments from harbour areas on the Pacific coast of Canada The major identified peaks in organisms and sediment samples are also present in creosote samples. When comparing compounds of similar molecular weights, the relative proportions of different PAH isomers also appear to be similar in the marine samples and in creosote. It is not yet however clear to what extent PAH pollution of such coastal waters is due to creosote contamination, as opposed to contamination from other sources having a similar pattern of hydrocarbons.

DISCUSSION PAH extraction and purification procedures similar to those reported here have been described by other workers. Pancirov and Brown (9)have reported a procedure for measuring PAHs in marine organisms which involves the use of radioactive internal standards and alkaline digestion of the sample followed by dimethyl sulfoxide/ isooctane partitioning and column chromatography on alumina. Purified extracts are subjected to gas chromatography, followed by trapping of peaks and quantitation by UV absorption. Black et al. (IO) have recently described procedures for the extraction and purification of PAHs which involve alkaline digestion, partitioning into cyclohexane, and column chromatography on Florisil. PAHs are then separated and measured by HPLC. Sample preparation schemes such as these which utilize alkaline digestion appear considerably simpler than the procedures reported by Hanus et al. (11)for the measurement of PAHs in oysters. In the latter method, samples are extracted by mechanical blending with acetonitrile. Sample lipids are saponified in a separate step requiring several changeovers of sample solvent. The use of reversed-phase high-pressure liquid chromatography columns to separate PAHs has been reported by a number of groups. Differences in the ability of various procedures to separate specific PAH isomers appear to reside in the manufacturer of the reversed-phase packing material and the precise chromatography conditions employed (solvents, gradients, and temperature). Ogan et al. (12) has reported that Perkin-Elmer reversed-phase columns are better able to separate selected pairs of PAHs than a number of similar packings from other manufacturers. He employed a solvent program consisting of 50% acetonitrile for 15 min followed by a steep gradient at 6.25%/min to 100% acetonitrile. We have employed a soivent program of 60% acetonitrile for 6 min followed by a gradient a t 3%/min to 99% acetonitrile. This program results in the complete or partial separation of all of the standards tested, with the exception of the pairs

benzo[e]pyrene/benzolilfluoranthene and benzo[ blfluoranthenelperylene. Ogan et al. (22) have reported the separation of the latter pair of compounds utilizing a Perkin-Elmer column eluted isocratically with 80% acetonitrile. However, it is not clear if the gradient elution program developed by Ogan et al. would separate the benzo[e]pyrene/benzoG]fluoranthene and benzo[ b]fluoranthene/perylene pairs, as benzolilfluoranthene and perylene were not included in the standards chromatographed using this procedure. A number of researchers have reported the use of stopped-flow fluorescence scanning (13,14) or scanning of trapped peaks (15-18) to confirm the identity of chromatographic peaks. Although such procedures work well for the detailed examination of the compounds occurring in one or two samples, they are time consuming and not well suited to identity confirmation when large numbers of samples are being processed. The use of two detectors for HPLC monitoring offers the possibility of post-run peak identity confirmation using the ratio in response between the two detectors. Such an approach has been reported for fluorescence detection using two sets of wavelength conditions (19) and UV combined with fluorescence detection (20). In contrast to confirmatory techniques using spectra, the detector response ratio is a simple number which should be readily usable in computerbased peak recognition systems. In view of the large number of PAH isomers even in samples consisting mainly of the parent nonalkylated compounds, it seems unlikely that a single column and gradient condition can ever be found which will resolve all isomers present. Potentially, the information needed for the quantitation of all compounds might be obtained by chromatographing a sample more than once, using columns of different selectivity. Alternately, in one run it may be possible to quantitate one compound in the presence of another by employing two or more detectors or detector wavelength settings which respond differentially to the two compounds in an unresolved pair. In the simplest form, this consists of finding a wavelength or set of wavelength settings which make a UV absorption or fluorescence detector highly or totally selective for one compound in an unresolved peak (13,19,21-23). Having determined the amount of this compound, we may calculate the amount of the other compound, if desired, from the response of a detector which responds to both compounds simultaneously. For example, in Figure 1, benzo[b]fluoranthene and perylene are unresolved. However, the UV detector is highly selective for benzo[ blfluoranthene (perylene absorbs poorly at 296 nm), and the amount of benzo[b]fluoranthene may be determined directly from the UV absorption chromatogram. Both benzo[b]fluoranthene and perylene respond in the fluorescence detector, and the amount of perylene may be determined from the fluorescence response by first correcting it for the contribution of the known amount of benzo[b]fluoranthene. In cases where two compounds each respond in both detectors, quantitation of each compound individually is still possible in most cases using simultaneous equations and appropriate response factors. The extraction and purification procedures reported in this paper were developed for the purpose of measuring PAHs in marine environmental samples such as tissues of marine organisms and sediments. However, the procedures may easily be adapted in part or in whole for the measurement of PAHs in other types of samples. Such samples may be introduced into toluene by an appropriate extraction or dilution scheme and then subjected to chromatography on Florisil followed by dimethyl sulfoxide/isooctane partitioning. Depending on the nature of the resulting chromatograms, the sample purification could be either simplified to save time or extended with further purification steps such as Sephadex LH-20

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chromatography (5),chromatography on Biobeads (24), charge-transfer complex formation (5),or high-pressure liquid chromatography on columns other than reversed phase (11, 14,15,25,26).

ACKNOWLEDGMENT We thank J. Fee for expert technical assistance and K. Ogan for helpful discussions. LITERATURE CITED (1) Dunn. B. P. Environ. Sci. Techno/. 1976, 10, 1018-1021. (2) Dun% B. P. In "Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment"; Afghan, B. K.. Mackay, D., Eds.; Plenum Press: New York, 1980; pp 109-119. (3) Kordan, H. A. Sclence 1965, 749, 1382-1383. (4) DePierre, J. W.; Moron, M. S.; Johannesen, K. A. M.; Ernsten, L. Anal. Biochem. 1975, 63. 470-484. (5) Giger, W.; Blurner, M. Anal. Chem. 1974. 46, 1663-1671. (6) Natusch, D. F. S.; Tomkins, B. A. Anal. Chem. 1978, 50, 1429-1434. (7) Dunn, 8. P.; Stich, H. F. Roc. Soc.Exp. Biol. M .1975, 750,49-51. (8) Dunn, B. P.; Stich, H. F. J. Fish. Res. Board Can. 1976, 33, 1469-1476. (9) Pancirov, R. J.; Brown, R. A. Envinm. Sci. Technd. 1977, 11, 989-991. (10) Black, J. J.; Dymerski, P. P.; Zapisek, W. F. Bull. Environ. Contam. TOX~COI.1979, 22, 278-284. (11) Hanus, J. P.; Guerrero, H.; Biehl. E. R.; Kenner, C. T. J . Assoc. Off. Anal. Chem. 1979, 62, 29-35.

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(12) w n . K.; Katz, E.; Slavln, W. Anal. Chem. 1979, 57, 1315-1320. (13) Nielsen, T. J . Chromatogr. 1979, 770, 147-156. (14) Wise, S. A.; Chesler. S. N.; Hertz, H. S.; Milpert, L. R.; May, W. E. Anal. Chem. 1977. 49, 2306-2310. (15) Eisenberg, W. C. J . Chromafogr. Sci. 1978, 76. 145-151. (16) Nakagawa. K.; Sato, Y.; Watabe, A.; Kawamura, T.; Morita. M. Bull. Environ. Contam. Toxkol. 1978. 19, 703-706. (17) Dong, M.; Locke, D. C.; Ferrand, E. Anal. Chem. 1976, 48, 368-372. (18) Fox, M. E.: Staley, S. W. Anal. Chem. 1976. 4 6 , 992-998. (19) Das, B. S.; Thomas, G. H. Anal. Chem. 1978, 5 0 , 967-973. (20) Marsh, S.; GrandJean, C. J . Chromatogr. 1978. 747, 411-414. (21) Srnlllie. R. D.; Wang, D. T.; Meresz, 0. J. Envkon. Sci. Heelth, Pert A 1978, A 73, 47-59: (22) Bcden, H. J . Chromatogr. Sci. 1976. 74, 391-395. (23) Fechner, D.; Seifert, B. In "Polynuclear Aromatic Hydrocabns"; Jones, P. W., Leber, P., Eds.; Ann Arbor Science: Ann Arbor, MI, 1979: pp 191-199. (24) Snook. M. E. Anal. Chim. Acta 1976, 81. 423-427. (25) Toussaint, G.; Walker, E. A. J . Chromatogr. 1979, 177, 448-452. (26) Lho. J. C.; Browner, R. F. Anal. Chem. 1978, 50, 1683-1686.

RECEIVED for review January 21,1980. Accepted July 28,1980. This work was supported by National Health Research and Development Project Grant No. 610-1138-40 and by a Research Scholar award to the principal author from the Department of National Health and Welfare, Canada.

Solvent Extraction of Scandium(II1) A. D. Langade and V. M. Shinde' Department of Chemistry, Shivaji University, Kolhapur 4 16 004, India

Separation of scandium(111) from iron( III), molybdenum(VI), vanadium( V), chromium(VI), tltanlum( IV), bismuth( III), rlrconium(IV), lanthanum(III), and thorium( I V ) is achieved by solvent extraction with mesltyi oxide from sodium sallcylate solution (0.1 M) adjusted to pH 4. Scandlun from the organic

phase Is stripped with water and determined photometrically as Its arsenazo complex at 570 nm. The extracted species is trisoivated, Le., Sc( HOC6H,C00),*3Me0.

Separation and purification of scandium are desired as it is associated with many cations in minerals. However, very few methods are available for the separation of scandium by liquid-liquid extraction. In this article we propose a simple and rapid method for the selective extraction of scandium(1II) from salicylate media by using mesityl oxide as an extractant. The metal ion is stripped with water and determined photometrically with arsenazo I (1) a t 570 nm. Various methods for the solvent extraction of scandium have been summarized and critically reviewed by different authors in their monographs (2-6), but a method for extractive separation of scandium(II1) is lacking. In this paper we describe quantitative extraction of scandium from sodium salicylate media (pH 4) into mesityl oxide which affords its quantitative separation from metal ions such as iron, vanadium, molybdenum, chromium, titanium, bismuth, zirconium, thorium, and lanthanum.

EXPERIMENTAL SECTION Apparatus and Reagents. Absorbance measurements were taken on a Zeiss spectrophotometer (German) using l-cm quartz cells; pH values were measured on a Philips pH meter (Precision type).

The stock solution of scandium(II1)was prepared by dissolving 0.15 g of scandium oxide (Koch-Light, England) in 3 mL of concentrated hydrochloric acid and evaporating to dryness. The residue was taken up in 100 mL of 0.1 M HCl and standardized complexometrically with EDTA (7).The diluted solution containing 25 pg/mL of scandium was prepared by suitable dilution. Mesityl oxide (bp 125-128 "C (BDH)) was used after double distillation. Arsenazo I ww used as a 0.1% aqueous solution. All other chemicals used in this work were of guaranteed grade. General Extraction Procedure. To an aliquot portion of solution containing 25 pg of scandium, 0.4 g of sodium salicylate was added to make a concentration 0.1 M in a total volume of 25 mL. The pH of the aqueous solution was adjusted to 4 by NaOH/HCl solutions by use of a pH meter and then the mixture was equilibrated for 20 s in a separatory funnel with 10 mL of undiluted mesityl oxide. The two layers were separated, and the metal ion from the organic phase was stripped with two 10-mL portions of water (containing a few drops of HC1 to avoid emulsion). The combined aqueous phase is evaporated to dryness, and scandium is estimated photometrically with arsenazo I at 570 nm against the reagent blank prepared analogously.

RESULTS AND DISCUSSION Effect of pH, Salicylate Concentraion, and Mesityl Oxide Concentration. The extraction of scandium was studied at various pH values, sodium salicylate concentrations (0.01-0.1 M), and mesityl oxide concentrations (35-100% using benzene as diluent). The results in Figure 1 indicate that the optimum pH for quantitative extraction of scandium salicylate species is 4. Beyond pH 4 the extraction decreases. Similarly the quantitative extraction of scandium(II1) occurs from 0.1 M sodium salicylate solution and extraction must be performed with 10 mL of undiluted mesityl oxide. The distribution ratio was calculated as usual (8). An attempt was made to ascertain the composition of the extracted species. Scandium was extracted a t fixed pH and

0003-2700/80/0352-2031$01.00/00 1980 American Chemical Society