ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
(8) I. P. Nathanson, "ConstructiveFunction Theory", Frederik Ungar Publishing Co., New York, N.Y., 1964. (9) I. Halasz, Anal. Chem., 36, 1428 (1964). (10) R. D. Condon et al., in " G a s Chromatography", R. P. W. Scott, Ed., Proc. 3rd Symp., Butterworths. London, 1960, p 30. (11) A . G. Zacchei and L. Weidner, J . Pharm. Sci., 64, 814 (1975). (12) A. Bromberg, private communication. (13) F. van de Craats, in "Gas Chromatography". D. H. Desty, Ed., Proc. 2nd Intl. Symp., A.P., 1958, p 248. (14) E. D. Pellizzari, J . Chromafogr., 98, 323 (1974). (15) L. J. Schmauch and R. A. Dinerstein, Anal. Chem., 32, 343 (1960).
(16) (17) (18) (19) (20) (21)
1429
L. CondaCBosh, J . Chem. Educ., 41, A235 (1964). I . J. Young, Am. Lab.. 7 (6), 37 (1975). I. J. Young, Am. Lab., 7 (a), 11 (1975). W. A. Dietz, J . Gas Chromatogr., 5 , 68 (1967). D. M. Rosie and R. L. Grob, Anal. Chem., 29, 1263 (1957). 0. Hainova, P. Bocek, J. Novak, and J. Janak, J . Gas Chromatogr., 5. 401 (1967).
RECEKED for review January 5, 1978. Accepted April 18, 1978.
Isolation of Polycyclic Organic Compounds by Solvent Extraction with Dimethyl Sulfoxide D. F. S. Natusch" Department of Chemistty, Colorado State University, Fort Collins, Colorado 80523
B. A. Tomkins' School of Chemical Sciences, University of Illinois, Urbana, Illinois 6 180 1
increasing polarity are used to elute the sample from a silica gel or alumina column. POM is thus separated and classified on the basis of its polarity. While the Rosen separation of POM has been widely used with considerable success (13-1 3, it does have several disadvantages. Thus, the procedure is time-consuming, requires considerable experience for successful operation, requires large quantities of solvents which must be subsequently removed, and is subject to loss of material by irreversible adsorption (17). In addition, the Rosen separation is designed to separate polycyclic aromatic hydrocarbons (PAH) so that their more polar derivatives may not be included in the so-called POM fraction. As a possible alternative to the Rosen separation we have developed a simple, rapid, solvent extraction procedure which cleanly separates POM from aliphatic material. This procedure, parts of which have been previously reported (8, 10, 18, 20, 21) involves extraction of P O M from a low-polarity solvent into dimethyl sulfoxide (DMSO) followed by displacement of the separated POM into fresh solvent by addition of water. In the following sections, we report an evaluation of the performance characteristics of this DMSO extraction procedure and of its compatibility with subsequent analytical steps. The partition ratios of a variety of both aliphatic and polycyclic aromatic compounds have been determined in order to establish the types of compounds that can be separated and to provide some insight into the POM-DMSO interactions which operate.
A rapid liquid-liquid extraction procedure has been developed for isolating polycyclic organic matter (POM) from complex mixtures of organic compounds present in an aliphatic hydrocarbon solvent. POM is first extracted into dimethyl sulfoxide (DMSO) and is then back-extracted into fresh pentane by addition of water to the DMSO. The procedure enables isolation of (a) aliphatic hydrocarbons, (b) alcohols, phenols, and low molecular weight aliphatic and aromatic acids, and (c) POM, phthalates, aromatic bases, and high molecular weight aliphatic acids. Partition ratios have been obtained for a variety of solute species in several solvent systems and the influence of temperature and the DMSO/water ratio has been established. Partition ratios are governed by the extent of interaction between the sulfur atom in DMSO and the aromatic 7r electron system. I n addition, a hydrogen bonding interaction involving the DMSO oxygen atom is indicated where hydroxyl groups are present.
I t is now well established that a large number of polycyclic aromatic compounds are produced during the combustion of carbonaceous materials and that they are widely distributed throughout t h e environment (1-10). Many of these compounds, which are collectively referred to as polycyclic organic matter (POM) exhibit pronounced carcinogenic, mutagenic, and teratogenic effects in biological systems. As a result considerable emphasis is currently being placed on the determination of POM in a variety of environmental systems ( 11-1 9). As encountered in the environment, POM usually constitutes only a small fraction of the organic material present. I t is frequently desirable, therefore, to separate the POM from a considerable excess of aliphatic material prior to analysis. T h e accepted method for such a separation is t h a t due to Rosen and Middleton ( I I , 1 2 ) . This involves ambient pressure liquid-solid chromatography in which solvents of gradually
EXPERIMENTAL Reagents a n d Chemicals. All solutes tested were obtained in 98% or better purity and were used as received. The solvents used were of either reagent grade or "distilled in glass" quality and were also used as received. Apparatus. The absorbance of a given solution was measured using a Cary 17 absorbance spectrophotometer and quartz cuvettes. Partition ratio measurements at elevated temperatures were performed using a constant temperature (fl "C) water bath. Fluorescence measurements were performed using an Aminco-Bowman Spectrophotofluorimeter, model 54-8940, with control module 54-8941. The excitation wavelength was optimized at 310
'Present address, Department of' Chemistry, Colorado State University, Fort Collins, Colorado 80523. 03O3.2700/78~03~0-'429$0 1 09/0
f
1978 American Chemical Society
1430
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
Table I. Gas Chromatographic Conditions Used in the Analysis of PAH instrument Tracor 500 Varian 2800 column 1 2 ft X 1 2 ft x in. 0.d. glass, in. 0.d. glass, nonsilanized nonsilanized packing material stationary phase 1.5%Dexsil 300-GC 2% Dexsil 300-GC support Chromosorb W-AW Gas Chrom Q, 80/lOO mesh 80/lOO mesh carrier gas and flow rate, mL/min nitrogen, 20 helium, 40 FID detector FID hydrogen flow, mL/min 45 45 air flow, mL/mino 300 300 operating temp., C 360 360 injector temp., " C 285 285 outlet temperature, C 340 340 oven temperatures, 'C isothermal for 2 min 165 100 5 rate, "C/min 8 300 final temperature 360 1024 X 8 X 10'" attenuation, amperes full scale nm, and the emission spectrum was scanned between 300 and 700 nm. Gas chromatography was used to analyze both synthetic mixtures of PAH and natural samples. The specific operating parameters are presented in Table I. Procedure. Standard solutions of aliphatic hydrocarbons. aliphatic acids, POM, phenols, phthalate esters, or aromatic bases dissolved in pentane were used to evaluate the DMSO liquidliquid partition. Typically, the standard solution is partitioned three times with equivolume amounts of fresh DMSO at room temperature. The DMSO layers are then separated from the pentane, combined, and diluted with two volumes of water. The POM is then hack-partitioned three times with equal volumes of pentane. The final solution is washed once with an equal volume of water to remove traces of DMSO, and can then be concentrated at low temperature for subsequent analysis by gas or liquid chromatography. The change in the partition ratio as the DMSO layer was diluted with water was observed by preparing solutions of constant pyrene concentration in which the volume/volume percentage of water was changed. (Caution: Considerable heat can be generated upon mixing DMSO and water.) All solutions were permitted to cool to room temperature before determining the partition ratio. Partition ratios for individual POM species were established by equilibrating individual solute species between two immiscible solvents in a separatory funnel using at least three volume ratios. The initial and final concentrations of solute were determined by UV-visible spectroscopy. The wavelengths chosen for quantitation were generally those exhibiting maximum absorbance; in a few cases, however, local maxima were employed t o avoid spectral interferences. The values of the partition ratio, D, reported represent the average of three determinations. The partition ratio, D, is calculated from POM species using the equation
D=-
AI* *CVII/VI) A: - AI
where A? and AI* are the absorbances of the initial and final solutions, respectively, of the solute in solvent I (either neat DMSO or DMSO/water), VI is the volume of solvent I, and VII is the volume of solvent I1 (an aliphatic hydrocarbon solvent). This equation, which uses two absorption measurements in the same solvent, permits a rapid calculation of D without prior evaluation of the absorptivity, but becomes imprecise at large values of D , where AI* approaches A t . The fraction of solute,,:$I recovered in solvent I after n discrete partitions is readily calculated using a known value of D from the relationship The DMSO/pentane system achieves phase equilibrium with normal shaking in 5 min or less; the layers separate cleanly in as little as 10 s. By contrast, DMSO-water/pentane may form an emulsion which takes about 10 to 15 min to clear. Adding ice
Varian 2800 6 ft x * / 4 in. 0.d. glass, nonsilanized 3% SP-2100 Supelcoport, 80/lOO mesh helium, 55 FID 44 300 350 275 350 15 4 325; hold 1 6 rnin 2x
lo-''
Table 11. Partition Ratios for Pyrene between DMSO and Several Solvents solvent
I)
cyclohexane N-heptane isooctane N-pentane N-hexane
4.6 5.1 8.7 9.0 11.5
or a small quantity (about 250 mg) of salt to the DMSO/water solution helps to inhibit emulsion formation. The performance of the proposed DMSO partitioning scheme was tested using a sample of fly ash which was collected in the plume of a coal-fired power plant using a Hi-Volume sampler. This sample was Soxhlel extracted with cyclohexane for 24 h. The extract was then concentrated and partitioned as described previously. Determination of Factors Affecting Partition Ratios. In order to evaluate the perforrnance of each stage of the separation procedure and to establish its limitations, the following series of experiments were performed. Solvent Type. Using pyrene as a test compound, partition ratios between DMSO and n-pentane, n-hexane, n-heptane, isooctane, and cyclohexane were determined. The results (Table 11) illustrate that, while n-hexane provides the largest partition ratio value, all of these solvents will enable quantitative removal of the test compound after three equivolume partitions. However, n-pentane is preferred for general use because of its high volatility and consequent ease of concentration. The difference in the partition ratios of pyrene between DMSO and the various alkanes listed is attributed to the differences in the solubility of the alkane solvents in DMSO (22). Temperature. Pyrene was partitioned between DMSO and n-heptane at four temperatures, ranging from ambient to 56 "C. No change in the distribution ratio was observed, within the precision of the analytical method. This conclusion agrees with the observation of Howard and Haenni (20) that the partition ratio of benzo[a]pyrene in the DMSO/n-heptane system was virtually unaffected at temperatures as high as 80 "C. Solute Species. In order to establish the types of POM which can be successfully partitioned into DMSO, a variety of compounds, including parent hydrocarbons, substituted polycyclic aromatic compounds, and polycyclic aromatic species containing heteroatoms was partitioned with n-pentane. Values of D and of the percentage recovery of POM from the pentane layer after three partitions with DMSO, 62, are presented in Tables 111-VI. It is apparent from these results that essentially all the compounds tested are amenable to separation by the proposed procedure. The D values listed in Table I11 also indicate that three equivolume partitions are sufficient to remove all these compounds quantitatively from a pentane extract. Efiect of Water: Evaluation of a Stripping Step. It is possible to strip POM from DMSO by adding water and extracting the
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11: SEPTEMBER 1978
Table 111. Partition Ratios for Polycyclic Aromatic Hydrocarbons Distributed between DMSO and N-Pentane compound ~ , n r n * logeb G~',%' D 278 301 361 295 338 323 282 305
naphthalene fluorene anthracene phenanthrene pyrene chrysene triphenylene coronene
3.77 3.97 3.88 3.65 4.65 4.07 4.27 5.23
2.3 3.0 4.9 4.4 9.0 19.7 28.5 36.7
97.3 98.5 99.5 99.4 99.9
=loo
= 100 = 100
1431
Table VI. Comparison of Partition Ratios for the DMSO/Pentane System as t h e 9-Position of Anthracene Is Changed compound 9,lO-dihydroanthracene xanthene thioxanthene anthracene acridine
a Wavelength at which absorbance measurements for D were made. Molar absorptivity at measurement wave= recovery of compound from pentane solulength. tion after three partitions with equal volumes of DMSO.
A,
log
nm 272 282 268 361 356
e
&I, %
l3.05 :3.42 3.98 3.88 4.10
94.9 98.8 99.4 99.5 -100
D 1.7 3.4 4.6 4.9 13.9
""h
v,= vn.
Table IV. Partition Ratios for Anthracene Derivatives Distributed between DMSO and N-pentane 1%
A,
compound nm e G~',% n-electron-poor substituents 9-methylanthracene 9-bromoanthracene 9-chloroanthracene 9,10-dimethylanthracene 9,lO-dibromoanthracene 9,lO-dichloroanthracene
371 395 372 383 385 383
3.90 3.85 3.90 3.97 4.00 4.03
D
99.5 99.3 99.0 98.4 95.8 89.5
I
Q
1
318 328 368 400 481 537
3.53 3.73 3.62 3.81 3.82 3.95
substituents affected by solvent association
-
9-anthracenemethanol 368 3.94 -100 9-anthracenecarboxylic acid 376 3.76 100 _
_
~
>50 39
~
Table V. Comparison of Partition Ratios for Polycyclic Aromatic Hydrocarbons and Their Aldehydes Distributed between DMSO and Pentane compound naphthalene
A,
nm
1% E
278 3.77
2-naphthalenecar boxaldehyde 328 4.10
fluorene 2-fluorenecarboxaldehyde phenanthrene
D
@31,%
97.3 99.9 98.5
phenanthrene-g-carboxalde-
301 320 295 318
3.97 4.44 -100 3.65 99.4 4.12 -100
hyde anthracene 9-anthraldehyde pyrene 1-pyrenecarboxaldehyde
361 400 338 395
3.88 99.5 3.81 -100 99.9 4.65 4.11 -100
> :
> :
2.3 9.3 3.0 35.7 4.4 50 4.9 50 9.0 49.3
--
DMSO/water layer with fresh aliphatic solvent (21). Again, n-pentane was used as the aliphatic stripping solvent. Partition ratios for pyrene were measured at different concentrations of water, as shown in Figure 1. The ratio of two volumes of water to one volume of' DMSO clearly reduces the value of D to a level such that pyrene can be quantitatively removed from DMSO/water by a single equivolume partition with pentane. The stripping procedure was rigorously tested by measuring the partition ratios of the anthracene analogues listed in Table IV between n-pentane and 2:l v/v water/DMSO. With the exception of 9-anthracenemethanol, 9-anthracenecarboxylic acid, and 9,10-dibromoanthracene, all rompounds either precipitated
\
1
4.7 4.3 3.6 3.0 1.9 1.1
99.9 11.1 99.9 9.67 =lo0 38.1 -100 >50 -100 >50 e100 >50
a\
0.1
1 t
n-electron-rich substituents anthrone anthraquinone 9-nitroanthracene 9-anthraldehyde 1-aminoanthraquinone 1-amino-4-hydroxyanthraquinone
\
"""0
\ 10
20 30 40 50 yo Water Content, vol/voI
60
7O
Figure 1. Distribution ratio of pyrene between DMSO/H,O and n-pentane as a function of DMSO/H20 volume ratio
or else exhibited partition ratios much less than 0.3. Of the three exceptions, the first two probably formed a solvent association complex with DMSO (22) and were retained in DMSO/water. 9,lO-Dibromoanthracene was present as a stable colloid in DMSO/H20 and strongly resisted partition into pentane. The addition of a small quantity of salt to the DMSO/H20 mixture dispersed this colloid and permitted rapid and quantitative extraction of 9,lO-dibromoanthracene into pentane.
OVERALL METHOD EVALUATION A N D RESULTS T h e overall method was evaluated on t h e basis of (a) the efficiency of the separation of POM from aliphatic hydrocarbons, and (b) the efficiency of separation of POM from other classes of compounds, such as aliphatic and aromatic acids, phenols, organic bases, and phthalate esters, which may be present in the extract. A pentane solution of polycyclic aromatic hydrocarbons (naphthalene, acenaphthylene, and anthracene) and aliphatic hydrocarbons (CI8, CI9, Cz0,CZ1,C22,and C2J was analyzed by gas chromatography, as shown in Figure 2a, then partitioned three times with equal volumes of DMSO. The DMSO layers were then combined. T h e residual pentane solution was washed once with water, then analyzed by gas chromatography, as illustrated in Figure 2b. Two volumes of water were added to the combined DMSO layers, and the resulting solution was partitioned three times with equal volumes of n-pentane. The pentane layers were combined, washed once with water, concentrated, and analyzed by gas chromatography, as illustrated in Figure 2c. Comparison of the three chromatograms in Figure 2 indicates that a clean separation of the PAH and aliphatic hydrocarbons was achieved. I n a similar fashion, pentane solutions of other organic compound classes were partitioned with DMSO. As shown in Table VII, neutral aromatic species, such as the organic
1432
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
ll
0
TIME, rnin
Figure 2. Gas chromatograms of test mixture of polycyclic aromatic and aliphatic hydrocarbonsat several stages of the proposed separation scheme. (a) Original mixture, (b) original mixture after three equivolume DMSO partitions and water wash, (c) pentane solution used to strip DMSOIH,O 1:2 vlv. Peaks: 1, naphthalene; 2, acenaphthylene; 3, anthracene; 4, n-octadecane; 5, n-nonadecane; 6, n-eicosane; 7, n-heneicosane; 8, n-docosane; 9, n-tricosane
bases and the phthalates, behave similarly to PAH. On the other hand, organic species which can be strongly associated with DMSO, such as the phenols and acids, can be readily partitioned into DMSO, but cannot be stripped easily by adding water. T h e foregoing separation scheme enables three groups of compounds to be separated in a matter of a few minutes. After a complete partition, involving three successive extractions from the original pentane solution into DMSO followed by three successive back-extractions from DMSO/water into fresh pentane, the original pentane solution retains aliphatic hydrocarbons; the DMSO/water solution retains species strongly associated with DMSO, such as alcohols, low molecular weight aliphatic and aromatic acids, and phenols; and the final pentane solution contains PAH, phthalates, aromatic bases,
la
2
d
t
b
I
Y
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6
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x
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b
Y
2 4 6 8
