(16) R. L. Gustafson and J. Paleos, “Organic Compounds in Aquatic Environments”, S. J. Faust and J. V. Hunter, Ed., M. Dekker, New York, 1971, p 213. (17) C. H. Chu and D. 4. Pletrzyk, Anal. Chem., 49, in press. (18) D. J. Pietrzyk, Talanfa, 16, 169 (1969). (19) R. Kunin, E. F. Meitzner, J. A. Oline, S. A. Fisher, and N. Frisch, Ind. fng. Chem., Prod. Res. Dev., I,140 (1962). (20) A. D. Wiiks and D. J. Pietrzyk, Anal. Chem., 44, 676 (1972). (21) R. L. Gustafson, R. L. Albright, J. Heisler, J. A. Lirio, 0. T. Reid, Jr., Ind. Eng. Chem., Prod. Res. Dev., 7 , 107 (1968). (22) J. Paleos, J. Colloid. Interface Sci., 31, 7 (1969). (23) L. R. Snyder, Anal. Chem., 46, 1384 (1974). (24) L. R. Snyder, “Principles of Absorption Chromatography”, Vol. 3, M. Dekker, New York, 1968.
(25) B. L. Karper, L. R. Snyder, C. Horvath, “An Introduction to Separatlon Science’ , J. Wiiey and Sons, New York, 1973. (26) D. J. Pietrzyk, Anal. Chem., 39, 1367 (1967).
RECEIVED for review August 12,1976. Accepted January 25, 1977. Part of this work was presented at the ACS Award in Chromatography Symposium at the Centennial American Chemical Society Meeting, New York, N.Y., April 4-9,1976. This investigation was by Grant Number CA 18555-01,awarded by the National Cancer Institute, DHEW.
Chromatography of Chlorinated Biphenyls on an Ion-Exchange Resin Toshlhiko Hanal and Harold F. Walton” Department of Chemistry, University of Colorado, Boulder, Colorado 80309
A calcium-form, 4 % cross-linked polystyrene-type cationexchange resin is a suitable stationary phase for the chromatography of chlorinated biphenyls, udng aqueous acetonitrile as the eluent. Substitution of chlorine atoms in the 2 position lessens the retention, but substitution in other positions increases k. Seventeen substituted biphenyls were studied over a range of temperatures and solvent compositions, and their ultraviolet absorption spectra are shown. The method Is applicable to traces of biphenyls in contaminated water.
with unsubstituted biphenyl, whereas substitution in the 4 position increased the retention. We ascribed this effect to the forcing of the two phenyl rings out of a common plane and to the consequent lessening of .rr-electron overlap. We have now extended this study to include a number of mono-, bi-, and trichlorobiphenyls. We have studied the effects of solvent composition and temperature, and have shown that a custom-made resin, synthesized from purified monomers, is superior to the ordinary mass-produced resin made for water conditioning.
EXPERIMENTAL For the analysis of mixtures of chlorinated biphenyls, gas chromatography is usually the method of choice. Recently a nematic liquid crystal stationary phase has been found to be effective in separating isomeric chlorinated biphenyls (I, 2), though excessive bleeding from these columns may cause problems. Liquid chromatography has been used by a few workers, and a definitive paper on high-speed liquid chromatography of polychlorinated biphenyls appeared recently (3). The authors of this paper used 5-bm silica gel as the absorbent and dry hexane as the solvent, with detection by ultraviolet absorption at 205 nm. Where the samples to be analyzed are partly or wholly aqueous, as is the case with environmental samples, it may be preferable to use an absorbent-solvent system that is compatible with water. Polluted waters that contain chlorinated biphenyls at parts-per-billion levels may be preconcentrated by pumping them through a column of a suitable absorbent, then they may be released and separated by chromatography with a mixed aqueous-nonaqueous solvent mixture, using a gradient if desired. Trace amounts of organic contaminants in water have been analyzed in this way, using methanol-water and acetonitrile-water gradients and a C18-bonded silica as the stationary phase (4). We have been exploring the uses of weakly cross-linked styrene-divinylbenzene ion-exchange resins as stationary phases for the chromatography of nonpolar aromatic compounds, and we reported the chromatography of polycyclic aromatic hydrocarbons on a 4% cross-linked cation-exchange resin (5). A separation of 2- and 4-substituted mono- and bichlorobiphenyls was reported. We noted that chlorine substitution in the 2 position lessened the retention, compared 764
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
Materials. The resin was a sulfonated styrenedivinylbenzene copolymer with 470 cross-linking, supplied by the Hamilton Company, Reno, Nev. The particle size was 10-15 km. Following our earlier experience (5),it was converted to the calcium form before use. The carbon-18bonded packing used for comparison tests was a prepacked column, Partisil O.D.S. from Whatman, Inc. Biphenyl and chlorinated biphenyls, including samples of Arochlors, were supplied by Analabs, Inc., North Haven, Conn. Acetonitrile was furnished by Burdick and Jackson Laboratories, Muskegon, Mich. Equipment and Procedure. These were essentially the same as those described earlier. Most work was done with waterjacketed glass columns, 6.3-mm i.d., supplied by Glenco Scientific, Inc., Houston, Texas; the resin beds were 20-25 cm long. The solvents were acetonitrile-water mixtures. Detection was by ultraviolet absorption at 254 nm. Absorption spectra were recorded in 1-cm cells with Cary Models 14 and 17 spectrophotometers.
RESULTS Comparison of Resins. Our first experiments ( 4 ) were made with the resin Aminex 50W-X4,20-30 bm, supplied by Bio-Rad Corp., Richmond, Calif. The Hamilton resin, used in the work now being reported, performed better in three respects. First, the back-pressure was much less; for 20-cm columns at a flow rate of 24 mL/h with 35% acetonitrile (v/v) a t 65 “C, the Aminex resin required 300 psi, the Hamilton resin only 120 psi, in spite of its smaller particle size. Second, the theoretical-plate heights for biphenyl for this solvent, temperature, and flow rate were 0.16 mm for the Aminex resin, 0.11 mm for the Hamilton resin. Third, the retention was somewhat greater with the Hamilton resin, but retention volumes depend so much on solvent composition and temperature (see below) that this comparison may be invalid. The
E
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0 4 c
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I
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U U U 40 55 70 40 55 70 40 55 70 TEMPERATURE 'C
Figure 1. Capacity factors, temperature, and sobent composition. The three sets of curves refer to 35 % , 30 % , and 25 % acetonitrile (v/v), respectively, Symbols: (0)2,2'-CI2;(X) 241; (A)unsubstituted biphenyl; (0)2,3-CI,; (+) 4-CI; (0) 341
Figure 2. Chromatogram of an 8-component mixture. Column, 0.63 cm X 24 cm; solvent, 30% (v/v) acetonitrile; temperature, 65 OC; flow rate, 12 rnL/h. Micrograms injected: 2,2'-, 2.7; 2-, 0.5; biphenyl, 0.1; 2,3-, 0.4; 4-, 0.08; 3-, 0.1; 4,4'-, 0.1; 3,3', 0.2
Table I. Capacity Factors and Elution Sequence, 35% CH,CN, 55 "C Capacity factor, k' Chlorine-substituted Ion-exchange C, ,-bonded packing, biphenyl resin 22 "C 2,2' 2
Biphenyl 294 2.5 2;4' 2,3 2,5.4' 2,4,4' 4 3 3,4,2' 4,4' 3,5 394 3,3' 3,4,3',4' 2,3,2',3'
1.23 1.53 1.73 1.80 1.91 1.97 2.07 2.27 2.31 2.35 2.73 3.01 3.03 3.25 3.50 3.90 5.72 6.87
2.61 2.28 1.74 4.72 4.34
4.04 3.80 7.66 7.63 3.02 2.96 7.02 4.82 5.98 5.24 5.03 12.00 6.26
makers of the Hamilton resins claim that their method of polymerization gives beads of homogeneous cross-linking,while large-scale polymerization gives beads that are relatively highly cross-linked a t the center and weakly cross-linked near the surface. It is known that styrene molecules combine faster with divinylbenzene molecules than with other styrene molecules (6) and inhomogeneous cross-linking is expected if the heat of reaction makes polymerization go faster in the center of the bead than near the surface. Beads that have a soft, weakly cross-linked surface zone will pack closely together and obstruct the solvent flow. Effects of Temperature and Solvent Composition. Figure 1 shows these effects, which are considerable, and points out the need for careful temperature control. The greatest differences in retention volumes are found at high acetonitrile concentrations and low temperatures. Lower temperatures give broader elution bands, however, and the optimum conditions, taking into account resolution and the running time, are probably 50-60 "C with 35% (v/v) acetonitrile. The slopes of the lines in Figure 1 show that the stronger the retention, the greater is the enthalpy of absorption. Elution Orders a n d Typical Chromatograms. The elution sequence of the compounds tested is shown in Table I; representative chromatograms appear in Figures 2 and 3. Figure 3 was recorded at a slow flow rate and under conditions
-
a1
I 20
10
1 30
I 40
VOLUME, m l
Figure 3. Chromatogram of a 9-component mixture. Column, 0.63 cm X 24 cm; solvent, 35% (v/v) acetonitrile; temperature, 25 OC; flow rate. 6 mL/h
10
20
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40
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60
V O L U M E , rnl
Figure 4. Chromatograms of Arochlors. Conditions as a Figure 3. Full scale absorbance, 0.32 unit
that give strong retention, and the running time (7 h) is too long for normal use, but 2,3-bichlorobiphenyl and 4-chlorobiphenyl are better resolved than they are in Figure 2. The peak of 3-chlorobiphenyl indicates a theoretical-plate height of 0.065 mm in Figure 2 and also in Figure 3. The running time in Figure 2 is 100 min. A peak separation that was nearly as good was obtained a t twice the flow rate, namely, at 24 mL/h, with running time 50 min. Figure 4 shows chromatograms of three Arochlors, mixtures of chlorinated biphenyls containing respectively 32 % , 42%, and 54% chlorine by weight. Arochlor 1232, with 32% C1, has an average of two chlorine atoms per molecule. The chromatogram, run under the same conditions as that of Figure 3, has five major peaks that correspond to the following substances: 2-chlorobiphenyl; biphenyl (or 2,4- or 2,5-bichlorobiphenyl); 2,3-bichloro-; 4-chloro- (or 2,4,4'- or 2,5,4'trichloro-); and 3-chloro-, with a low flat peak beyond that could be 3,4-bichloro-. The problem of identifying overlapping ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
765
0 9
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0 v)
0 4
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W A V E L E N G T H , nrn
Figure 5. Absorption spectra in 100% acetonitrile. Concentrations in W / L : biphenyl, 9; 2-chloro, 10; 2,2’-bichloro,8 0 2,3-bichloro, 12.5; 4-chloro and 4,4’-bichloro,each 5.0; 3-chlor0, 3.5. The spectrum of 313’-bichlorobiphenyI(3.5 mg/L) was so close to that of $&br&iphenyl that it is not shown. Cell thickness, 1 cm
peaks is obvious from the elution volumes given in Table I. Spectra. Peak resolution and identification might, however, be helped by using different wavelengths for detection. The spectra shown in Figures 5 and 6 show very clearly the effect that 2 substitution has on the ultraviolet absorption. One could not distinguish 2,4,4’- from 2,5,4’trichlorobiphenyl, but one could easily distinguish either of these from 4-chlorobiphenyl, whose elution peak overlaps the peaks of these trichlorobiphenyls. Likewise 3,4,2’-trichlorobiphenyl and 4,4’-bichlorobiphenyl, whose elution volumes are very close together, could be distinguished by their spectra. The analysis of mixtures of chlorinated biphenyls would be an interesting exercise in multiparameter monitoring. Figures 6 and 6 show the “n. bands” of the absorption spectra, not the main bands that occur in the range 200-220 nm (3). The main bands are more intense, and were used by Brinkman in the work cited, but in our case they were hidden by the absorption of the acetonitrile solvent. Ref. (3)shows the spectra of four chlorinated biphenyls in hexane and a table of absorption maxima in the same solvent. The a bands of different isomers differ considerably in their intensities; 2 substitution not only shifts the absorption to shorter wavelengths, but it also weakens it considerably (5). Collection of Trace Amounts from Water. The idea that our chromatographic system might be used to analyze ,traces of chlorinated biphenyls in contaminated water was tested in a crude way by preparing a solution of 0.25 mg of Arochlor 1232 in 1 L of 10% acetonitrile, then pumping this solution through the resin column a t 24 mL/h for 105 min with the column maintained at 50 “C. At the end of this period the influent was changed to 35% (v/v) acetonitrile, and the absorption of the effluent a t 254 nm was recorded. A chromatogram very much like that in Figure 4 was obtained, indicating that the technique is basically sound. The solubility of the more highly chlorinated biphenyls in water is so small that, in making synthetic solutions for testing, one must beware of precipitation and adsorption on the walls of glass containers. Comparison of Resin with Microparticulate CISBonded Silica. The familiar reverse-phase C18-bonded packing can also be used for the chromatography of chlorinated biphenyls. Tests were made with aqueous ethanol, 766
ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
‘230
240
250 260 2 7 0 2 8 0 WAVELENGTH, nm
290
Figure 6. Absorption spectra in 100% acetonitrile. Concentrations in mg/L: 2,4- and 2,5-bichloro, 12.5; 3,4- and 33-, 10; 2,5,4’-, 2,4,4‘-, and 3,4,2’-trichlorobiphenyl, 10. Cell thickness, 1 cm
methanol, and acetonitrile at room temperature. The elution sequences with all these solvents were nearly the same, and were very different from the sequence found on the cationexchange resin. Data for 35% acetonitrile (v/v) are shown in Table I. The desorbing effect of 2 substitution is not absent in the c18 packing (probably it is a solvent effect) but it is far less pronounced than it is on the resin. The (218 packing is not aromatic in character, and a-orbital overlap is less important. DISCUSSION Compared with the liquid chromatographic technique of Brinkman et al. 031,our method has less sensitivity and lower resolution. We tried to increase the resolution by using a longer column, 60 cm instead of 25 cm, but found that the longer column gave considerably more than twice the back pressure of the shorter one and that the resin became compressed and compacted under flow. Two 25-cm columns in series gave no better plate numbers than a single column, presumably because the end fittings contributed significantly to the band broadening. Nevertheless, the theoretical-plate heights of 0.05-0.07 mm that were obtained with the 25-cm columns were highly satisfactory. The advantages of this method, compared with that of Brinkman et d.,are the compatibility with water and the selective effect of 2 substitution, as well as the ease of column preparation. ACKNOWLEDGMENT We thank Maria-Elena Gonzalez for help in the laboratory, and James V. Benson, of the Hamilton Company, for his suggestions. LITERATURE CITED (1) G. M. Janlnl, K. Johnston, and W.L. Zillnski, Anal. Chem., 47, 670 (1975). (2) Analabs. Inc., technical literature. (3) U. A. Th. Brlnkman, J. W. F. L. Seetz, and H. G. M. Reymer. J . Chromatogr., 116, 353 (1976). (4) J. N. Llttle and G. J. Falllck, J . Chromatogr., 112, 389 (1975). (5) D. M. Ordemann and H. F. Walton, Anal. Chem., 48, 1728 (1976). (6) R. H. Wiley and E. E. Sale, J . Polym. Scl., 42. 491 (1960).
RECEIVED for review October 7,1976. Accepted January 21, 1977. Support is acknowledged from the National Science Foundation, Grants MPS 73-08592 and CHE 76-08933.
