Anal. Chem. 1980, 52, 2275-2277
screening and quantitating mirex levels down to the 500-fg level. The multiple-ion detection technique extends the determination to include related degradation products and provides a high probability for correct identification when one considers four separate ion ratios and retention times fit. T h i s procedure does not suffer from the interferences previously reported by Laseter et al. (6) and can be employed for a broad range of samples.
LITERATURE CITED (1) Millard, 8. J. "Quantitative Mass Spectrometry"; Heyden Publishing Co.: London, 1978; Chapter 6. (2) Ciaeys, M.; Markey, S. P.; Maehaut, W. Bbmed. Mass Spectrom. 1977, 4 (2),122-128.
(3) (4) (5) (8) (7)
(8) (9) (10)
2275
Van Vaeck, L.; Van Cauwenbergha, K. Anal. Len. 1977, 10, 467-482. FuJIl,T. Anal. Chlm. Acta 1977, 92, 117-122. Buckle, W. L.; Elchelberger, J. W.; Anal. Chern. 1979, 57, 567A. Laseter, J. L.; Deleon, J. R.; Remel, P. C. Anal. Chem. 1978, 50, 1189-1 172. Deleon, J. R.; Warren, V.; Laseter, J. L. Quant. Mass Spectrom. Life Sei. 1978, 2 , 483. Norstrom, R. J.; Hallet. D. J.; Onuska, F. I.; Comba, M. E. Environ. Scl. Techno/. 1980, 74, 860-866. Hallett, D. J.; Norstrom, R. J.; Onuska, F. I.; Comba, M. E.; Sampson, R. J . Agric. FocdChem. 1976. 2 4 , 1186-1193. "Analytical Method Manual-1979"; Environment Canada, Inland Waters Directorate-WQB: Ottawa, Aug 1979.
for review September 13, 1979. Accepted August 20, 1980.
Determination of Trace Amounts of Alkylbenzenesulfonates by High-Performance Liquid Chromatography with Fluorimetric Detection Atsuo Nakae," Kazuro Tsujl, and Makoto Yamanaka Tochigi Research Laboratories, Kao Soap Co., Ltd., 2606, Akabane, Ichikai-machi, Haga-gun, Tochigi, Japan
Trace amounts of aikylbenzenewlfonates (ABS) In river water were determined by reversed-phase hlgh-performance liquid chromatography (HPLC) without pretreatment. The fluorhetrk detector was operated at 225 nm (excitation) and at 295 nm (emission). Alkyl chaln distributions and partial phenyl isomer compositions of the determined ABS were also Obtained. By addltion of a large amount of sodium dodecyi sulfate to the collected samples and the standard ABS solution, the adsorption loss of ABS was negllgible. Relative standard deviation for the river water, contalning 0.097 Fg/mL of ABS, was 2 % .
Trace amounts of alkylbenzenesulfonates (ABS) in the environment have been determined by colorimetry with methylene blue ( I ) , infrared spectrometry (2, 3 ) , gas chromatography (GC) (4-6), atomic absorption spectrometry (7, 8),and high-performance liquid chromatography (HPLC) ( S I 2 ) . Colorimetric methods based on methylene blue have long been used but often give erroneous results due to interference by many organic and inorganic materials. Colorimetric results, therefore, have been presented not in terms of ABS but of methylene blue active substances. Infrared spectrometric methods based on the extraction of amine salts of ABS require several different separation steps and a concentration step. The atomic absorption spectrometric method based on the extraction of an ion-association compound with t h e bis(ethylenediamine)copper(II) or t h e tris(1,lOphenanthroline)copper(II) cations is a highly sensitive method, but ABS is determined indirectly. GC has very high efficiencies for the analysis of individual components of ABS. Moreover, specific and highly sensitive detectors such as flame photometry (13),electron capture ( 5 ) ,and mass spectrometry (6) enable trace amounts of ABS to be determined accurately without interferences. GC, however, requires the coversion of ABS into volatile derivatives such as the methylsulfonates or sulfonyl chlorides before the analysis, and i t is not appli0003-2700/80/0352-2275$01 .OO/O
cable to multisample analysis. HPLC is the most suitable method for the analysis of ABS, because it does not require the conversion of ABS into volatile derivatives. In a previous paper (9),we reported that 1-5 hg of ABS was determined by HPLC employing UV detection a t 225 nm, with silica gel as a stationary phase and n-hexanelethanol (8:2, v/v) containing sulfuric acid as a mobile phase. Since ABS eluted under the above chromatographic conditions giving a single peak,information on the alkyl chain distributions and the isomer compositions was not obtained. We also reported that even- or odd-carbon-numbered ABS were separated by HPLC using porous microspherical poly(styrene-divinylbenzene) gel as a stationary phase and 0.5 M perchloric acid in methanol as a mobile phase (14). But this method was not applicable to the determination of trace amounts of ABS in the environment, because commercial ABS gave a complicated chromatogram. In this paper, we describe improved chromatographic conditions for determining ABS in river water with improved results.
EXPERIMENTAL SECTION Apparatus. The liquid chromatograph consisted of a Hitachi 635 pump with a Hitachi 650-10 LC fluorescent spectrophotometric detector (Hitachi Scientific Instruments, Inc., Tokyo, Japan), a NS NHV-5000-6MP injection six-way valve with a 500-fiL loop (Nippon Seimitsu Co., Tokyo, Japan), an ATTO perista minipump (ATTO Co., Tokyo, Japan), and a Haake Model FE circulator (Haake Inc., Karlsruhe, West Germany) for column temperature control. Peak areas and retention times were obtained with a Shimadzu Chromatopac-E1A data processor (Shimadzu Scientific Instruments, Inc., Kyoto, Japan). Reagents. Deionized water was distilled once in an all-glass apparatus. ABS and sodium dodecyl sulfate (SDS) were obtained from our company and were purified to remove unreacted and inorganic substances. All other reagents were of analytical-reagent grade. Standard ABS Solution. ABS solutions (0.01-0.50 pg/mL) containing 0.5% (v/v) formalin, which prevents the biodegradation of ABS, and SDS (100 gg/mL) were prepared. Sample Preparation. A 0.5-mL formalin solution containing 10 mg of SDS was added to 100 mL of collected samples from 0 1980 American Chemical Society
2276
ANALYTICAL CHEMISTRY, VOL. 5 2 , NO. 14, DECEMBER 1980
river water, and the mixture was filtered through a 0.45-pm Millipore filter (Millipore Co., Sive City, MA). Chromatographic Procedure. Analyses were carried out on a 15 X 0.46 cm column packed with 5-pm LiChrosorb RP-18 (Merck, Darmstadt, West Germany). The column was slurrypacked under the following conditions: slurry liquid, carbon tetrachloride containing 5% w/v poly(oxyethy1ene) dodecyl ether (Kao Soap Co., Tokyo, Japan); slurry concentration, 16% w/v; packing pressure, 400 kg/cm2;pump, Hitachi 635, maximum flow; pressurizing liquid, methanol. The mobile phase is 0.1 M sodium perchlorate in methanol/ water (8:2, v/v). The column effluent was monitored at 225 nm of exciting wavelength with 10 nm of slit width and 295 nm of emission wavelength with 15 nm of slit width. The flow rate was maintained a t 1.0 mL/min. The sample loop of the injection valve was washed with methanol and water, respectively,for 1min by using the peristaltic pump as a solvent supplier at flow rate 3 mL/min. After the sample loop was washed, standard ABS or sample solutions were introduced to the sample loop with the peristaltic pump for 1 min. The injection valve was rotated, and the solutions in the sample loop were injected into the chromatograph. The data processor was started simultaneously. RESULTS AND DISCUSSION Fluorescence S p e c t r u m of ABS. ABS in water absorbs light of wavelength 210-240 nm and fluoresces at 27Ck320 nm. By use of 0.1 M sodium perchlorate in methanol/water (8:2) as the mobile phase, the operating conditions of the fluorimetric detector were optimized to correct for a solvent effect and an instrumental constant. When each component of ABS was eluted in the flow cell of the detector, the flow of the mobile phase was stopped and both the excitation and emission spectrums were measured. Under these chromatographic conditions, all components of ABS absorb light of maximum wavelength at 225 nm and fluoresce a t a maximum wavelength of 295 nm. Chromatography. When water was used as a mobile phase, ABS was strongly held to the column and was not eluted. With methanol as a mobile phase, ABS was eluted at the solvent front. By use of various ratios for methanol/ water solutions as a mobile phase, ABS gave complicated chromatograms, and the elution peaks were not identified. In a previous work, homologous ABS were separated by using poly(styrene-divinylbenzene) gel as a stationary phase with a methanolic solution of inorganic acids or their salts as a mobile phase (14). In this study, a methanolic solution containing sodium perchlorate was applied as the mobile phase for the separation, and satisfactory results were obtained. The elution order of ABS follows that of increasing alkyl chain length and, for the same alkyl chain length, that of decreasing distance of the substituent phenyl group from the end of the alkyl chain. With 0.1 M sodium perchlorate in methanol/ water (82) as a mobile phase, ABS was well resolved according t o the alkyl chain length. Moreover, partial separations of positional isomers of ABS were obtained as shown in Figure 1. The logarithms of the capacity factors of 2-phenyl isomers in ABS were directly proportional to their alkyl chain lengths, and similar relationships for the other phenyl isomers of ABS were obtained. By use of these linear relationships, the elution peaks can be identified and ABS in river water can be determined accurately. In the absence of SDS, the alkyl chain distributions and the phenyl isomer composition of the standard ABS were influenced by the time required to introduce the sample with the peristaltic pump to the sample loop of the injection valve. By increasing the introduction time, the longer alkyl chain ABS and its 2-phenyl isomer contents were increased. It seems that the adsorption of ABS to the sample loop is affected by the ABS composition. Therefore, it is difficult to determine
(-4 1 3-6$
O I I
=
10
Ismin
Flgure 1. Chromatogram of standard alkylbenzenesulfonates. Conditions: column 15 X 0.46 cm, 5 pm LiChrosorb RP-18; eluent, 0.1 M sodium perchlorate in methanol/water (8:2); flow rate, 1 mL/min; pressure, 100 kg/cm2;cdwnn temperature, 40 O C ; detector, fluorimetric detector (excitaton at 225 nm and emission at 295 nm); injection volume, 500 pL; sample concentration, 0.10 pg/mL. 4 is t h e position of t h e phenyl group from t h e terminal methyl group on the chain.
Table I. Calibration Data alkyl chain length and
isomer distribution
concn of standard ABS solution, pg/mL 0.50 0.25 0.10 0.05 0.025 0.01
Total Peak Area ( x counts) 331.1 165.4 65.7 29.2 16.4 5.5 Peak Area % C,, ABS
3- to 5-Ph 2-Ph C,, ABS 3- to 6-Ph
2-Ph C,, ABS 3- to 6-Ph 2-Ph
9.7 3.3
8.9 3.8
9.9 4.2
11.0
3.2
12.1
11.0
3.6 ND'"
31.3 12.6
32.8 34.6 38.2 35.0 45.6 12.2 10.0 10.9 9.9 ND'"
22.4
22.2 6.4
6.8
20.8 6.1
25.1 6.2
29.0 43.3 4.5 NDa
11.4 12.4
5.4
6.0 NDa ND'" ND'"
C , , ABS
3- t o 7-Ph 2-Ph a
ND
=
11.7 2.4
2.4
2 . 2 NDa
not determined.
the trace amounts of ABS with accurate results. Due to the addition of a large amount of SDS t o the standard ABS solutions and the collected samples, these adsorption effects were negligible, because SDS adsorbs to the various surfaces instead of ABS and prevents the adsorption loss of ABS. Moreover, SDS does not influence the detection and the chromatographic separations. It was necessary t o inject 500 p L of sample solutions for the determination of 0.01-0.50 pg/mL ABS. This injection volume was remarkably larger than that of conventional reversed-phase HPLC. However, ABS in the sample solution was concentrated to the top of the column since ABS is not eluted with water, and it was well resolved as shown in Figure 1. When the injection volume was above 500 p L , the peaks observed were broad and the 2-phenyl isomers were not separated from the other isomers. Injection of distilled, deionized, or nonpolluted river water did not show any peaks in the ABS eluted region due to organic impurities in the water. Calibration Curve. Standard ABS solutions were analyzed by the chromatographic procedure. The calibration data are listed in Table I. A linear relationship was obtained between the concentration and the total peak area calculated by the summation of areas of individual components. Above 0.10
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
Tama R i v e r
Ta River
Table 11. Determination of Sodium
Alkylbenzenesulfonates in River Water amt found,a pg/mL
(I
c11
I
range, pg/mL RSD, o/o
Ta River,
0.097
0.096-0.100
2.0
Taiso Bridge Tama River, M a r u k o Bridge
0.377
0.371-0.381
1.4
Mean of five replicated analyses.
Table 111. Composition of the Determined ABS in River Watera alkyl chain
length and isomer distribution
peak area Ta River
2277
%
Tama River
3-6$
c11
c12
i
3-6@ 3-7p
3-70
C,, ABS
3- to 5-Ph
16.2 2-Ph 2.9 C,, ABS 3- to 6-Ph 41.7 2-Ph 5.9 C , , ABS 3- to 6-Ph 24.9 2-Ph 1.9 C,, ABS 3- t o 7-Ph 6.4 2-Ph N D ~ a Mean of five replicated analyses. ND mined.
33.7
'
3-50
N D ~
49.2 ND 15.0
N D ~
Ta Tama
0.187 0.439
0.182 0.450
3-6p
=
c13
3- 7'
not deter-
Table IV. Recovery Test of ABS river
"
2.2
ND
1 r
0
amt amt present,a found,b pg/mL ccg/mL
CI 2
recovery, range, pg/mL
%
0.178-0.186 0.436-0.456
97.3 102.5
Test solutions were prepared by the addition of 10 mL of 1.0 pg/mL standard ABS solution to 90 mL of the collected river water. Mean of five replicated analyses. pg/mL ABS solution, alkyl chain distributions and the isomer compositions were determined accurately, but minor components of ABS were not detected below 0.10 pg/mL standard ABS solution. Analysis of River Water. ABS in T a River (sampling point: Taiso Bridge, Utsunomiya, Tochigi) and Tama River (sampling point: Maruko Bridge, Tokyo) was determined by the proposed method. Reproducibility within 2% relative standard deviation was obtained as shown in Table 11. The alkyl chain length and the isomer distribution of the determined ABS are also shown in Table 111. ABS in both the river water samples had low or no 2-phenyl isomer content and short alkyl chain lengths compared with the standard ABS. It was suggested that biodegradation of ABS took place in the river stream. Recovery tests were made on both the river water samples by adding known amounts of ABS. The results are summarized in Table IV and the chromatograms are shown in Figure 2. By the proposed HPLC method, ABS in the polluted environment is determined accurately without preconcentration
5
10
15n ,i
C
5
10
15min
Figure 2. Analysis of river water: (A) chromatograms of ABS originally found in Ta River (0.097 pg/mL) and Tama River (0.377 pg/mL); ( 8 ) chromatograms of 10 m L of 1.0 p g / m L standard ABS solution added to 90 m L of the river water.
and derivatization. This method could be applied to the biodegradation test of ABS. Biodegradated intermediates such as w- and P-oxidated ABS could possibly be determined by using gradient elution HPLC.
ACKNOWLEDGMENT T h e authors wish to acknowledge the technical assistance of Nobuko Hanawa. LITERATURE CITED Abbott. D. C. Analyst (London) 1962, 8 7 , 286-293. Sallee, E. M.; Fairing, J. D.; Hess, R. W.; House, R.; Maxwell, P. M.; Melpolder, F. W.; MMdleton, F. M.; Ross, J.; Woeifei, W. C.; Weaver, P. W. Anal. Chem. 1956, 28, 1822-1826. Arnbe. Y.; Hanya, T. Bunseki Kagaku 1972, 27,252-256. Watanabe, S.;Nukiyama, M.; Takagi, F.; Iida, K.; Kalse, T.; Wada, T. Nippon Shokuhin Eiseigaku Zasshi 1975, 16, 212-217. Tsukioka, T.; Oka, H.; Maruyama, M.;Ogiwara, K.; Nagase, K. presented at the 37th meeting of Japanese Society of Public Health: Tokyo, Oct 19, 1978; No. 518. Hon-narnl, H. T.; Hanya T. J. Chromatogr. 1978, 161, 205-212. Lebiharn, A.; Courtot-Coupey, J. Anal. Left. 1977, 10, 759-769. Crisp, P. T.; Eckert, J. M.; Gibson, N. A.; Kirkbright, G. F.; West, T. S . Anal. Chim. Acta 1976, 8 7 , 97-101. Kunlhiro, K.; Nakae, A.; Muto, G. BunsekiKagaku 1975, 24, 188-192. Takano, S.;Yagi, N.; Kunihiro, K. Yukagaku 1975, 24,389-394. Takano, S.;Takasaki, C.; Kunihiro, K.; Yarnanaka, M. Yukagaku 1976, 25,31-34. Hashirnoto, S.;Sakurai, K. T.; Nagai, T. Bunseki Kagaku 1978, 25. 640-643. Imaida, M.: Sumirnoto, T.; Yada, M.;Yoshida, M.; Koyama, K.; Kunita. N. Nippon Shokuhin Eiseigaku Zasshi 1975, 16, 218-224. Nakae, A.; Kunihiro, K. J. Chromatogr. 1978, 752,137-144.
RECEIVED for review April 8,1980. Accepted August 19,1980.
