Anal. Chem. 1997, 69, 3119-3123
Extraction of Elemental Sulfur from an Aqueous Suspension for Analysis by High-Performance Liquid Chromatography Paul F. Henshaw,* Jatinder K. Bewtra, and Nihar Biswas
Department of Civil and Environmental Engineering, University of Windsor, Windsor, Ontario, Canada N9B 3P4
A bioreactor is being developed that produces elemental sulfur suspended in aqueous bioreactor contents. The concentration of elemental sulfur must be measured explicitly in order to study the efficiency of the conversion of sulfide to elemental sulfur. Extracting the sample with ethanol gave erroneous results when sulfide was present in solution. The extraction of aqueous elemental sulfur into petroleum ether prior to colorimetric determination was tested. When the aqueous matrix was simply deionized water, the extraction was poor. The development of a method of extraction of the sulfur into chloroform prior to quantification by high-performance liquid chromatography is described. The efficiency of the extraction was found to be greater than 90% in all matrixes tested and linear for aqueous elemental sulfur concentrations up to 200 mg/L. Hydrogen sulfide is a toxic gas which is a by-product of petroleum refining. Sulfide (S2-) has a high oxygen demand, and thus its concentration in petroleum refinery wastewater is regulated. Elemental sulfur (S0) is a nontoxic and useful form of sulfur. A bioreactor system is being developed to remove hydrogen sulfide from wastewater and convert it to elemental sulfur.1 Green sulfur bacteria produce extracellular orthorhombic elemental sulfur.2 Since elemental sulfur is essentially insoluble in water (saturation at 5 × 10-6M),3 it forms a suspension in water, giving the bioreactor a “cloudy” appearance. The concentration of elemental sulfur in the bioreactor must be quantified in order to optimize its production from sulfide. An elemental sulfur assay has been reported in which the aqueous sample is diluted 1:24 or 1:405 with 95% ethanol, refluxed for 2 h, and centrifuged and the optical density measured at 264 nm. In experiments by the authors, the presence of 69 mg of S2-/L in the sample resulted in only 9-18% of the absorbances expected for the S0 present in the sample.6 Due to the yellow color of the mixture, it was suspected that the sulfide reacted with elemental sulfur to form polysulfides (Sx2-), which are known to (1) Henshaw, P. F.; Bewtra, J. K.; Biswas, N.; Franklin, M. Water Sci. Technol. 1992, 25, 265-267. (2) Truper, H. G.; Hathaway, J. C. Nature 1967, 215, 435-436. (3) Chen, K. Y.; Morris, J. C. Environ. Sci. Technol. 1972, 6, 529-537. (4) Cork, D. J. Kinetics of sulfate conversion to elemental sulfur by a bacterial mutualism: a hydrometallurgical application. Ph.D. Dissertation, University of Arizona, Tuscon, 1978. (5) Maka, A. Control of oxidative sulfur metabolism in Chlorobium. Ph.D. Dissertation, Illinois Institute of Technology, Chicago, 1986. (6) Henshaw, P. F. Biological removal of hydrogen sulfide from refinery wastewater and conversion to elemental sulfur. M.A.Sc. Thesis, University of Windsor: Windsor, ON, Canada, 1990. S0003-2700(96)01149-3 CCC: $14.00
© 1997 American Chemical Society
absorb light at 285-290 nm.7 The addition of acid during refluxing, to remove the sulfide interference as H2S(g), resulted in recoveries of 130-138%.6 Because of this variable recovery, another assay was sought for elemental sulfur in aqueous suspensions. Methods for measuring elemental sulfur dissolved in organic liquids include colorimetry8 and the use of high-performance liquid chromatography (HPLC).9 In the colorimetric method, a petroleum ether sample containing dissolved elemental sulfur is converted to thiocyanate. The thiocyanate reduces Fe3+, and the Fe2+ formed is measured colorimetrically. An aqueous solution of mercury is added to the original sample to precipitate HgS and remove any sulfide interference.8 This method was adapted by the authors to measure aqueous sulfur by stirring the sample, an Hg2+ solution, and pure petroleum ether together for 45 min to allow the S0 to partition into the organic phase.6 A portion of the petroleum ether layer was then carried through the described method. Four replicates at each of four S0 concentrations were performed in each of three aqueous matrixes: deionized water (deH2O), 186 mg of S2-/L, and the contents of the bioreactor. With the exception of the deH2O matrix, the slope and intercept of the calibration curves (response versus concentration) developed using aqueous suspensions of sulfur were not significantly different from those of the standards prepared by dissolving S0 in petroleum ether. Therefore, the extraction of elemental sulfur from matrixes containing sulfide or bioreactor contents was exhaustive. The absorbances measured after extractions from deH2O were only 70% of those expected. In addition, the coefficients of variation of absorbances measured in processed samples in all matrixes averaged 10%. This method is also relatively labor intensive. Lauren and Watkinson9 performed elemental sulfur analysis using several HPLC columns. Of special interest is the use of a C18 reversed-phase column with methanol as the mobile phase for elemental sulfur samples dissolved in chloroform. The development of a procedure for the extraction of elemental sulfur from aqueous dispersions prior to analysis by HPLC is described in this paper. EXPERIMENTAL SECTION (A) Apparatus, Reagents, and Solutions. HPLC Equipment. The apparatus included a Waters 501 solvent delivery system, intelligent sample processor (WISP) Model 712, Nova(7) Chen, K. Y. In Chemistry of Water Supply, Treatment and Distribution; Rubin, A. J., Ed.; Ann Arbor Science: Ann Arbor, MI, 1974; pp 109-135. (8) Bartlett, J. K.; Skoog, D. A. Anal. Chem. 1954, 26, 1008-1011. (9) Lauren, D. R.; Watkinson, J. H. J. Chromatogr. 1985, 348, 317-320.
Analytical Chemistry, Vol. 69, No. 15, August 1, 1997 3119
Pak C18 column (3.9 × 150 mm), 486 tunable absorbance detector set at 254 nm,9 system interface module, and Maxima 820 software. The guard column was a Zorbak ODS (4.0 × 12.5 mm). The eluent was methanol (Baker, HPLC grade) at 1.0 mL/min. Dissolved Sulfur Standards. Elemental sulfur (Aldrich 99.999%) was refluxed in HPLC CHCl3 (Aldrich) for 2.5 h, cooled, and made to the mark in a volumetric flask with CHCl3. This stock solution was serially diluted with HPLC CHCl3. These operations were performed in a fume hood. Sulfur Suspension. Approximately 200 mg of elemental sulfur (Aldrich, 99.999%) was weighed and refluxed in 150 mL of 95% ethanol for 1 h. The ethanol solution was poured hot into a 1 L volumetric flask and made up with the matrix. The three matrixes tested were deH2O, sulfide solution, and bioreactor contents. Dilute Sulfur Suspension. The sulfur suspension was diluted 1:500 with matrix/ethanol solution. Matrix/Ethanol Solution. A 15% solution of 95% ethanol in the matrix was prepared. Sulfide Matrix. A weighed quantity of rinsed, patted-dry Na2S‚9H2O crystals was dissolved in deaerated (boiled for a few minutes and then covered and cooled rapidly) deH2O water. The sulfide content was measured by the methylene blue colorimetric method.10 Bioreactor Contents Matrix. Green sulfur bacteria were grown in a batch bioreactor.1 Before the bioreactor liquid was used, its sulfide content was measured to be sure that it was approximately zero. (B) Recommended Procedure. Chloroform (2 mL, Aldrich, HPLC grade), 0.5 mL of 10% (v) nitric acid, and a 5.00 mL sample were added to a screw-capped culture tube (Pyrex, No. 99447, 16 × 125 mm) and shaken with a Burrell wrist action shaker (Model 75) for 15 min. For calibration purposes, the sample consisted of dilute sulfur suspension or a combination of sulfur suspension and matrix/ethanol solution totaling 5.00 mL. After shaking, the tube was centrifuged (International Equipment Co., Centra 8) at 2500 rcf (relative centrifugal force) or 3000 rpm. A 1 ml aliquot of the CHCl3 layer was pipetted into a WISP vial containing 3.00 mL methanol (Baker, HPLC grade) inverted and injected into the HPLC. A 1 mL sample of each standard was added to 3.00 mL of HPLC methanol in a WISP vial, inverted, and injected into the HPLC. The HPLC run time was 5 min, and the sulfur peak retention time was ∼3 min. (C) Experiments. Earlier tests were performed on solutions of elemental sulfur (J. T. Baker, sublimed sulfur, recrystallized in benzene) dissolved in HPLC CHCl3. Chloroform/methanol ratios of 3:1, 1:1, and 1:3 were used. The integrated HPLC detector responses for the peaks were 503 695, 506 561, and 557 538 µV‚s, respectively. Therefore, the lowest CHCl3 content gave the highest response at the same sulfur concentration. In order to minimize the use of CHCl3, further tests were performed using 1:3 chloroform/methanol. A test of the effect of varying shake and settling times was performed. A 5 mL aliquot of a 200.6 mg of S0/L suspension and 2.00 mL of CHCl3 were shaken for 0.5-4 h and allowed to settle for either 1 or 20 h. Figure 1 shows that all of the extractions except those with the shortest shake and settling times gave (10) Truper, H. G.; Schlegel, H. G. Antonie van Leeuwenhoek 1964, 30, 225238.
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Figure 1. Detector response as a function of aqueous elemental sulfur concentration at various shake and settling times. Hollow bars represent experiments where 1 h was allowed for settling. Bars with hatched fill represent experiments where 20 h were allowed for settling.
roughly equal HPLC detector responses. At a minimum, 1 h shaking followed by 1 h settling is required to extract the elemental sulfur into the organic phase. A test of reproducibility was conducted in which seven samples at each of five concentrations (0-200 mg of S0/L) were extracted from each of three matrixes (deH2O, sulfide solution, bioreactor contents). To provide a margin of safety, these tests used a shake time of 1 h and a minimum settling time of 20 h. Simultaneously, unextracted S0 standards were analyzed by HPLC. The responses of the samples where the S0 was extracted from deH2O and from sulfide solution were significantly less than those where S0 was extracted from reactor contents or from the dissolved S0 standards (Figure 2). In addition, there were significant variations in the responses between extractions from the same concentration of sulfur suspension for the deH2O and sulfide matrixes. Experiments were performed to eliminate the position of the tube in the shaker, the sulfur source, and the volume of CHCl3 as the source of the variability in replicates of the same extraction. Finally, centrifuging of the sample was performed to eliminate a foam layer that formed during shaking. Centrifuging of the samples in the deH2O matrix resulted in recoveries similar to those in which there was overnight settling, but with less variability (Table 1). A series of experiments was performed at various shake and centrifuge times to reduce the process time for each sample. The experiments performed on day 1 (Figure 3) where the shake time was 60 min suggested that higher centrifuge times resulted in higher detector responses. However, this conclusion is not supported if the point with a response/ concentration value of 8745 µV‚s‚L/mg is excluded. The results from those experiments performed on day 1 cannot be directly
Table 1. Effect of Centrifuge (“spin”) and Settling (“sett”) Times on Elemental Sulfur Extraction from Aqueous Suspension
} }
response/[S0] (mV‚s‚L/mg)
spin time (h)
sett time (h)
detector response (µV‚s)
aqueous [S0] (mg/L)
E5-2A E5-2B E5-4A E5-4B U5-2A U5-2B U5-4A U5-4B
0 0 0 0 0 0 0 0
23 23 23 23 23 23 23 23
1 269 476 1 998 161 791 180 786 867 978 969 1 605 377 145 039 235 400
200.4 200.4 100.2 100.2 200.4 200.4 100.2 100.2
6 335 9 971 7 896 7 853 4 885 8 011 1 447 2 349
E5-2CA E5-2CB E5-4CA E5-4CB U5-2CA U5-2CB U5-4CA U5-4CB
1 1 1 1 1 1 1 1
0 0 0 0 0 0 0 0
1 661 828 1 621 703 736 420 788 712 1 677 687 1 693 487 819 273 826 687
200.4 200.4 100.2 100.2 200.4 200.4 100.2 100.2
8 293 8 092 7 350 7 871 8 372 8 451 8 176 8 250
U5-2BA U5-2BB U5-4BA U5-4BB
1 1 1 1
23 23 23 23
1 703 251 1 715 388 853 381 858 338
200.4 200.4 100.2 100.2
8 499 8 560 8 517 8 566
lab label
}
jx ) 6093; σn-1 ) 2980
jx ) 8107; σn-1 ) 354
jx ) 8536; σn-1 ) 33
Figure 2. Detector response as a function of aqueous elemental sulfur concentration. Results plotted with downward triangles (3) and a solid regression line are from dissolved sulfur standards (no extraction) where the concentration in chloroform has been divided by 2.5. Results plotted with circles (O) and a dotted line have been extracted from a matrix of deionized water. Results plotted with squares (0) and a dashed line have been extracted from a matrix of 150 mg of S2-L. Results plotted with upward triangles (4) and a dotdash line have been extracted from a matrix of bioreactor contents.
Figure 3. Extraction of aqueous elemental sulfur at various shake and centrifuge times. Filled symbols represent experiments performed on day 1. Results plotted with filled upward triangles (2) are for 60 min shaking. Results plotted with filled downward triangles (1) are for 30 min shaking. Hollow symbols represent experiments performed on day 2. Results plotted with open circles (O) are for 30 min shaking. Results plotted with hollow upward triangles (4) are for 15 min shaking. Results plotted with hollow downward triangles (3) are for 5 min shaking.
compared with those from day 2, because of slight day-to-day differences in the response of the HPLC system. The data from experiments performed on day 2 show no pattern between detector response and shake or spin times. Therefore, minimum shake and spin times of 15 and 5 min, respectively, were used.
However, at reduced shake and spin times the responses were still low when sulfide was in the matrix, presumably due to the formation of polysulfides, which prevented the extraction of sulfur into the organic layer. The addition of varying amounts of Hg2+ and HCl was tested as a means to alleviate the sulfide interference, Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
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Table 2. Effect of Additives To Remove Sulfide Interference in Elemental Sulfur Extraction from Aqueous Suspension Containing Sulfide solution added compd
vol (mL)
detector response (µV‚s)
response/[S0] (µV‚s‚L/mg)
deH2O deH2O deH2O deH2O HgCl2 HgCl2 HgCl2 HgCl2 HCl HCl HCl HCl
0.5 0.5 0.2 0.2 0.5 0.5 0.2 0.2 0.5 0.5 0.2 0.2
3 594 2 551 6 886 4 030 827 752 664 280 16 884 12 796 1 913 385 1 906 475 1 948 161 1 970 250
18 13 34 20 4126 3311 84 4 9538 9504 9712 9822
Table 3. Statistical Analysis of Replicates from HPLC Assay for Elemental Sulfur in Various Matrixesa matrix none (stds) jx CV n deH2O xj CV n 305 mg of S2-/L jx CV n reactor contnts jx CV n 146 mg of S2-/L jx CV n all
sulfur concn (mg of S0/L) 9.988 85 458 1.44 5 0.4016 4168 20.9 5 0.4012 42 018 1.33 5 0.4006 3642 17.5 5 0.4014 32 194 58.7 5
49.94 425 936 1.66 5 100.4 832 519 0.581 5 100.3 948 417 1.75 5 100.2 778 800 0.632 5 100.4 862 364 1.20 5
249.7 2 110 734 1.85 5 200 1 658 421 0.783 5 200.6 1 828 545 1.47 5 200.3 1 551 068 0.771 5 200.7 1 713 541 2.12 5
all data for matrix s ) 8444.2 i ) 2537.6 r2 ) 0.9995 s ) 8254.8 i ) 1815.3 r2) 0.9999 F* ) 9.56 s ) 8923.7 i ) 43423.5 r2 ) 0.9994 F* ) 87.63 s ) 7741.0 i ) 1541.9 r2 ) 0.9999 F* ) 129.7 s ) 8394.26 i ) 25879.2 r2 ) 0.9990 F* ) 2.53 s ) 8359.4 i ) 25739.6 r2 ) 0.9923 F* ) 0.64
aj x mean response (µV‚s); CV, coefficient of variation (%); n, number of data points; s, slope (µV‚s/mg S0/L); i, intercept (µV‚s); r2, coefficient of determination; F*, calculated F statistic value.
with HCl addition giving the highest detector responses. Table 2 shows that acid addition gave the highest and most consistent response per unit of sulfide concentration. RESULTS AND DISCUSSION The extractions of aqueous S0 from all matrixes were retested using the recommended procedure. The results are shown in Table 3 and Figure 4. In estimating the accuracy of this method, it is best to compare, by use of the F test,11 the slope and intercept of the linear regression line obtained in each matrix with that obtained from the standards. The F test reveals that the results in the 146 mg of S2-/L matrix only are sufficiently close to those of the standards to be considered from the same pool of data. (11) Neter, J.; Wasserman, W.; Kutner, M. H. Applied Linear Statistical Models, 2nd ed.; Richard D. Irwin Inc.: Homewood, IL, 1985.
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Figure 4. Detector response as a function of aqueous elemental sulfur concentration using the recommended procedure. Results plotted with diamonds ()) and a solid regression line are from dissolved sulfur standards (no extraction) where the concentration in chloroform has been divided by 2.5. Results plotted with circles (O) and a dotted line have been extracted from a matrix of deionized water. Results plotted with downward triangles (3) and a dot-dash line have been extracted from a matrix of 305 mg of S2-/L. Results plotted with upward triangles (4) and a dot-dot-dash line have been extracted from a matrix of 148 mg of S2-/L. Results plotted with squares (0) and a dashed line have been extracted from a matrix of bioreactor contents.
The matrixes with deH2O, 305 mg of S2-/L, reactor liquid, and reactor liquid with sulfide yielded F values greater than the 99% critical value, meaning that the calibration curves of these matrixes are significantly different from that determined using dissolved S0 standards. The subtle differences in the regressed lines in different matrixes turn out to be significant because of the small variations in responses of replicate samples. The source of the negative bias exhibited in a matrix of bioreactor contents as compared to the calibration standards is not clear. Elemental sulfur did not exist in the bioreactor contents as evidenced by the low detector responses that resulted from the addition of small amounts of S0. Besides, this would cause a positive bias. Similarly, sulfide was driven out of the samples by acidification, thus not allowing polysulfides to form. A possible explanation is that some of the enzymes from the bacteria were present in the extract and facilitated oxidation of the S0. Further investigation into this phenomenon by repeating the test with different bacteria concentrations is warranted. In practice, there are two ways to use these calibration curves. The first way is to assume the matrix has no effect on the detector response and accept a large uncertainty in the concentration of S0 that is predicted by a given detector response. In the second way, the calibration curve used to interpret the detector response will be from either (a) the reactor contents regression line or (b)
a linear interpolation between the deH2O, 146 mg of S2-/L, and 300 mg of S2-/L lines, depending upon the source and sulfide content of the sample. During operation of the S0-producing bioreactor, the sample size for sulfur analysis was typically 5.00 mL. At this sample size and using the regression line for the reactor contents, the maximum elemental sulfur concentration that could be measured was 200 mg/L with an uncertainty in the final S0 concentration of (1 mg/L. CONCLUSION The recommended procedure for extracting elemental sulfur from aqueous suspensions into chloroform was found to be
effective for aqueous sulfur concentrations up to 200 mg/L. The detector response was a linear function of the aqueous S0 concentration within this range, with little scatter from replicate analyses at the same concentration. ACKNOWLEDGMENT This research was supported by funding from the Natural Sciences and Engineering Research Council of Canada. Received for review November 13, 1996. Accepted April 17, 1997.X AC961149P X
Abstract published in Advance ACS Abstracts, July 1, 1997.
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