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ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
the 228Thregion due t o background. T h e count rates of sample and background are simply:
R1 =
Cl -
tl
a n d R3 =
c3 -
(4)
t3
and t h e estimate of t h e standard deviation:
where C1 is the total number of counts in the *'*Th region in counting time tl.
R2 = FR,
(6)
where F is the ratio * * 8 T h / * T hestablished from measurement of the tracer ( F = 0.0814 and s ( F ) = 0.0038 for this work). and R, is the count rate in the * * T hregion, and C, = total counts in the 229Thregion in time to. Again propagating t h e error for Equation 6 gives:
R3 is the background count in -1000 min (the average counting time used for background); background counts are less than 1, so that s 2 ( R 3 )is of the order of and can be neglected for our system. By the standard rules for propagation of error for sums and differences, the random error associated with R from Equation 3 is:
s 2 ( R )= s 2 ( R J + s2(R2)+ s2(R3)
(8)
a n d s*(R) can now be readily calculated by combining Equations 5 a n d 7 with 8. T h e cpm observed/dpm added is the product E Y , so that if E is known, then Y may be calculated. In fact,
R y=0 ET The error is propagated in the usual fashion for products and quotients. In short, the error in A (Equation 2) consists of two terms, comprising the contributions from the error in observed count rate and the product of yield and efficiency. T h e relative importance of the two as source of error depends on the activity in the sample and t h e amount of added tracer.
ACKNOWLEDGMENT T h e authors thank Friedrich Steinhausler for his help in propagating the error and Sarah Hlavka for her technical assistance in sample preparation.
LITERATURE CITED (1) H. G. Petrow. A. Cover, W. Schiessle. and E. Parsons, Anal. Chem.. 36, 1600 (1964). (2) H. G. Petrow and C.Strehlow, Anal. Chem., 39, 265 (1967). (3) C. W. Sill, K. W. Puphal, and F. Hindman, Anal. Chem.,46, 1725 (1974). (4) C. W. Sill, Anal. Chem.. 49, 618 (1977). ( 5 ) D. R . Percival and D. B. Martin, Anal. Chem., 46, 1742 (1974). (6) N. P. Singh, S. A. Ibrahim, N. Cohen, and M. E. Wrenn, Anal. Chem., 5 0 , 357 (1978). (7) M. E. Wrenn, N. P. Singh, S. A. Ibrahim, and N. Cohen, Anal. Chem.. 5 0 , 1712 (1978). (8) "Health and Safety Manual", Health and Safety Laboratory, U S . Energy Research and Development Administration, 1976. (9) M. E. Wrenn and N. Cohen, Health Phys., 13, 1075 (1967). ( 1 0) M. E. Wrenn, Proc. Int. Symp. Areas of High Natural Radioactivity. Pocos de Caldas. Brazil, 131-157 (1975).
RECEIVED for review J u n e 21, 1978. Accepted November 3, 1978. Research supported by Contract No. AT(49-24) 0358 from the US.Nuclear Regulatory Commission, Contract No. EY-76-S02-2968 from the US.Department of Energy and is part of a center program supported by Grant No. ES 00260 from the National Institute of Environmental Health Sciences and Grant No. CA 13343 from the National Cancer Institute.
Determination of Benzidine, Dichlorobenzidine, and Diphenylhydrazine in Aqueous Media by High Performance Liquid Chromatography R. M. Riggin" and C. C. Howard Organic, Analytical, and Environmental Chemistry Section, Battelle ' s Columbus Laboratories, 505 King A venue, Columbus, Ohio 4320 1
A high performance liquid chromatographic method is described for the determination of benzidine, 3,3'-dichlorobenzidine, and 1,2-diphenyIhydrazine in aqueous media. These compounds can be assayed either by direct injection, or by solvent extraction or resin adsorption of the aqueous sample prior to analysis with detection limits of less than 1 pg/L. Linearity, precision, and specificity of the method was excellent and no interferences were encountered in the several wastewater samples analyzed. Diphenylhydrazine was found to be extremely unstable (half time for disappearance approximately 15 min) in wastewater, thus making its analysis extremely difficult and of limited practical value.
T h e discharge of hazardous industrial effluents into t h e environment has focused a great deal of attention toward the monitoring and control of such effluents. In this vein, the U S . Environmental Protection Agency (EPA) has established a 0003-2700/79/0351-0210$01 .OO/O
list of hazardous organic chemicals (priority pollutants) which are likely to be present in certain industrial aqueous effluents, and thus should be monitored in these effluents. For this reason, there is need for relatively specific, sensitive procedures which can be utilized in t h e routine analysis for these compounds in aqueous media. This paper deals specifically with the analysis of the three basic priority pollutants: benzidine, 3,3'-dichlorobenzidine (DCB), and 1,2-diphenylhydrazine ( D P H ) . Benzidine and DCB, as well as various other aromatic amines, have been used extensively in the synthesis of dyes and D P H is an intermediate in the synthesis of benzidine. All three of these compounds are suspected carcinogens and thus their presence in the aqueous environment is of particular concern t o human health ( I ) . Various methods have been published for the analysis of benzidine and DCB while no methods for D P H analysis have been reported. In one study, colorimetric, thin-layer, and gas chromatographic procedures were evaluated for the analysis 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979 of benzidines in natural waters (2). Gas chromatography with flame ionization detection (FID) was found to be the preferred method, with a detection limit of 2-3 pg/L. More recently, gas chromatography (FID) and spectrophotofluorimetry were used for benzidine analysis in a variety of matrixes, with detection limits in the low microgram per liter region ( 3 ) . Recent application of best available technology to remove toxic materials from industrial effluents has resulted in a need for more sensitive analytical techniques having submicrogram per liter detection limits for the analysis of benzidine, DCB, and DPH. Several approaches could be used t o extend the detection limits of the gas chromatographic methods reported (colorimetric procedures are too nonspecific a t low concentrations and will not be considered further). The use of a more sensitive and specific detection system such as alkali flame detection has been applied t o certain amines a t lower levels t h a n are possible with FID ( 4 ) . Fluoracylation of amines followed by gas chromatography with electron capture detection has also been employed a t picogram levels ( 5 ) . Neither of the above approaches has been reported for the compounds of interest. However, we have evaluated both of these approaches in our laboratory and have found: (1)that the free amines are not readily chromatographed a t levels below 10 ng on column (and D P H decomposes instantaneously to azobenzene in the injection port), and (2) while DCB is easily derivatized by most fluoroacylating reagents, benzidine and DPH could not be satisfactorily derivatized. An alternate approach which we have found far superior is the use of reversed phase high performance liquid chromatography (HPLC) with electrochemical detection. We have used this technique extensively for various compounds in biological media (e.g., catecholamines) and found it t o be extremely sensitive (low picogram detection limits) and specific for readily oxidizable compounds (6). Since the three compounds of interest here are readily oxidized a t a glassy carbon electrode, it was felt t h a t this approach should be useful. The specific operational details of this technique have been reviewed and will not be described here ( 7 ) . EXPERIMENTAL Apparatus. The HPLC system used in this study was assembled from modular components consisting of an Altex Model llOA Liquid Chromatographic Pump, a Rheodyne 7010 injector with a 50-pL loop, a 4.6-mm i.d. X 25 cm stainless steel column
packed with Lichrosorb RP-2 (5-pm particle diameter), and an electrochemical detector (Model LC-SA) equipped with a thinlayer glassy carbon electrode (Model TL5) available from Bioanalytical Systems, West Lafayette, Ind. Data were recorded on a strip chart recorder and quantitative measurements were based on peak height. The mobile phase reservoir consisted of a single neck flask maintained at 30 "C to reduce the amount of dissolved air. A vortex evaporator (Model 3-2200, Buchler Instruments) was used for concentrating small volumes of organic solutions. The liquid chromatographic columns were slurry packed using established methods (8). Procedure. In order to ascertain the stability of stock solutions of the three amines, 100-ppm solutions of each of the amines were prepared and 4-mL aliquots were sealed in 10-mL glass ampules. The ampules were stored in the dark a t room temperature and a t 0, 30, 60, and 90 days three ampules of each solution were opened and assayed by HPLC by comparing to freshly prepared standard solution. Figure 1 shows the separation of benzidine, DCB, and DPH by HPLC and lists the chromatographic conditions used throughout this study. The extractability of the three compounds from aqueous solution was investigated using two different organic solvents and three different pH levels. Aqueous solutions were 0.1 M phosphate buffers at the appropriate pH. A 500-mL portion of the aqueous solution was spiked with 10 ppb of the amines and extracted with 50 and then 30 mL of organic solvent in a 1-L separatory funnel. The combined extracts were washed with 20 mL of water, mixed with 20 mL of methanol, and concentrated to 5-10 mL on a
211
Benzl'le
Figure 1. Separation of DCB, DPH, and benzidine by reversed phase liquid chromatography. Volume injected, 25 pL; amount injected, 3 ng each; flow rate, 0.8 mL/min; mobile phase, 50/50 acetonitrile/pH 4.7, 0.1 M sodium acetate buffer: stationary phase, 4.6 mm i.d. X 25 cm RP-2 (5 pm): electrode potential, 0.9 V
rotating evaporator at room temperature. The solution was then transferred to a 15-mL conical centrifuge tube and concentrated t o 2 mL on a vortex evaporator at 35 "C. The solution was then diluted to 4 mL with acetate buffer and analyzed by HPLC. Benzidine and DPH were investigated separately throughout this study since DPH can be converted to benzidine at low pH. An alternate method which can be used for extraction of wastewater in the field was evaluated. Aqueous solutions were adjusted to pH 7 with 0.2 M phosphate buffer. Ten milliliters of the aqueous sample was passed through an ODS reversed phase cartridge (Sep Pak C-18, Waters Associates) at a flow rate of approximately 10 mL/min using a glass syringe. The cartridge was then washed with 5 mL of distilled water (discarded) and then eluted with 3 mL of methanol which was stored at -70 "C. When the sample was to be analyzed, the methanol was concentrated to 0.5 mL in a vortex evaporator, 1 mL of 0.1 M pH 4.7 acetate buffer was added, and the sample reconcentrated to 1 mL and analyzed by HPLC. The stability of the three amines in dilute aqueous solution was investigated under various conditions including three pH levels, with or without chlorine, and two temperatures. The samples were spiked with 10 pg/L of the three amines (benzidine and DPH were studied separately as before) and stored a t the appropriate temperature for 7 days. The samples were then extracted as described earlier. One half of the samples were spiked with 2 ppm of NaOC1. The samples stored at pH levels other than 7 were adjusted to pH 7 prior to extraction by the addition of either 0.1 M H2S04or NaOH. A preliminary study was conducted to determine the feasibility of monitoring the benzidines in water effluents by direct injection (50 pL) onto the HPLC system. Several actual wastewater samples (1L) were collected in half-gallon bottles containing 200 mL of methylene chloride, 5 g of KHS04, and 75 g of NaCl. The water samples were -pH 2 (as a result of the KHS04) and were maintained at 4 "C until analyzed. Five milliliters of the water sample was spiked with 50 ng of benzidine and DCB, filtered through a 0.2-pm filter (Millipore type GS), and injected onto the HPLC system. RESULTS A N D DISCUSSION
T h e H P L C conditions shown in Figure 1 were found to be quite satisfactory. The RP-2 column was found t o be superior to any other reversed phase or ion-exchange packing materials (e.g., VYDAC TP SCX, Whatman Partisil SCX, F-Bondapak-18, and Lichrosorb RP-8 and RP-18). Column efficiencies of 5000 theoretical plates per 25-cm column for benzidines were commonly achieved using RP-2 whereas none of the other packing materials exhibited greater than 2500 theoretical
212
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
Table I . Solvent Effect on Stability % recoverya day 0
benzidine 85 87 84 -
85 30
i
2b
108
94 95 __ 60
90
0
99 = 8 92 95 93 93 i 2 93 97 95 95 i 3 100
98
30
60
90 90 90 90 105 93 105 __ 101 + 7 87 87
2
96 102 93 97 i 5 100 100 103 __
DPH 81 81 81 81 0 I
i /
0
/
86 86i 1 92 90 90 91 i 1 acetonitrile 102 95 98 __
101 -
loot
DCB methanol
98 I4 88 92 99 93i 5 98 98 98 -
0
Figure 2. Linearity of response for DPH, DCB, and benzidine (See Figure
1 for conditions) 109 100 80 -
9 6 i 15 0
Table XI. Extraction Studiesb % recovery
pH
benzidine DC B methylene chloride
2 0
98 98 101 0 99 100 98 101 98 * 1 101 i 1 a Stability was determined by HPLC analysis after various storage {eriods by comparison t o freshly prepared standards. Average + standard deviation.
~
0
7
68 81 81 76 __
101 i 2
90
plates. Column lifetime for the RP-2 was found to be greater t h a n 6 months under t h e conditions used. Linear response for t h e three amines was achieved over the range of 1-1000 ng as shown in Figure 2. Repeatability of eight replicate injections was *2.8% for each of the components at the jO-ng level a t a signal to noise ratio of 10. The results from the solvent stability study are summarized in Table I. I t is readily apparent t h a t benzidine and DCB are quite stable in both acetonitrile and methanol. D P H was found to be extremely unstable (half time of disappearance less than 24 h) in all the solvents evaluated, including benzene, methylene chloride, methanol, triethylamine, acetonitrile, and acetic acid. Therefore it was necessary t o prepare D P H solutions fresh daily. The extraction methodology was found to be a very delicate area since numerous unanticipated problems developed which led to low recoveries, especially for benzidine and DPH. The stability of D P H was found t o be a major problem. T h e following are some of t h e other problems encountered. (1)Benzidine is substantially adsorbed on N a 2 S 0 4during t h e drying of organic extracts. Although use of K2C0, corrected this problem, elimination of the drying step was found t o be useful when using H P L C analysis. (2) Benzidine is heat labile so t h a t Kuderna Danish concentration techniques gave unacceptable results. T h e use of
0 0 0
10
2
7
87 89 88
92 __ 10
80
9
69 77 __ 75 99
33 0 i
7a
84
99 104 __
76+ 8 96 i 9 92 101 103 104 80 82 __ 92+ 13 9 6 + 12 chloroform 0 0 0 __ 0
89i 2 79 90
DPH
93
14 i 1 6 75 82 58 60 68 i 17 75 79 82 79 i 4
84 89 __
35 28 41 -
92 i 3 32 92 95 99 94 + 4
35 i 19 69 70 64 37 60 I 24
88
81
98 65 88 94 67 __ 86i 7 93+ 5 67 i 1 6 a Average i standard deviation. Five hundred-milliliter volumes of aqueous solutions at the stated pH were extracted with the organic solvent and the extracts assayed by HPLC. Triplicate analyses were conducted in all cases. rotary evaporation and vortex evaporation eliminated this problem. ( 3 ) Decomposition of benzidine occurred when concentrating it in methylene chloride or chloroform solutions. The addition of 15% MeOH prior to concentration stabilized the benzidine.
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
213
Table 111. Preservation Date (after 7 Days Storage) %
benzidine
DPH
94 -
DCB 90 85 -
4 "C
87a 80 80 -
88 82 82 -
0 0 0 -
room temperature
80 64 55 -
PH 2
7
room temperature
4 *c
80
60
79 56 68
10
room temperature 4
4.7
recoveryb
cc
4 'C
70 73 72 82 38 -
82 110 89 __
99 97 83 -
90 80 82 81
93 44 __
0 0 -
0 0
0 0 0 0 0 0 0 0 0 -
64 72
74
0
81
-.
86
91 -
79
86
10 28 19
IC-
JL
0
:.,ecl#or
Figure 4.
:n,e:j
Cn
i-#ec!m
Chromatograms for solvent extraction of municipal sewage.
(A) Unspiked. (B) Spiked with 10 ppb benzidine and DCB. (C) Standard corresponding to 100 % recovery of spike
Z e r o recovery was realized for the samples a Average. spiked with 2 ppm NaOCl and therefore this data is not reported in the table. Five-mjllilitpr volumes of distilled water a t the stated pH were stored a t either room temperature or 4 'C, in the dark, for seven days. The solutions were then extractedand assayed by HPLC.
t
Figure 5. Chromatogram for resin concentration of municipal sewage. (A) Unspiked. (B) Spiked with 10 ppb benzidine and DCB. (C) Standard corresponding to 100% recovery of spike
nl ,_. "
.,* -*.
Figure 3. Chromatograms for direct injection of aqueous effluent from an organic chemical plant. (A) Spiked with 10 ppb DCB and benzidine. (B) Unspiked
The results of the extraction experiments are summarized in Table 11. Chloroform a t p H 7 was found to be the most satisfactory extraction condition and was used for all further work. D P H extraction was somewhat irreproducible due to its instability during concentration. However chloroform extraction a t p H 7 gave an extraction efficiency greater than 50%. T h e results of the preservation study are shown in Table 111. DPH was not stable ( < l o % remaining) for longer than 1 day under any of the conditions studied and thus it is not included in the table. A t each pH, DPH degrades to different
components (based on HPLC retention times) indicating that several competing reactions are taking place in solution. At p H 10, D P H apparently decomposes primarily to azobenzene; a t p H 2, it degrades to benzidine; and a t p H 7 , still a third unidentified (oxidizable) component is formed. Kone of the amines were detectable in the solutions to which chlorine was added. A p H of 2 was found t o give the best results for benzidine and DCB. However, at pH 2, D P H degrades to benzidine thus creating an undesired artifact. This problem was overcome by employing 0.1 M p H 3.7 acetate buffer where both benzidine and DCR are well preserved, and D P H degrades to two unidentified components, not to benzidine. T h e three analytical approaches, direct injection, solvent extraction, and resin adsorption were applied t o actual wastewater and/or surface water samples and the results are shown in Figures 3, 4, and 5 , respectively. Each of the approaches was found to be quite effective. Direct injection, while most susceptable to interferences, is very rapid and has a detection limit of approximately 1 pg/L. Solvent extraction serves to clean up and concentrate the sample to give a detection limit of 50 ng/L or better. However, D P H is not
214
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
"-
I
I3
23
-,-e
33 :"E'
E:
43 1
5:
63
-: h",.fE
Figure 6. Stability curve for DPH in municipal sewage
efficiently recovered by this technique (as described previously). Resin adsorption offers a detection limit of approximately 100 ng/L and offers some degree of cleanup. The primary advantages of resin adsorption are its speed and the efficient recovery of D P H (at least from distilled water). D P H was found t o be quite unstable in aqueous solution a n d even more unstable in wastewater samples. Figure 6 shows the stability of D P H in a municipal sewage effluent a t t h e 100 Fg/L level. As shown, D P H disappears with a half time of approximately 15 min without removal of oxygen and a half time of 60 min when oxygen is removed by a nitrogen purge. This result indicates that D P H analysis in wastewater is virtually impossible and perhaps meaningless since it is so unstable. At least, the analytical result obtained will represent t h e portion of intact D P H remaining a t the time of analysis, which will be quite different from the D P H level in the original effluent.
CONCLUSIONS T h e results of this study clearly show t h a t H P L C with electrochemical detection is a sensitive method for the analysis
of benzidine, DCB, and D P H in aqueous samples. These compounds can be assayed by direct injection, solvent extraction, or resin adsorption techniques a t the submicrogram per liter level. Direct injection is satisfactory in most cases where 1 pg/L sensitivities are adequate. Use of solvent extraction results in a detection limit of 50 ng/L, whereas resin adsorption affords a detection limit of 100 ng/L. T h e resin adsorption technique has the advantage of being used in t h e field, thus eliminating t h e need t o preserve dilute aqueous solutions of the compounds of interest and also avoids t h e emulsion problems frequently encountered during solvent extraction of certain wastewater samples. While benzidine and DCB are relatively stable compounds, D P H is extremely unstable and none of the approaches described herein gave entirely satisfactory results.
ACKNOWLEDGMENT We thank Peter Mondron for packing the HPLC columns for this study.
LITERATURE CITED T. J. Haley, Clin Toxicol., 8, 13 (1975). R. L. Jenkins, and R. B. Baird, Bull. Environ. Contam. Toxicol.. 13, 436 ( 197 5). M. C. Bowman, J. R. King, and C. L. Holder, Int. J . Environ. Anal. Chem., 4 , 205 (1976). H. B. Hucker, and S. C. Stauffer, J . Chromatogr., 138, 437 (1977). M. Makita, S.Yamamoto, and M. Kono, Clin. Chim. Acta, 61, 403 (1975). P. T. Kissinaer, R. M. Riaain, R. L. Alcorn. and L. D. Rau, Bochem. M e d . , 13, 299 (1575). P. T. Kissinger, Anal. Chem., 49, 447A (1977). P. J. Mondron, and P. G. Bonnett, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Clevehnd, Ohio, Abstract No. 437, 1978.
--
for review August 18, 1978. Accepted October 27, 1978. Work supported by the U.S. Environmental Protection Agency, EMSL, Cincinnati, Ohio. RECEIVED
