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RECEIVEDfor review August 25, 1977. Accepted October 27, 1977. Presented in part at the Electrochemical Society Meeting, Toronto, Ontario, May 1975 (Abstract No. 399). Financial support by the National Science Foundation, Grant No. GP-12221, and by the Wisconsin Alumni Research Foundation through the University of Wisconsin Graduate School are gratefully acknowledged.
Determination of Trace Quantities of Aromatic Amines in Dyestuffs Janos Schulze," Charles Ganz,' and Douglas Parkes Dyestuffs and Chemicals Division, CIBA-GEIGY Corporation, Post Office Box
A method is described for the simultaneous determination of trace amounts of benzidine, a- and $-naphthylamine, 3,3'dichlorobenzidine, and 4-dimethyiaminoazobenzenein dyestuff formulations utilizing gas-liquid chromatography and thin-layer chromatography. The detection limit for the analysis was 100 pg/g. Recovery of aromatic amines from "spiked" dyestuffs was generally found to be greater than 9 0 % with a relative standard deviation of less than 10%.
dition, effluent standards have been enacted for benzidine in wastewater ( 2 ) . Since these amines are sometimes intermediates in dyestuff manufacture, the possibility arises that residual amounts may be present as contaminants in dyestuffs and dye intermediates. Thus, for both worker and user protection, accurate but reasonably simple analytical methods should be used in order to detect the aforementioned compounds in dyestuffs and dye intermediates. T h e Environmental Protection Agency accepted the Chloramine T complexation method as the official method for the analysis of benzidine in effluent ( 2 ) . Because of interferences, the method is not applicable to dyestuffs. Other chromatographic and spectrophotometric methods have been published describing the determination of one or more of these aromatic amines in wastewater, air, urine, tissue, cigarette smoke, and food dyes (3-12). Methods have been also described for the determination of benzidine and Jnaphthylamine in dyes by thin-layer chromatography (13, 14) and for the determination of 4-dimethylaminoazobenzene in dyes by paper chromatography (15). To our knowledge, no publications have appeared on the simultaneous determination
Five aromatic amines, benzidine, a- and d-naphthylamine, 3,3'-dichlorobenzidine and 4-dimethylaminoazobenzene, were included in a list of 14 carcinogens by the Occupational Safety and Health Administration ( I ) . The concentrations of these compounds in various matrices t o which workers may be exposed cannot exceed 1 7 ~ in the case of n-naphthylamine and 0.1% in the case of the other four compounds. In adPresent address, EN-CAS Analytical Laboratories, Winston-Salem, N.C. 27101. 0003-2700/78/0350-0171$01.00/0
7 1422, Greensboro, North Carolina 27409
C
1977 American Chemical Society
172
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
314
40 -
BENZIDINE 30 -
20
-
w ALPHA NAPHTHYLAMINE
10 -
k’ I 0
I
I 8
4
U I
12
16
BETA NAPHTHYLAMINE Migration Distance [cm)
CI
CI
Thin-layer chromatogram of aromatic amines, System B, methylene chloride/acetone 90: 10. Concentration of each amine, 0.05 mg/mL. Sample volume spotted, 10 pL. (1) Benzidine; (2) finaphthylamine; (3) 3,3’-dichlorobenzidine;(4) a-naphthylamine; (5) 4-dimethylaminoazobenzene 3,3’ .DICHLOROBENZIDINE
’:da1 3
4-DIMETHYLAMINOAZOBENZENE
Figure 1. Chemical structures of aromatic amines
1
20
10
0
I 4
I 8
Time (Mmutari
Figure 4. Gas-liquid chromatogram of aromatic amines. Concentration of each amine, 0.05 mg/mL. Sample volume injected, 3 pL. Column oven temperature, 140 O C . (1) a-Naphthylamine; (2) @-naphthylamine 0
4
8
12
16
Migration Distance lcm)
Figure 2. Thin-layer chromatogram of aromatic amines, System A, hexane/ethyl acetate/ghcial acetic acid 80:20:5. Concentration of each amine, 0.05 mg/mL. Sample volume spotted, 10 pL. (1) Benzene; (2) @-naphthylamine;(3)3,3’-dichlorobenzidine;(4) a-naphthylamine; (5) 4-dimethylaminoazobenzene
of t h e above five compounds in a single dyestuff. I n this paper, we describe complementary thin-layer chromatographic (TLC) and gas-liquid chromatographic (GLC) methods for the simultaneous determination of benzidine, a- and @-naphthylamine,3,3’-dichlorobenzidine, and 4-dimethylaminoazobenzene in dyestuffs and dye intermediates. T h e detection limit of the analysis has been established to comply with current governmental regulations (1) and can be lowered if needed.
EXPERIMENTAL Apparatus. Gas-liquid chromatography was performed on
a Tracor model MT-220 gas chromatograph equipped with a flame ionization detector. Direct evaluation of thin-layer chromatograms was carried out with a Perkin-Elmer MPF-2A fluorescence spectrophotometer equipped with a thin-layer scanning accessory. The spectrophotometer was operated in the reflectance mode with an emission fdter passing only the light above 430 nm. Excitation and emission wavelengths were maintained at 264 nm and 524 nm, respectively. An ultrasonic cell disruptor (model W185-Heat Systems U1trasonics, Inc.), equipped with a microtip accessory, was used to enhance the extraction of aromatic amines-particularly from insoluble dyestuffs. The output control was maintained at a setting sufficient to give a reading of approximately 50 W on the power meter. Laboratories were illuminated with yellow light (devoid of UV components) to prevent photochemical degradation of the more labile amines. Thin-Layer Chromatography. Thin-layer plates were 20 X 20 cm, precoated with a 250-rm layer of silica gel G-F254, (EM Laboratories, Inc., Catalogue No. 5767). The TLC plates were
ANALYTICAL CHEMISTRY, VOL. 50, NO 1, JANUARY 1978
173
Table I. Determination of Aromatic Amines in Dyestuffs by TLC, Accuracy and Precision
i
!I
Av %
Amine Benzidine
2
&-Naphthylamine 0-Naphthylamine 3,3'-Dichlorobenzidine I
I
4
O
4-Dimethylaminoazobenzene
I 8
Time Iminuterl
Figure 5. Gas-liquid chromatogram of aromatic amines. Concentration of each amine, 0.05 mg/rnL. Sample volume injected, 3 FL. Column temperature, 240 O C . (1) Benzidine; (2) 4-dirnethylarninoazobenzene; (3) 3,3'-dichlorobenzidine
not activated before use. Thin-layer tanks were not saturated and were used immediately after introduction of the appropriate solvent mixture. Aliquots (10 pL) of standard solutions or of the sample extracts were applied to the TLC plates by means of 5-pL capillary pipets (Microcaps, Drummond Scientific Company). In order to ensure small, uniform spot shapes, one 5-pL portion was spotted, allowed to dry, and then a second 5-pL portion was spotted on top of the first. Thin-layer plates were developed in the appropriate solvent system until the solvent migrated a distance of 15 cm up the plate. The TLC plates were allowed to air dry for approximately 1 h before scanning. The measured parameter was fluorescence quenching. Solvent systems for thin-layer chromatography were as follows: System A = hexanelethyl acetatelglacial acetic acid 80:20:5 System B = methylene chloride/acetone 90:10
a
Added,
Found,
!Jg/g
Mg/g
400 400 400 400 400 400 400 400 400 400 400 400 400 400 400
400 420 420 400 400 420 410 410 380 390 400 390 360 370 360 400 390 390 390 360
400 400 400 400 400
RecoverY
+ 10
103
410t 10
103
3 9 0 i 10
98
370-r 10
93
3805 10
95
410
SD = standard deviation.
Gas-Liquid Chromatography. The column used to separate inch x 3.5 mm i.d., glass U the aromatic amines was a 6 ft x tube, packed with 370 OV-1 on 100/1:!0 Gas Chrom Q. Temperatures of the inlet and detector were maintained at 250 and 270 "C, respectively. The column oven was maintained at 140 "C for the separation of a- and 3-naphthylamine or at 240 "C for the separation of benzidine, 3,3-dichlorobenzidine, and 4-dimethylaminoazobenzene. Carrier gas was helium at the flow rate of 60 mL/min. Aliquots (3 pL) of extracts were injected directly into the gas-liquid chromatograph. Reagents a n d Chemicals. Organic solvents (spectrograde) were obtained from Matheson Coleman and Bell. Aromatic amine standards (reagent grade) were purchased from Pfaltz & Bauer, Inc. (Flushing, N.Y.) and used without further purification. Standard solutions in chloroform were prepared in a controlled area and stored at 4 "C in amber glassware. Gas chromatographic column packing, 3% OV-1 on 100/120 Gas Chrom Q was obtained
1
4-DIMETHYL4MINOAZOBENZENE
CONCENTRATION OF AROMATIC AMINE I m g / m l )
Flgure 6. Analysis of aromatic amines by TLC. Typical standard curves
Av M g / g found + S Da
174
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
Table 11. Determination of Aromatic Amines in Dyestuffs b y CLC, Accuracy and Precision Av %
Amine Benzidine
&-Naphthylamine @-Naphthylamine 3,3'-Dichlorobenzidine 4-Dimethylaminoazobenzene
Added, wglg
Found, Pglg
400 400 400 400 400 400 400 400 400 400 400 400 400 400 4 00 400 400
391 400 400 373 450 370 420 440 430 370 410 430 443 353 353 393 437 337 347 437
400 400 400
a
Av pglg found + SD"
RecoverY
3 9 1 t 20
98
420
t
40
105
410
t
30
103
386
+
40
96
390 + 6 0
97
SD = standard deviation.
from Applied Science Laboratories, State College, Pa. Procedure. Three representative 1-g portions of dyestuff were added to each of three 45-mL centrifuge tubes fitted with ground glass stoppers. To one tube was added 400 pg each of the compounds under investigation. The dyestuff samples were dissolved or suspended in 4 mL of 0.5 N NaOH and 20 mL of chloroform was added. The mixtures were sonified for 6 min, then centrifuged at 1500 rpm for 15 min. Five-mL aliquots of the resulting organic phases were transferred into 15-mL centrifuge tubes, washed with 5 mL of 6 N NaOH solution, and the washed organic phases were transferred to small screw cap vials. Aliquots of the extracts were then analyzed by both gas-liquid chromatography and thin-layer chromatography as described above.
&naphthylamine eluted much earlier than the other three materials and could he separated a t 140 "C (Figure 4). However, in order to separate benzidine, 4-dimethylaminoazobenzene, and 3,3'-dichlorobenzidine within a reasonable time, a column temperature of 240 "C was required (Figure 5 ) . At this temperature, the naphthylamines merged with the solvent band. By temperature programming, all five compounds can be separated in a single injection. In the absence of interferences, the minimum detection limit by TLC was 100 ng of each amine (spotted in 10 FLof solvent) which is equivalent to 0.02% (200 ppm) with the quantities of dyestuff specified in the method. Similarly, the GLC detection limit was 15 ng (injected in 3 1L of solvent) or 0.01% (100 ppm). T h e detection limits can be decreased by concentrating the extracts. In our case, a concentration step was avoided so that the analysis could be expedited. Recovery and precision data are shown in Tables I and 11. The recovery of aromatic amines from "spiked'' dyestuffs was generally found to be greater than 9 0 % . T h e standard deviation from the mean was less than 1070.In some cases, recoveries greater than 100% were found, probably because of interfering components present in some of the dyestuffs. T h e relationship of peak height to aromatic amine concentration was linear when determined by GLC, but nonlinear when determined by TLC (Figure 6). The latter was probably due to light scattering since measurements were carried out in the reflectance mode (16). No attempt was made to apply mathematical corrections to the TLC calibration data since t h e precision and accuracy obtained with the nonlinear standard curves (see Table I) were adequate for the purposes of the assay. In summary. a combined GLC-TLC method has been described for the simultaneous qualitative and quantitative determination of five regulated aromatic amines in dyestuffs. T h e method is simple, fast, and is capable of determining aromatic amine concentrations at least ten times less than the minimum OSHA standard.
RESULTS AND DISCUSSION Complete separation of the five aromatic amines (see Figure 1)was possible by utilizing one or more combinations of TLC and GLC. T h e TLC developer hexane/ethyl acetate/glacial acetic acid 80:20:5 (System A) was able to resolve all five compounds (Figure 2). Benzidine, however, remained at the origin. T o ensure accurate qualitative and quantitative TLC determination of benzidine, it was necessary to use a second developer system. Methylene chloride/acetone 9O:lO (System B) proved satisfactory for benzidine (Rf = 0.4) but in this developer, a-naphthylamine co-chromatographed with 3,3'dichlorobenzidine (Figure 3). Because of these differences in separation, System A was used for the determination of aa n d P-naphthylamine, 3,3'-dichlorobenzidine, and 4-dimethylaminoazobenzene while System B was used for the determination of benzidine. Similarly, the conditions for gas-liquid chromatography varied depending on the compounds to be analyzed. a - and
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8)
(9) (IO) (11) (12) (13) (14) (15) (16)
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RECEIVED for review July 1, 1977. Accepted October 27, 1977.
