# Dyes, Benzidine-, o-Tolidine-, o-Dianisidine- (5013)

​DYES, BENZIDINE-, o-TOLIDINE-, o-DIANISIDINE-
Table 1
MW: Table 1
METHOD: 5013, Issue 2
CAS: Table 1
RTECS: Table 1
EVALUATION: PARTIAL
OSHA : no PEL NIOSH: lowest feasible; carcinogen ACGIH: carcinogen
PROPERTIES:
5013
Issue 1: 15 May 1989 Issue 2: 15 August 1994
colored powders; water soluble; VP not significant
SYNONYMS: Table 1.
SAMPLING SAMPLER:
FILTER (5-µm PTFE membrane)
MEASUREMENT TECHNIQUE:
HPLC, UV DETECTION
ANALYTE:
benzidine, o-tolidine, o-dianisidine
DESORPTION:
2 mL H 2O; ultrasonic
REACTION:
reductive cleavage of dye to free amine with sodium hydrosulfite
FLOW RATE: 1 to 3 L/min VOL-MIN: -MAX:
150 L @ 0.1 mg/m 3 500 L
SHIPMENT:
keep samples dry and cool; protect from light
SAMPLE STABILITY: FIELD BLANKS: BULK SAMPLE:
INJECTION VOLUME:
10 µL
at least 7 days @ 25 °C in the dark MOBILE PHASE:
60% methanol/40% phosphate buffer; ambient temperature
COLUMN:
10 cm x 8-mm ID, Waters Radial Pak C 18, 10-µm particles, with Radial Compression Module or equivalent
DETECTOR:
UV detector @ 280 nm
CALIBRATION:
solutions of benzidine, o-tolidine and odianisidine in methanol
RANGE:
ca. 15 to 250 µg per sample (Table 1)
2 to 10 field blanks per set
include 1 bulk sample for each dye present on the filter ACCURACY
RANGE STUDIED:
not studied
BIAS:
none identified
ˆ ): not evaluated OVERALL PRECISION (S rT ACCURACY:
not determined
ESTIMATED LOD: 3 µg benzidine per sample [1] PRECISION (Sr):
0.04 to 0.08 [2,3]
APPLICABILITY: The working range is ca. 0.06 to 8 mg/m 3 for a 250-L air sample. This method determines benzidine, o-tolidine or o-dianisidine from dyes based on these amines, but will not distinguish different dyes based on the same amine. The metho d will not distinguish benzidine obtained from reduction of the dyes and free benzidine in the dye formulation.
INTERFERENCES: Aniline, azobenzene, p-aminophenol, p-phenylenediamine or p-nitroaniline do not interfere in the measurement when present in equimolar amounts.
OTHER METHODS: This revises P&CAM 325 [2,3]. P&CAM 234 [4,5], a general colorimetric method for diazonium salts and azo dyes, has not been revised.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94 ​DYES, BENZIDINE-, o-TOLIDINE-, o-DIANISIDINE-: METHOD 5013, Issue 2, dated 15 August 1994 - Page 2 of 5
EQUIPMENT:
REAGENTS: 1. 2. 3. 4. 5. 6.
7. 8. 9. 10.
11.
12.
Water, deionized and distilled. Methanol, HPLC Grade. Benzidine.* o-Tolidine.* o-Dianisidine.* Calibration stock solutions. Dilute the following amounts to 10 mL with methanol. Stable one month at 4 °C. a. Benzidine, 15.6 mg b. o-Tolidine, 15.3 mg c. o-Dianisidine, 5.6 mg Disodium hydrogen phosphate, Na 2HPO 4. Potassium dihydrogen phosphate, KH 2PO 4. Sodium hydrosulfite, Na 2S2O 4. HPLC mobile phase buffer. Dilute 3.39 g KH 2PO 4 and 4.30 g Na 2HPO 4 to 1 L with water. Prepare daily. Reduction buffer. a. Solution A. Dilute 1.179 g KH 2PO 4 and 4.30 g Na 2HPO 4 to 1 L with water. Prepare daily. b. Solution B. Dilute 11.79 g KH 2PO 4 and 43.00 g Na 2HPO 4 to 1 L with water. Prepare daily. Reducing solution. Dilute 200 mg Na 2S2O 4 to 10 mL with the appropriate reduction buffer of Solution A or B (see Table 1) immediately before use.
1. Sampler: PTFE filter, 5-µm, 37-mm (Millipore Mitex or equivalent), with backup pad in a three-piece plastic cassette filter holder. 2. Personal sampling pump, 1 to 3 L/min, with flexible connecting tubing. 3. High pressure liquid chromatograph equipped with 280-nm UV detector, integrator and column (page 5013-1), Waters Model RCM 100 Radial Compression Module with Waters Radial Pak C 18 column or equivalent. 4. Syringe or autosampler for HPLC injection. 5. Syringes, volumetric, 10-, 25- and 50-µL. 6. Flasks, volumetric, 10- and 100-mL, 1-L. 7. Vials, 4-mL, with screw caps. 8. Pipets, volumetric, 1-mL. 9. Tweezers. 10. Beakers, 50-mL. 11. Ultrasonic water bath.
SPECIAL PRECAUTIONS: NIOSH recommends that benzidine- and benzidine-congener-based dyes be recognized and handled as human carcinogens because they can be reduced to the free aromatic amine in living systems and eliminated by the usual metabolic pathways [4,6]. Benzidine is a recognized human carcinogen and regulated by the Occupational Safety and Health Administration. o-Tolidine and o-dianisidine are currently suspect carcinogens and should be handled accordingly [4,6]
SAMPLING: 1. 2.
Calibrate each personal sampling pump with a representative sampler in line. Sample at an accurately known flow rate between 1 and 3 L/min for a total sample size of 150 to 500 L.
SAMPLE PREPARATION: 3. 4. 5.
Remove the filter from the cassette with clean tweezers and place it face up in a 50-mL beaker. Add 1.0 mL H 2O to the beaker and swirl to wet the entire deposit. Add another 1.0 mL H 2O to the beaker and swirl. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94 ​DYES, BENZIDINE-, o-TOLIDINE-, o-DIANISIDINE-: METHOD 5013, Issue 2, dated 15 August 1994 - Page 3 of 5
6. 7. 8.
9.
Turn the filter sample side down and place the beaker in an ultrasonic bath for 15 min. Pipet 1.0 mL desorbed sample solution to a 4-mL vial. Add 1.0 mL freshly prepared reducing solution. NOTE: Where the composition of the sample dye is unknown or different from the dyes listed in Table 1, reduce a 1-mL aliquot of the desorbed dye solution with the reducing solution made from buffer A. If precipitation of the dye from solution is observed at this point, reduce the remaining 1-mL aliquot of the desorbed dye solution with the reducing solution made with buffer B. Cap the vial and allow to stand with occasional shaking for at least 1 h or until the change in solution color is completed.
CALIBRATION AND QUALITY CONTROL: 10.
11.
Calibrate daily. a. Dilute aliquots of the calibration stock solutions for each of the amines (benzidine, o-tolidine, o-dianisidine) to 10 mL with methanol in volumetric flasks. Prepare at least six working standards to cover the ranges of interest (0.38 to 16 µg/mL benzidine; 0.77 to 15.3 µg/mL o-tolidine; 0.56 to 11.7 µg/mL o-dianisidine). b. Analyze the working standards together with samples and blanks (steps 12 through 14). c. Prepare separate calibration graphs (peak area vs. µg per sample) for benzidine, o-tolidine. and o-dianisidine. At the completion of step 9, the total effective volume of the solution containing the entire sample is 4 mL. Determine recovery if the filter sample is known to contain only one dye. a. Prepare a solution of a known concentration of the bulk sample of the dye in 10 mL H 2O. b. Add known volumes of this solution to PTFE filters using a microliter syringe and allow to dry. Prepare three filters at each of five concentration levels. c. Analyze the filters by steps 3 through 9 and 12 through 14. d. On the day of analysis of the filters, add the same volume of dye solution which was spiked onto the filters to 2.0 mL H 2O. Reduce this solution (steps 7 through 9) and analyze together with samples and standards (steps 12 through 14). e. Calculate recovery (µg benzidine, o-tolidine, or o-dianisidine recovered from the filter/µg from step 11.d). f. Prepare graph of recovery vs. µg benzidine, o-tolidine or o-dianisidine recovered.
MEASUREMENT: 12. 13. 14.
Set the HPLC to conditions given on page 5013-1 and in Table 1. Inject 10 µL sample into the HPLC. Make duplicate injections of samples and standards. Measure peak areas.
CALCULATIONS: 15.
16.
Read the mass, µg (corrected for recovery, if applicable) of the free amine corresponding to each peak area for the sample (W) and average media blank (B) from the appropriate calibration graph. Calculate the concentration, C, of benzidine, o-tolidine or o-dianisidine in the air volume sampled, V (L):
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94 ​DYES, BENZIDINE-, o-TOLIDINE-, o-DIANISIDINE-: METHOD 5013, Issue 2, dated 15 August 1994 - Page 4 of 5
EVALUATION OF METHOD: This method was laboratory evaluated over the ranges listed in Table 1 [2]. The concentration of dye remaining after reduction varied from 0 to 6% as determined by visible spectrophotometry. The reduced free amine was confirmed by GC/MS. The lower end of the measurement range was defined as the level which gave at least 75% recovery with approximately 10% Sr. For C. I. Direct Red 28, the range covered 27.3 to 273 µg dye per sample. The lower level of this range gave 94.4% recovery ( Sr = 6.3%) based on free benzidine for six replicate samples. For the other three dyes the results were as follows: C. I. Direct Blue 6, 15.0 to 300.0 µg dye per sample, 100.3% recovery ( Sr = 4.34%); C. I. Direct Brown 95, 24.3 to 243 µg dye per sample, 78.5% recovery ( Sr = 6.7%); C. I. Direct Black 38, 12.5 to 250.0 µg dye per sample, 78.1% recovery ( Sr = 12.8%). The pooled precisions ( Sr) for samples at three concentrations in the measurement range for each dye are as follows: Direct Red 28, 0.045; Direct Blue 6, 0.061; Direct Brown 95, 0.072; Direct Black 38, 0.078. Since the method does not distinguish between various benzidine-based dyes, recovery correction factors cannot be applied unless a single known dye is contained on the filter. Recovery studies must be performed on the bulks collected with the samples because of variability in sample purity and variety of benzidine-based dyes. Recovery studies on spiked samples stored at 75% relative humidity indicated no change due to humidity in the recoveries or sample stability. A user check of this method [1] gave estimated LODs for benzidine (3 µg per sample; 0.7 µg/mL) and dianisidine (4.5 µg per sample; 1.1 µg/mL). The LODs could be lowered to 0.06 µg/mL for benzidine and 0.07 µg/mL for dianisidine by using fluorescent detection with excitation at 285 nm and emission at 375 nm.
REFERENCES: [1] [2] [3]
[4] [5] [6]
User check, Wisconsin Occupational Health Laboratory (NIOSH, unpublished, September 27, 1984). NIOSH Manual of Analytical Methods, 2nd ed., Vol. 6, P&CAM 325, U.S. Department of Health and Human Services, Publ. (NIOSH) 80-125 (1980). Kennedy, Eugene R. and Martha J. Seymour. Development of an Analytical Method for Benzidine-Based Dyes, Chemical Hazards in the Workplace Measurement and Control, ACS Symposium Series 149, American Chemical Society, Washington, DC, 21-35 (1981). Special Occupational Hazard Review for Benzidine-Based Dyes, U.S. Department of Health, Education, and Welfare, Publ. (NIOSH) 80-109 (1979). NIOSH Manual of Analytical Methods, 2nd ed., Vol. 1, P&CAM 234, U.S. Department of Health, Education, and Welfare, Publ. (NIOSH) 77-157-A (1977). Preventing Health Hazards from Exposure to Benzidine Congener Dyes, U.S. Department of Health and Human Services, Publ. (NIOSH) 83-105 (1983).
METHOD WRITTEN BY: Eugene R. Kennedy, Ph.D., Martha J. Seymour and Charles E. Neumeister, NIOSH/DPSE.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94 ​DYES, BENZIDINE-, o-TOLIDINE-, o-DIANISIDINE-: METHOD 5013, Issue 2, dated 15 August 1994 Page 5 of 5 TABLE 1. BENZIDINE- AND BENZIDINE-CONGENER-BASED DYES.
Color Index Name
C.I. Number
Molecular Formula
M.W.
Synonyms [4]
Range a µg per sampler µg/mb3 r
BENZIDINE-BASED: C.I. Direct Red 28
22120
C32H22N6S2O 6Na 2
696.67
Congo Red; CAS #573-58-0 RTECS QK1400000
27.3-273
546.5 -46
C.I. Direct Blue 6
22610
C32H20N6S4O 14Na 4
932
Amidine Blue 2B; CAS #2602-46-2 RTECS QJ6400000
15.0-300
30.6 -00
C.I. Direct Brown 95
30145
C31H18N6O 9Na 2Cu
759
Chrome Leather Brown; CAS #16071-86-6 RTECS GL7375000
24.3-242
486.4 -85
C.I. Direct Black 38
30235
C34H25N9S2O 7Na 2
781
Amidine Black GA; CAS #1937-37-7 RTECS QJ6160000
12.5-250
250.5 -00
o-TOLIDINE-BASED: C.I. Direct Red 2
23500
C34H26N6S2O 6Na 2
724.74
Benzopurpurine 4B; CAS #992-59-6 RTECS QK1765000
16.7-334
334.6 -68
24140
C34H24N4S2O 10Na 2
758.69
Direct Azurine G; Benzo Azurine G; CAS #2429-71-2
10.2-204
204.4 -08
o-DIANISIDINE-BASED: C.I. Direct Blue 8 a b
Air concentrations calculated from analytical results with an air volume of 500 L assumed. The letters "A" and "B" refer to the reduction buffer solutions described in the REAGENTS section, items 11.a and b.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94