(1) BENZIDINE and (2) 3,3'-DICHLOROBENZIDINE: METHOD 5509, Issue 2, dated 15 August 1994 - Page 2 of 4
REAGENTS: 1. 2. 3. 4. 5. 6. 7.
8. 9.
EQUIPMENT:
Methanol, HPLC grade. Acetonitrile, HPLC grade. Triethylamine. Water, distilled, deionized. Benzidine.* 3,3'-Dichlorobenzidine.* Eluent: 0.17% (v/v) triethylamine in methanol. Dilute 170 µL triethylamine to 100 mL with methanol. Calibration stock solution, 0.5 µg/µL. Dissolve 50 mg analyte in 100 mL eluent. Recovery (R) stock solution, 0.5 µg/µL. Dissolve 50 mg analyte in 100 mL methanol.
See SPECIAL PRECAUTIONS.
1. Sampler: 13-mm, Type AE, glass fiber filter (Gelman, or equivalent) in a 13-mm filter holder (Swinney, Gelman, or equivalent). 2. Personal sampling pump, 0.2 L/min, with flexible connecting tubing. 3. High-performance liquid chromatograph, UV detector, integrator and column (page 5509-1). 4. Test tubes, 1-mL, with polyethylene stoppers. 5. Syringes, glass, 10- and 25-µL, readable to 0.1 µL. 6. Pipets, delivery, 0.5- and 5-mL, graduated in 0.1 mL. 7. Flasks, volumetric, 10- and 100-mL. 8. Centrifuge. 9. Test tube shaker, vortex type.
SPECIAL PRECAUTIONS: Benzidine is a recognized human carcinogen and can be absorbed through the skin [4,5,6]. 3,3'-Dichlorobenzidine is a carcinogen [4,6]. Take appropriate precautions to avoid personal and area contamination.
SAMPLING: 1. 2. 3. 4.
Calibrate each personal sampling pump with a representative sampler in line. Sample at 0.2 L/min for a total sample size of 20 to 100 L. Cap the sampler. Ship and store samples at -15 °C if benzidine may be present. NOTE: Samples may be stored at room temperature if only 3,3'-dichlorobenzidine is present.
SAMPLE PREPARATION: 5. 6. 7. 8.
Place glass fiber filter in a test tube. Add 0.5 mL eluent to each test tube. Seal each test tube and shake them on a test tube shaker. Allow samples to stand for 1 h with intermittent shaking. Centrifuge each sample for 10 min.
CALIBRATION AND QUALITY CONTROL: 9.
10.
Calibrate daily with at least six working standards over the range 0.05 to 7 µg analyte per sample. a. Deliver aliquots of calibration stock solution with microliter syringe to eluent in 10-mL volumetric flasks and dilute to the mark. b. Analyze together with the samples and blanks (steps 12 through 14). c. Prepare calibration graph (peak area vs. µg analyte). Determine recovery (R) at least once per year for each lot of filters. Prepare four filters at each of five levels, plus three media blank filters. a. Place sample filters into separate test tubes. b. Inject an aliquot of R stock solution, or a dilution thereof in methanol, directly onto the filter. c. Cap the test tubes. Allow to stand overnight. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94
