Page:NIOSH Manual of Analytical Methods - 8306.pdf/2

 BENZIDINE in urine: METHOD 8306, Issue 2, dated 15 August 1994 - Page 2 of 4 EQUIPMENT:

REAGENTS: 1. 2.

Hydrochloric acid, conc. (sp. gr. 1.18). Sodium hydroxide, 10 N. Dissolve 400 g NaOH in 1:1 methanol:water (v/v) to make 1 L solution. CAUTION: EXOTHERMIC REACTION. 3. Sodium hydroxide, 5 N. Dilute 10 N with 1:1 methanol:water 4. Acetone, hexane, methanol, and benzene *, pesticide grade. 5. Hexane:benzene, 3:2 (v/v). 6. Sodium sulfate (anhydrous) extracted in Soxhlet extractor for 24 h with benzene. 7. Trimethylamine stock solution. a. Dissolve 2 g (CH 3)3NHCl in 5 mL 5 N NaOH. CAUTION: EXOTHERMIC REACTION b. Extract four times with 5-mL portions of benzene. c. Percolate each 5 mL extract through a 2cm anhydrous sodium sulfate column. d. Keep in the dark and refrigerated when not in use. Reagent stability unknown. 8. Trimethylamine working solution. Dilute trimethylamine stock solution 1:20 with benzene. Reagent stability unknown. 9. Benzidine stock solution, 100 µg/mL.* Dissolve 10 mg benzidine, accurately weighed, in benzene to make 100 mL solution. Refrigerate. Discard if a color develops. 10. Methylene dianiline stock solution, 100 µg/mL. Dissolve 10 mg methylenedianiline,, accurately weighed, in benzene to make 100 mL solution. Refrigerate. Discard if color develops. 11. Derivatized methylenedianiline stock solution, 1 µg/mL. a. Add 0.1 mL methylenedianiline stock solution to 1.5 mL benzene and 0.5 mL trimethylamine working solution. b. Derivatize and wash (steps 12 and 13). c. Percolate the solution through 2 cm anhydrous sodium sulfate. d. Dilute to 10.0 mL with benzene. Stable at least one month. 12. Florisil, PR grade, 60/100 mesh; activated at 650°C. a. Extract 24 h in a Soxhlet extractor with benzene. b. Heat to 130°C for 24 h. c. Cool and add 10 g distilled water/100g Florisil. Shake overnight.

1. Bottles, polyethylene, 125-mL. 2. Gas chromatograph with 28Ni63 electron capture detector, temperature programming, capillary column option, Grob-type injection system, and electronic integrator. 3. Shaker and rotator. 4. Evaporator and vortex mixer. 5. Centrifuge (refrigerated optional). 6. Waterbath with a temperature range 30 to 100°C. 7. Soxhlet extractor. 8. Culture tubes, 25 × 200-mm, 20 × 125-mm, and 16 × 150-mm, with PTFE-lined screw caps.** 9. Pipettes, 1-, 5- and 10-mL and Pasteur (disposable).** 10. Graduated centrifuge tubes, 15-mL, glass-stoppered.** 11. Volumetric flasks.**

Wash with detergent, clean with chromic acid, rinse with distilled water, acetone, and benzene.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94