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
