Page:NIOSH Manual of Analytical Methods - 5516.pdf/3

SAMPLE PREPARATION:

6. Add 25 μL acetic anhydride to acetylate the 2,4- and 2,6-toluenediamine and excess 1-(2-methoxy­ phenyl)piperazine. Allow 4 hours for completion of reactions. Evaporate sample to dryness under a gentle stream of nitrogen while warming to 40 to 50 °C on a hotplate. Redissolve residue in 1.5 mL methanol. NOTE: The acetylation reaction for 2,4-toluenediamine to produce 2,4-bisacetamidotoluene is:

CALIBRATION AND QUALITY CONTROL:

7. Calibrate daily with at least six working standards.

a. Using aliquots of calibration stock solution, prepare working standards of 2,4- and 2,6- bisacet­ amidotoluene in methanol covering the range 0.1 to 3 μg/mL each.

b. Analyze these with the unknown and blank samples (steps 9 through 11).

c. Prepare calibration graphs (peak height vs. μg 2,4- and 2,6-toluenediamine per sample). Multiply the concentration (µg/mL) of bisacetamidotoluene by 0.889 mL to obtain the quantity (µg) of toluenediamine per sample.

NOTE: The factor 0.889 includes the MW of toluenediamine (122.17), the MW of bisacetamidotoluene (206.24), and the 1.5-mL solution volume from step 6.

8. Prepare three quality control samples by adding known quantities of 2,4- and 2,6-toluenediamine to 15 mL of sampling medium and analyze (steps 9 through 11).

MEASUREMENT:

9. Set up the HPLC system according to the manufacturer’s recommendations and to the conditions given on page 5516-1. The mobile phase program is:

a. Linear gradient 100% A to 90% A over t = 0 to 8 min.

b. 90% A to 40% A over t = 8 to 19 min following the convex gradient.

c. Hold at 40% A for 1 min, or as long as necessary to clear the column.

d. Return to 100% A and hold for 7 min before the next run.

NOTE: If only 2,4- and 2,6-toluenediamine are to be quantified, the mobile phase program may be modified to hasten elution of the ureas derived from the isocyanates.

10. Inject a 10-μL aliquot of solution from step 6 or step 7b.

11. Measure the peak heights. Adjusted retention times for some compounds of interest are:

CALCULATIONS:

12. Using the calibration graphs, determine the mass, μg, of 2,4- and of 2,6-toluenediamine in each sample (W) and in the average media blank (B). 13. Calculate the concentration, C, of 2,4- and of 2,6-toluenediamine in the air volume sample, (L):

$${\displaystyle C={\frac {(W-B)}{V}},{\text{mg}}/{\text{m}}^{3}}$$