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These procedures were designed and tested for internal use by OSHA personnel. Mention of any company name or commercial product does not constitute endorsement by OSHA.
|Target concentration:||10 mg/m3 (ACGIH TLV)|
|Procedure:||Collection using a bubbler containing isopropanol. Analysis is by high performance liquid chromatography with ultraviolet detection.|
|Recommended air volume and sampling rate:||250 L at 1.0 L/min|
|Reliable quantitation limit:||1.0 µg/m3|
|Standard error of estimate at the target concentration:
|Status of method:||A sampling and analytical method which has been subjected to the established evaluation procedures of the Organic Methods Evaluation Branch.|
Date: August 1980
Chemist: Warren Hendricks
Organic Methods Evaluation Branch
OSHA Salt Lake Technical Center
Sandy UT 84070-6406
Diphenylamine (DFA) has been determined using thin layer (Refs. 5.1. and 5.2.), liquid (Ref. 5.3.), and gas (Ref. 5.4.) chromatographic techniques. Spectrophotometric analytical procedures are also available for the compound. (Ref. 5.5.)
A literature search resulted in no citations regarding air sampling methods for DFA. Since the vapor pressure of DFA is 1 mm Hg at 108°C, both aerosol and vaporous DFA may be encountered in the field. A general sampling procedure should provide a means to collect both phases. It was determined by sampling over an open container of DFA, that glass fiber and PTFE filters would not retain DFA vapors. Florisil tubes did collect DFA vapors, but since the collection efficiency of aerosols on adsorption tubes has not been well established, it was decided not to pursue this approach. Therefore, the use of a bubbler containing a collection medium was evaluated for the sampling procedure. Isopropanol was selected as the collection medium because DFA is very soluble in that solvent.
The LD50 for DFA was 300 mg/kg when administered orally to the guinea pig. The lowest published oral lethal dose for the rat and for the hamster was 3000 and 500 mg/kg respectively. The lowest published oral toxic dose administered to rats 17 to 22 days pregnant was 7500 mg/kg and the toxic effects were teratogenic in nature. The lowest published oral lethal dose for humans was 500 mg/kg. (Ref. 5.6.)
The probable human oral lethal dose was estimated to be 0.5 to 5 g/kg. DFA, when given by mouth (in an oil solution) to laboratory animals, causes loss of appetite, diarrhea, emaciation, hypothermia, and general physical weakness, probably from protracted inflammation of the membrane lining of the stomach and the intestines. Death may not result until 2 to 3 weeks after a single lethal dose. (Ref. 5.7.)
Male and female albino rats fed 0.5 to 1.0% dietary concentrations of DFA for 2 years showed growth arrests which, in part, were due to decreased food intake. Moderate degrees of anemia were also observed, but return to normal hemoglobin levels followed feeding of the control diet. Leukocyte numbers and percentages remained within normal ranges in anemic rats. DFA did not increase proteinuria or cause glycosuria. An apparent decrease in litter sizes, number of pups weaned, and decreased weights of pups at weaning, observed in gestating and lactating rats fed DFA was probably due to reduced food intake. The 2-year ingestion of DFA caused lesions only in the urinary tract, namely, cystic dilatation of renal tubules with inflammation between the tissues. The glomeruli were never altered. Lower tubules were sometimes filled with a fluid. The incidence of tumors was due to the age of the rats at autopsy, not because of treatment with DFA. (Ref. 5.8.)
DFA was reported to be less toxic and less readily absorbed through the skin and respiratory tract than aniline, but the acute and chronic systemic toxicity potentials were considered to be high for both compounds. Industrial poisoning with DFA has been encountered and reported symptoms included bladder and skin problems, abnormal heartbeat, and high blood pressure. It was also reported that animals exposed to DFA dust developed definite changes in their liver, spleen and kidneys. Based on industrial experience, a TLV of 10 mg/m3 was suggested for DFA dust because that value was known to be sufficiently low to prevent systemic poisoning. (Ref. 5.9.)
While there is no experimental or epidemiological evidence that DFA is a carcinogen, commercial DFA usually contains 4-aminodiphenyl (a very potent carcinogen) as an impurity. The expanding use of DFA requires the continuing investigation of this compound to be maintained. (Ref. 5.10.)
Diphenylamine is widely used as a rubber antioxidant and accelerator, an insecticide (directly and by fusion with sulfur), solid fuel rocket propellant, stabilizer for explosives, preparation of azo dyes (Acid Yellow 36, Acid Yellow 63, and Acid Orange 5), pharmaceuticals, veterinary medicine, storage preservation of apples, and as a reagent in analytical chemistry. (Refs. 5.11 and 5.12.)
|synonyms:||aniline, N-phenyl; anilinobenzene; Big Dipper; NCI 10355; DFA; DPA; NO SCALD; N-phenylaniline; Scaldip|
|molecular structure:||Figure 1.1.5.|
|physical appearance:||white to grayish crystals|
|vapor pressure:||1 mm Hg at 108.3°C|
|solubility:||Soluble in carbon disulfide, benzene, alcohol, and ether. Insoluble in water.|
|boiling point:||302°C, 179°C at 22 mm Hg|
|flash point:||307°F (closed cup)|
|autoignition temp.:||1173°F (combustible)|
|l max: 208 nm (log e = 4.33)
l max: 286 mm (log e = 4.29)
The detection limit of the analytical procedure is 0.42 ng per injection. This is the amount of analyte which will give a peak whose height is about five times the height of the baseline noise. (Section 4.1.)
The detection limit of the overall procedure is 0.26 µg per sample (1.0 µg/m3). This is the amount of analyte spiked into the sampling device which allows recovery of an amount of analyte equivalent to the detection limit of the analytical procedure. (Section 4.2.)
The reliable quantitation limit is 0.26 µg per sample (1.0 µg/m3). This is the smallest amount of analyte which can be quantitated within 95% confidence limits of ±25%. (Section 4.3.)
The reliable quantitation limit and detection limits reported in the method are based upon optimization of the instrument for the smallest possible amount of analyte. When the target concentration of an analyte is exceptionally higher than these limits, they may not be attainable at the routine operating parameters.
The sensitivity of the analytical procedure over a concentration range representing 0.5 to 2 times the target concentration based on the recommended air volume is 178.9 area units per µg/mL. The sensitivity is determined by the slope of the calibration curve. (Section 4.4.) The sensitivity will vary somewhat with the particular instrument used in the analysis.
The pooled coefficient of variation obtained from replicate determinations of analytical standards at 0.5, 1, and 2 times the target concentration is 0.033. (Section 4.6.)
The overall procedure must provide results at the target concentration that are ±25% or better at the 95% confidence level. The precision at the 95% confidence level for the 16-day ambient temperature storage test is ±11.6%. (Section 4.7.) This includes an additional ±5% for sampling error.
Isopropanol, HPLC grade.
Because of present laboratory limitations, test atmospheres of DFA could not be generated to determine collection capacity. The following data were gathered in order to approximate the 5% breakthrough air volume.
Two isopropanol bubblers were placed in series and 2 mg of DFA was added to the first bubbler. Air, at about 75% relative humidity and 22°C, was drawn through the train at 1 L/min. The volume of isopropanol in each bubbler was maintained at 10 to 15 mL by adding pure HPLC grade isopropanol as required. Small samples were removed from the second bubbler at various intervals and an amount of DFA equivalent to 5% of the DFA added to the first bubbler was found in the second bubbler after about 315 L of air had been sampled.
A short piece of silanized glass tubing containing a plug of silanized glass wool was butted to the inlet of a glass bubbler with Teflon tubing. DFA crystals were placed on the glass wool plug. A second bubbler was placed in series and then air at about 80% relative humidity and 21°C was drawn through the device at 1 L/min. The volume of isopropanol in each bubbler was maintained at 10 to 15 mL by adding pure HPLC grade isopropanol as required. Small samples were removed from both bubblers at various intervals and after 400 L of air had been sampled, breakthrough from the front to the rear bubbler was about 2%. The amount of DFA in the front bubbler was determined to be about 360 µg.
It is unknown if there are interferences with the collection of DFA in isopropanol bubblers.
Measure to ±0.1 mL, the volume of isopropanol in the sample transport vial.
|column:||DuPont Zorbax CN (4.6 mm x 25 cm)|
|mobile phase:||methanol/water 60/40 (v/v)|
|flow rate:||1 mL/min|
|UV detector:||280 nm (fixed wavelength)|
|injection volume:||25 µL|
DFA, mg/m3 = (A)(B)/C
|where||A = µg/mL from Section 3.7.2.
B = volume (in milliliters) of isopropanol from Section 3.4.
C = air volume in liters
The detection limit for DFA was 0.42 ng (25 µL x 0.017 µg/mL) per injection. This amount of analyte gave a peak whose height was about 5 times the amplitude of the baseline noise. (Figure 4.1.)
The detection limit of the overall procedure was 0.26 µg (15 mL x 0.017 µg/mL) per sample.
The reliable quantitation limit was the same as the detection limit of the overall procedure since the interval about the detection limit was less than ±25% at the 95% confidence level. This was determined by replicate injections from a standard solution.
Reliable Quantitation Limit Data
peak height, mm
The data in Table 4.6. is presented graphically in Figure 4.4. This is the calibration curve and the slope of the regression line is a measure of the sensitivity of the analytical method.
A typical chromatogram for DFA is presented in Figure 4.5.
This data represents multiple injections from standard solutions. The injection volume was 25 µL and the concentrations of the standards were 83.4, 166.7, and 333.4 µg/mL.
Precision of the Analytical Method
|x target conc.
CV = 0.033
The data in Table 4.7. represent the results of storage tests conducted at ambient (20 to 25°C) and reduced (-5°C) temperatures. The samples were prepared by placing 15-mL aliquots of a solution containing 166.7 µg/mL DFA in isopropanol into 20-mL glass scintillation vials. Three separate vials were analyzed on the day indicated.
The data in Table 4.7. are presented graphically in Figures 4.7.1. and 4.7.2.
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