# Polynuclear Aromatic Hydrocarbons by HPLC (5506)

​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC
Formulae: Table 1
MW: Table 1
METHOD: 5506, Issue 3
RTECS: Table 2
EVALUATION: PARTIAL
OSHA : Table 3 NIOSH: Table 3 ACGIH: Table 3 Compounds
CAS: Table 2
PROPERTIES:
acenaphthene acenaphthylene anthracene benz[a]anthracene benzo[b]fluoranthene benzo[k]fluoranthene
5506
Issue 1: 15 May 1985 Issue 3: 15 January 1998 Table 1
benzo[ghi]perylene benzo[a]pyrene benzo[e]pyrene chrysene dibenz[a,h]anthracene fluoranthene
fluorene indeno[1,2,3-cd]pyrene naphthalene phenanthrene pyrene
NAMES & SYNONYMS: Polycyclic aromatic hydrocarbons, PAHs; also see Table 2.
SAMPLING SAMPLER: FILTER + SORBENT TUBE (37-mm, 2-µm, PTFE + washed XAD-2, 100 mg/50 mg) FLOW RATE:
2 L/min
VOL-MIN: -MAX:
200 L 1000 L
SHIPMENT:
transfer filters to culture tubes; wrap sorbent and culture tubes in Al foil; ship @ 0 C
MEASUREMENT TECHNIQUE:
HPLC, FLUORESCENCE/UV DETECTION
ANALYTE:
compounds listed above
EXTRACTION:
5 mL acetonitrile; ultrasonic bath, 30 to 60 minutes
INJECTION VOLUME:
SAMPLE STABILITY:
unknown; protect from heat and UV light
FIELD BLANKS: MEDIA BLANKS:
3 to 10 field blanks per set 6 to 10 media blanks per set
10 to 50 µL
MOBILE PHASE:
acetonitrile/water gradient @ ambient temperature, 1 mL/min
COLUMN:
250 x 4.6-mm, reversed-phase, 5-µm C18
DETECTOR:
UV @ 254 nm; fluorescence @ 340 nm (excitation), 425 nm (emission)
CALIBRATION:
standards in acetonitrile
RANGE:
see EVALUATION OF METHOD
ACCURACY RANGE STUDIED: not determined BIAS: OVERALL PRECISION ( ACCURACY:
not determined
rT
not determined
ESTIMATED LOD: see EVALUATION OF METHOD PRECISION ( r):
see EVALUATION OF METHOD
not determined
APPLICABILITY: This method is applicable to samples that can be extracted with acetonitrile. This method is not applicable to samples that require a different extraction solvent or contain large amounts of highly adsorptive particulate matter, e.g., fly ash or diesel soot; also, this method is not applicable to asphalt fume samples.
INTERFERENCES: Any compound that elutes at the same HPLC retention time may interfere. Heat, ozone, NO2, or UV light may cause sample degradation.
OTHER METHODS: This revises P&CAM 206 and 251 [1]. Method 5515 uses the same sampling technique, with gas chromatographic measurement [2]. Method 5800 uses the same sampling technique, and a flow-injection method to determine total polycyclic aromatic compounds at two different sets of fluorescent wavelengths [3].
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC: METHOD 5506, Issue 3, dated 15 January 1998 - Page 2 of 9
REAGENTS:
EQUIPMENT:
1. Water, distilled, deionized, degassed. 2. Acetonitrile, HPLC grade, degassed. 3. PAH test mixture,* a liquid standard containing the PAHs except benzo[e]pyrene (EPA 610 Polynuclear Aromatic Hydrocarbons,Supelco, Cat. No. 4-8743; or equivalent). 4. Benzo[e]pyrene,* solid (Supelco, Cat. No. 442475; or equivalent).
1. Sampler: a. Filter. 37-mm, 2-µm pore size, PTFE membrane filter laminated to PTFE, (Zefluor, Pall Gelman Sciences, Cat. No. P5PJ037; SKC Inc., Cat. No. 225-17-07; or equivalent filter), cellulose spacer ring, 37-mm OD, 32-mm ID, (SKC Inc., Cat. No. 225-23; or equivalent) in a 37-mm cassette filter holder. NOTE: If sampling is to be done in bright sunlight, use opaque or foil-wrapped cassettes to prevent sample degradation. b. Sorbent tube, washed XAD-2 resin (front = 100 mg; back = 50 mg) (ORBO 43, Supelco, Cat. No. 2-0258; or equivalent), connected to filter with minimum length of PVC tubing. Plastic caps are required after sampling. NOTE: If pressure drop is excessive or pump fails, use a larger diameter sorbent tube with XAD-2 resin (ORBO 42 Large, Supelco, Cat. No. 2-0264U; or equivalent). 2. Personal sampling pump capable of operating for 8 h at 2 L/min, with flexible connecting tubing. 3. Aluminum foil. 4. Refrigerant, bagged. 5. Culture tubes, PTFE-lined screw cap, 13-mm x 100-mm. 6. Forceps. 7. Syringe filters, 0.45-µm, 25-mm, PTFE (Acrodisc-CR, Pall Gelman Sciences, Cat. No. 4219; or equivalent). 8. Pipet, 5-mL. 9. Syringe or micropipets, 1- to 100-µL. 10. Ultrasonic bath. 11. HPLC, with gradient capability, fluorescence (excitation @ 340 nm, emission @ 425 nm) and UV (254 nm) detectors in series, electronic integrator, and a 250 x 4.6-mm C 18 column (Vydac 201TP, The Separations Group, Hesperia, CA, Cat. No. 201TP54; or equivalent). 12. Volumetric flasks, 10- and 100-mL. 13. Recommendation: lighting in laboratory should be incandescent or UV-shielded fluorescent.
SPECIAL PRECAUTIONS: Treat all polynuclear aromatic hydrocarbons as carcinogens. Samples and unused standards are considered toxic waste. Dispose of in an appropriate manner. Counter tops and equipment should be checked regularly with a "black light" for fluorescence as an indicator of contamination by PAHs.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC: METHOD 5506, Issue 3, dated 15 January 1998 - Page 3 of 9
SAMPLING: 1. Calibrate each personal sampling pump with a representative sampler in line. 2. Take personal samples at 2 L/min for a total sample size of 200 to 1000 L. 3. Immediately after sampling, transfer the filter carefully with forceps to a culture tube. Hold filter at edge to avoid disturbing the collected sample. Cap the tube and wrap in aluminum foil. NOTE: This step is necessary to avoid loss of analytes by sublimation. 4. Cap the sorbent tube and wrap in aluminum foil. 5. Ship to laboratory in insulated container with bagged refrigerant.
SAMPLE PREPARATION:
6. 7.
8.
9.
NOTE: UV light may degrade PAHs; therefore, recommend using yellow, UV-absorbing shields for fluorescent lights or use incandescent lighting. Refrigerate samples upon receipt at laboratory. Extract PAH from filters. a. Add 5.0 mL of acetonitrile to each culture tube containing a filter. Similarly, add 5.0 mL of acetonitrile to each culture tube containing the media and reagent blanks. Cap the tubes. b. Place capped tubes in an ultrasonic bath for 30 to 60 min. Desorb PAH from sorbent. a. Score each sorbent tube with a file in front of the front (larger) sorbent section. Break tube at score line. b. Transfer front glass wool plug and front sorbent section to a culture tube. Transfer back sorbent section, and the middle glass wool plug to a second culture tube. c. Add 5.0 mL acetonitrile to each culture tube. Cap the tubes. d. Place capped tubes in an ultrasonic bath for 30 to 60 min. Filter all sample extracts through an 0.45-µm syringe filter.
CALIBRATION AND QUALITY CONTROL: 10. Calibrate daily with at least six working standards. NOTE: If a benzo[e]pyrene standard is needed, weigh desired amount and add to a known volume of the PAH test mixture. a. Dilute aliquots of thePAH test mixture (containing benzo[e]pyrene if needed) with acetonitrile in 10-mL volumetric flasks. The concentration range should cover most of the PAH concentrations in the samples. b. During analysis, intersperse working standards with samples and blanks. c. Prepare calibration graphs (peak area vs. µg of each PAH per sample). 11. Recovery and desorption efficiency. a. Determine recovery (R) from filters and desorption efficiency (DE) from sorbent tubes at least once for each lot of filters and sorbent tubes used in the range of interest. (1) Filters. Using a microliter syringe or a micropipette, spike four filters at each of five concentration levels with a mixture of the analytes. Allow the filters to dry in the dark overnight. Analyze the filters (steps 7, 9, and 13 through 15). Prepare graphs of R vs. amounts found. (2) Sorbent tubes. Transfer an unused front sorbent section to a culture tube. Prepare a total of 24 culture tubes in order to measure DE at five concentration levels plus blank in quadruplicate. Using a microliter syringe or micropipette, add calibration stock solution directly to sorbent. Cap culture tubes and allow to stand overnight. Desorb and analyze (steps 8, 9, and 13 through 15). Prepare graphs of DE vs. amounts found. b. Check R and DE at two levels for each sample set, in duplicate. Repeat determination of R or DE graphs if checks do not agree to within ±5% of R or DE graph. 12. Analyze at least three field blanks for each sample medium.
MEASUREMENT: NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC: METHOD 5506, Issue 3, dated 15 January 1998 - Page 4 of 9
13. Set HPLC according to manufacturer’s instructions, conditions on page 5506-1, and steps 14 and 15. 14. Inject sample aliquot (10 to 50 µL). Start mobile phase gradient: a. Linear gradient from 60% acetonitrile/40% deionized water to 100% acetonitrile at 1 mL/min over 20 min. b. Hold at 100% acetonitrile for 20 min. c. Linear gradient to initial condition, 5 min. 15. Measure peak areas for each analyte using the appropriate detector as specified in Table 1. NOTE 1: The order of elution for the PAHs appears in Table 4. NOTE 2: If peak area is above the calibration range, dilute with acetonitrile, reanalyze, and apply dilution factor in calculations. NOTE 3: If sample has many interferences, additional sample cleanup may be necessary.
CALCULATIONS: 16. Read the mass, µg (corrected for R or DE) of each analyte found on the filter (W) and front sorbent (Wf) and back sorbent (Wb) sections, and on the average media blank filter (B) and front sorbent (B f) and back sorbent (Bb) sections from the calibration graphs. 17. Calculate concentration, C (mg/m3), as the sum of the particulate concentration and the vapor concentration in the actual air volume sampled, V (L).
W
C
Wf
Wb
B V
Bf
Bb
, mg/m 3
mg/m3
NOTE 1:
µg/mL
NOTE 2:
Wf and Wb include analyte originally collected on the filter as particulate, then volatilized during sampling. This can be a significant fraction for many PAHs (e.g., anthracene, fluoranthene, fluorene, naphthalene, phenanthrene).
EVALUATION OF METHOD: The UV detector is used to analyze for some PAHs (see Table 1), and the remaining PAHs are analyzed by a fluorescent detector, which gave better sensitivity for some PAHs. The ranges of the limit of detection (LOD) and the limit of quantitation (LOQ) values for the 17 PAHs are reported in Table 4 [4]. The LOD and LOQ values varied because of differences in the detectors used and the concentrations of the standards. Therefore, it is important that the LOD and LOQ values be determined for each set of samples. The LOQs are the lower end of the analytical ranges. The upper end of the analytical ranges were not determined. This method was evaluated by means of a user check [5]. An independent laboratory prepared spiked filters and sorbent tubes for a recovery and desorption efficiency study (see Table 4). For the filters, except naphthalene, the recovery results were greater than or equal to 75%. Since naphthalene is fairly volatile under ambient conditions, this may account for the poor recovery results. For the sorbent tubes, only four of the 17 analytes had desorption efficiencies that were greater than or equal to 75%. During the user check, the sorbent tubes were extracted by adding 5 mL acetonitrile and were allowed to stand for 30 minutes with occasional swirling. In more recent quality control experiments, the desorption efficiencies were often better for some analytes (see Table 4) [4]. These results were achieved using an ultrasonic bath for 30 to 60 minutes. The results indicated the importance of preparing media spikes for recovery and desorption efficiency studies for each set of samples; moreover, the results reenforce this need when using new lots of media.
REFERENCES:
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC: METHOD 5506, Issue 3, dated 15 January 1998 - Page 5 of 9
[1]
NIOSH [1977]. Total particulate aromatic hydrocarbons: Method P&CAM 206. NIOSH Manual of Analytical Methods, 2nd ed., Vol. 1. Cincinnati, OH: National Institute for Occupational Safety and Health, DHEW (NIOSH) Publication No. 77-157-A. [2] NIOSH [1994]. Polynuclear aromatic hydrocarbons by GC: Method 5515. In: Eller PM, Cassinelli ME, ® eds. NIOSH Manual of Analytical Methods (NMAM ), 4th ed. Cincinnati, OH: National Institute for Occupational Safety and Health, DHHS (NIOSH) Publication No. 94-113. [3] NIOSH [1998]. Polycyclic aromatic compounds: Method 5800. In: Eller PM, Cassinelli ME, eds. NIOSH Manual of Analytical Methods (NMAM®), 4th ed., 2nd Supplement. Cincinnati, OH: National Institute for Occupational Safety and Health, DHHS (NIOSH) Publication No. 98-119. [4] NIOSH unpublished data. [5] User Check Report for Method 5506, Analytical Report for NIOSH Sequence 4170 (NIOSH, DPSE, MRSB, unpublished, March 16, 1984). [6] Handbook of Chemistry and Physics, 78th ed. [1997], Boca Raton, FL: CRC Press. [7] IARC [1983]. IARC monographs onthe evaluation of the carcinogenic risk of chemicals to humans: polynuclear aromatic compounds, Part1, chemical, environmental and experimental data. Vol. 32. Lyon, France: World Health Organization, International Agency for Research on Cancer. [8] Lenga RE, Votoupal KL, eds. [1993]. The Sigma-Aldrich library of regulatory and safety data, Volume 1. Sigma-Aldrich Company. [9] NIOSH [1998]. Registry of toxic effects of chemical substances data base. Cincinnati, OH: National Institute for Occupational Safety and Health. Unpublished data base. [10] NIOSH [1997]. NIOSH pocket guide to chemical hazards. Cincinnati, OH: National Institute for Occupational Safety and Health, DHHS (NIOSH) Publication No. 97-140. [11] ACGIH [1997]. 1997 TLVs® and BEIs®, Threshold limit values for chemical substances and physical agents, Biological exposure Indices. Cincinnati, OH: American Conference of Governmental Industrial Hygienists (ACGIH). [12] NIOSH [1992]. NIOSH recommendations foroccupational safety and health, compendium of policy documents and statements. Cincinnati, OH: National Institute for Occupational Safety and Health, DHHS (NIOSH) Publication No. 92-100.
METHOD REVISED BY: L. D. Olsen, B. R. Belinky, C. E. Neumeister, L. B. Jaycox, and D. D. Dollberg, NIOSH/DPSE.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC: METHOD 5506, Issue 3, dated 15 January 1998 - Page 6 of 9 TABLE 1. FORMULAS AND PHYSICAL PROPERTIES.
FORMULA
WEIGHT
DETECTOR
MELTING POINT ( C)
1. NAPHTHALENE
C10H8
128.17
UV
80.2
218
[6]
2. ACENAPHTHYLENE
C12H8
152.20
UV
92.5
280
[6]
3. ACENAPHTHENE
C12H10
154.21
UV
93.4
279
[6]
4. FLUORENE
C13H10
166.22
UV
115
295
[6]
5. ANTHRACENE
C14H10
178.23
UV
215
340
[6]
6. PHENANTHRENE
C14H10
178.23
UV
99.2
340
[6
7. FLUORANTHENE
C16H10
202.26
FL
108
384
[6]
8. PYRENE
C16H10
202.26
FL
151
404
[6]
9. BENZ[a]ANTHRACENE
C18H12
228.29
FL
167
435
[7]
10. CHRYSENE
C18H12
228.29
UV
258
448
[6]
11. BENZO[b]FLUORANTHENE
C20H12
252.32
FL
168
[7]
12. BENZO[k]FLUORANTHENE
C20H12
252.32
FL
217
480
[6]
13. BENZO[a]PYRENE
C20H12
252.32
FL
177
495
[6, 8]
14. BENZO[e]PYRENE
C20H12
252.32
FL
178
311
[6]
15. BENZO[ghi]PERYLENE
C22H12
276.34
FL
278
[7]
16. INDENO[1,2,3-cd]PYRENE
C22H12
276.34
FL
164
[7]
17. DIBENZ[a,h]ANTHRACENE
C22H14
278.35
FL
270
524
[7, 8]
COMPOUND (by M.W.)
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
BOILING POINT ( C)
REFERENCE ​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC: METHOD 5506, Issue 3, dated 15 January 1998 - Page 7 of 9 TABLE 2. SYNONYMS, CAS AND RTECS NUMBERS. COMPOUND (alphabetically)
SYNONYMS, CAS and RTECS Numbers*
1. ACENAPHTHENE
CAS # 83-32-9; RTECS # AB1000000
2. ACENAPHTHYLENE
acenaphthalene; CAS # 208-96-8; RTECS # AB1254000
3. ANTHRACENE
CAS # 120-12-7; RTECS # CA9350000
4. BENZ[a]ANTHRACENE
1,2-benzanthracene; benzo[b]phenanthrene; 2,3-benzophenanthrene; tetraphene; CAS # 56-55-3; RTECS # CV9275000
5. BENZO[b]FLUORANTHENE
3,4-benzofluoranthene; 2,3-benzofluoranthene; benz[e]acephenanthrylene; B(b)F; CAS # 205-99-2; RTECS # CU1400000
6. BENZO[k]FLUORANTHENE
11,12-benzofluoranthene; CAS # 207-08-9; RTECS # DF6350000
7. BENZO[ghi]PERYLENE
1,12-benzoperylene; CAS # 191-24-2; RTECS # DI6200500
8. BENZO[a]PYRENE
3,4-benzopyrene; 6,7-benzopyrene; B(a)P; BP; CAS # 50-32-8; RTECS # DJ3675000
9. BENZO[e]PYRENE
1,2-benzopyrene; 4,5-benzopyrene; B(e)P; CAS # 192-97-2; RTECS # DJ4200000
10. CHRYSENE
1,2-benzophenanthrene; benzo[a]phenanthrene; CAS # 218-01-9; RTECS # GC0700000
11. DIBENZ[a,h]ANTHRACENE
1,2,5,6-dibenzanthracene; CAS # 53-70-3; RTECS # HN2625000
12. FLUORANTHENE
benzo[jk]fluorene; CAS # 206-44-0; RTECS # LL4025000
13. FLUORENE
CAS # 86-73-7; RTECS # LL5670000
14. INDENO[1,2,3-cd]PYRENE
2,3-phenylenepyrene; CAS # 193-39-5; RTECS # NK9300000
15. NAPHTHALENE
naphthene; CAS # 91-20-3; RTECS # QJ0525000
16. PHENANTHRENE
CAS # 85-01-8; RTECS # SF7175000
17. PYRENE
benzo[def]phenanthrene; CAS # 129-00-0; RTECS # UR2450000
Data from [6, 8, and 9].
TABLE 3. EXPOSURE LIMITS.* COMPOUND
OSHA†
NIOSH†
ACGIH‡
0.2 mg/m3
2. BENZ[a]ANTHRACENE
suspect human carcinogen
3. BENZO[b]FLUORANTHENE
suspect human carcinogen
1. ANTHRACENE
4. BENZO[a]PYRENE 5. CHRYSENE
3
0.2 mg/m
3
suspect human carcinogen §
0.2 mg/m
potential occupational carcinogen
animal carcinogen
10 ppm; STEL 15 ppm
10 ppm; STEL 15 ppm
10 ppm; STEL 15 ppm
7. PHENANTHRENE
0.2 mg/m3
8. PYRENE
0.2 mg/m3
6. NAPHTHALENE
† Information from [10]. ‡ Information from [11]. § Information from [12].
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC: METHOD 5506, Issue 3, dated 15 January 1998 - Page 8 of 9 TABLE 4. LOD AND LOQ VALUES, AND RECOVERY DATA. Recoveries (%)†
Range of values* COMPOUND (by elution order)
LOD (µg per sample)
LOQ (µg per sample)
Filters
Sorbent tubes
1. NAPHTHALENE
0.20 - 0.80
0.39 - 2.6
49.6
68.5
2. ACENAPHTHYLENE
0.090 - 2.0
0.28 - 6.6
98.2
98.2
3. ACENAPHTHENE
0.20 - 5.0
0.58 - 16.
0.030 - 0.30
0.099 - 0.26
95.0
95.0
5. PHENANTHRENE
0.0070 - 0.060
0.023 - 0.19
99.0, 90.4*
84.0, 92.5*, 82.6*
6. ANTHRACENE
0.0010 - 0.090
0.023 - 0.30
81.8, 94.4*
72.8, 96.2*, 72.9*
7. FLUORANTHENE
0.0020 - 0.090
0.0066 - 0.30
94.9, 90.4*
73.0, 93.5*, 81.7*
8. PYRENE
0.0010 - 0.30
0.0036 - 0.99
94.4, 76.1*
84.9, 77.0*, 75.9*
9. BENZ[a]ANTHRACENE
0.0010 - 0.090
0.0042 - 0.30
86.6, 92.7*
62.4, 95.0*, 72.3*
10. CHRYSENE
0.0070 - 0.20
0.023 - 0.37
94.6, 89.9*
62.7, 89.8*, 74.0*
11. BENZO[e]PYRENE
0.0060 - 0.80
0.020 - 2.6
110
48.3
12. BENZO[b]FLUORANTHENE
0.0030 - 0.20
0.011 - 0.66
94.8
64.2
13. BENZO[k]FLUORANTHENE
0.0020 - 0.040
0.0054 - 0.13
103
53.2
14. BENZO[a]PYRENE
0.0020 - 0.10
0.0051 - 0.33
101, 88.1*
50.4, 91.6*, 68.4*
15. DIBENZ[a,h]ANTHRACENE
0.0040 - 0.60
0.014 - 2.0
76.5
61.0
16. BENZO[ghi]PERYLENE
0.0030 - 0.50
0.011 - 1.7
76.5
61.0
17. INDENO[1,2,3-cd]PYRENE
0.0090 - 0.20
0.027 - 0.66
91.6
36.5
4. FLUORENE
† Data from [5]
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition ​POLYNUCLEAR AROMATIC HYDROCARBONS by HPLC: METHOD 5506, Issue 3, dated 15 January 1998 - Page 9 of 9
ACENAPHTHENE
BENZ[a]ANTHRACENE
ACENAPHTHYLENE
BENZO[b]FLUORANTHENE
BENZO[g h i]PERYLENE
BENZO[a]PYRENE
CHRYSENE
DIBENZ[a,h]ANTHRACENE
FLUORENE
PHENANTHRENE
INDENO[1,2,3-c d]PYRENE
ANTHRACENE
BENZO[k]FLUORANTHENE
BENZO[e]PYRENE
FLUORANTHENE
NAPHTHALENE
PYRENE
1. Structure of the PAHs.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition
Figure