Sampling is performed using a high volume sampler. Particle bound polycyclic aromatic hydrocarbons (PAHs) are collected on a glass fibre filter, and more volatile PAHs are adsorbed to plugs of polyurethane foam (PUF) placed behind the filter. The filter and the plugs are soxhlet extracted with cyclohexane after sampling. The pre-concentrated extracts are cleaned using liquid/liquid extraction and HPLC before analysis using high resolution gas chromatography combined with mass spectrometry (GC/MS).
Autosampler | : | Hewlett-Packard 7673 or similar |
Detector | : | Hewlett-Packard 5970 Mass Selective Detector (MSD), 5973 MSD or similar |
Gas chromatography | : | Hewlett-Packard 5890 or similar with splitless injector |
GC-column | : | Capillary column, 25-30 m x 0.25 cm, CP-Sil 8CB, SE 52 or similar, 0.1 µm film thickness |
Integrator system | : | Hewlett-Packard Pascal 3.2 ChemStation or similar |
Injector | : | Rheodyne 7125 with 2 ml sample loop |
Column | : | Lichrosorb Si-60-5, 5 mm, 4.6 mm x 25 cm |
Pump | : | LDC Constametric model III |
Syringe | : | 1000 ml, Hamilton no. 1001 |
UV-detector | : | LDC UVIII monitor model 1203 |
Valve for switching solvent | : | Hamilton no. 86414 |
Extractors | : | 60 ml, male glass joint 24/29 and female
glass joint 34/35 500 ml, male glass joint 24/29 and female glass joint 60/48 2000 ml, male glass joint 34/35 with flat lid, size 34/35 |
Extraction thimbles | : | 28 x 80 mm, cellulose, Schleicher &
Schuell 53 x 145 mm, cellulose, Schleicher & Schuell 60 x 180 mm, cellulose, Schleicher & Schuell |
Coolers | : | double surface, 345 mm long with male glass
joint 34/35 cooler, 260 mm long with male glass joint 34/35 cooler, 330 mm long with male glass joint 24/29 |
Connector | : | female glass joint 34/35 to male glass joint 60/48 |
Desiccator | : | internal diam. 30 cm, lid with vacuum connection |
Micro pipettes | : | 10, 20, 25, 50, 100 ml, < +0.25%, Brand or similar |
Graduated cylinders | : | 100, 200 and 500 ml |
Pasteur pipettes | : | 150 and 230 mm long |
Sample vials | : | 1.5 ml (Brown cat.no. 150900) with screw cap, Teflon lined (Brown cat.no. 150930) |
Round bottomed flasks | : | 250, 500 and
1000 ml, ground glass joint 24/29 3000 ml, ground glass joint 34/35 |
Centrifuge tube | : | 15 ml, conical with ground glass plug 14/15, graded to 10 ml |
Analytical
balance, 0-160 g, precision ± 0.02 mg
Gloves, thin polyethylene, KEBO
Gloves, solvent resistant of PE/EVOH/PE
Membrane vacuum pumps with Teflon membrane
(solvent resistant), 2.4 m3/h
Metal cartridges (metal cylinders), for active
carbon/ molecular sieve filter
Micro balance, capacity 3000 mg, precision + 1 mg
Millipore, MilliQ plus, water purifier
Pressure valve GA 2 (L'Air Liquide) with needle
valve ALG 2B (L'Air Liquide)
Porcelain dish
TurboVap 500 pre-concentrator, Zymark
Tissue paper Kimwipes, Kimberly-Clark
Ultrasonic bath
Heater mantles for round bottomed flasks for
500, 1000 and 3000 ml flasks
Oven, 50500°C
Helium,
4.6 99.996%, Hydro
Nitrogen Hydro Ultra, 5.0 99.999%, Hydro
Active
carbon, 1.5 mm diam., Merck no. 2514
Chrompack
Gas Clean moisture filter, no. 7971
Chrompack
Gas Clean oxygen/charcoal filter, no. 7972
Molecular
sieve, 0.5-2 mm diam., Merck no. 5707
Acetone,
Merck no. 12
Acetonitrile,
Ratburn no. RH1016
Diethylether,
Ratburn no. RG2013
2,2-Dimethoxypropane,
Merck no. 802936
Dimethylformamide,
Rathburn no. RG2014
Acetic
acid, Merck no. 62
Hexane,
Merck no. 4371
Chloroform,
Merck no. 2445
Methanol,
Merck no. 6011
Sodium
sulphate, Merck no. 6649
PAH-standards,
see 6.1
RBS
25, lab detergent
Cyclohexane,
Merck no. 2817
Toluene,
Merck no. 8389
All glass equipment must be decontaminated before use. Leave the equipment in 2.5% RBS in water for 16 hours. Flush well with hot tap water followed by MilliQ water. Leave to dry on a clean surface.
Put ca 50 filters (Gelman-Type A/E, 142 mm) on an Al-foil and heat to 450°C for 8 hours. After cooling to room temperature wrap each filter in Al-foil.
Extract thimbles for 8 hours ("1 day") with cyclohexane in a 600 ml soxhlet extractor. Dry in a desiccator connected to a vacuum pump (capacity 2.4 m3/h, and 80 kPa (0.8 bar) at 100°C. Connect pump outlet to a cooler to condense solvent. Wrap dry thimbles in Al-foil.
Dismantle the sampler. Wash glass, metal parts and gaskets with warm detergent (2.5% RBS 25 in water). Flush all parts except the gaskets, with warm water, distilled water and acetone. Flush the gaskets with warm water, distilled water, not acetone.
Heat ca. 100 g sodium sulphate in a porcelain dish at 600°C for ca. 20 hours. Store in a 250 ml Pyrex bottle with tight screw cap. Label the bottle with date for cleaning. Max. storage time is 1 month.
Toluene
Clean
new PUF-plugs with toluene (Merck no. 8389) in a 2000 ml soxhlet extractor. The
extractor can take up to 8 plugs simultaneously. Use a 3000 ml round bottomed
flask and fill toluene into the extractor until it empties the content into the
round-bottomed flask. Add ca. 500 ml toluene and mount the lid and cooler. Turn
on the heater and the cooling water. Extract the plugs for
24 hours.
Acetone, cyclohexane
Squeeze
toluene out of the plugs (solvent resistant gloves!) and transfer the plugs to
another 2000 ml soxhlet extractor. Acetone is added as prescribed for toluene
and the plugs are extracted for 8 hours.
Finally, extract with cyclohexane (new extractor) for 8 hours.
Observe! Used plugs (which previously have passed trough the whole cleaning procedure, toluene included) can be cleaned as follows:
1) Soxhlet extraction with
acetone for 8 h
2) Soxhlet extraction with
cyclohexane for 8 h
After final extraction squeeze the cyclohexane out of the plugs. Place the plugs in a desiccator. Put the desiccator in an oven at 60°C, and connect desiccator to a vacuum pump. Dry for 16 hrs and wrap the plugs in Al-foil individually. Store pairs of plugs and a filter in zip-shut plastic bags.
Fill XAD-2 in a thimble and put it in a soxhlet extractor. Extract for 8 h with each of the following solvents: Methanol, acetonitrile and diethyl ether. Leave the wet adsorbent on an Al-foil in a fume hood until it appears dry. Dry in an oven at 35°C overnight.
Helium GC carrier gas cleaning:
Nitrogen used for final sample blow-down/pre-concentration is cleaned using a metal cartridge filled with active carbon. Exchange adsorbent when exchanging the gas bottle.
Chrompack filters are discarded after use.
Re-activation of molecular sieves: Fill molecular sieves in a metal cartridge and activate at 300°C (3 h) in an oven, flushing the cartridge with 20 ml/min. pre-filtered helium.
Active carbon is discarded after use.
Sampling, extraction, pre-concentration and weighing is described in chapters 3.14.4 - 3.14.7.
Depending of sample type the internal standard is added in an amount similar to the expected concentration level of the sample.
The extract is cleaned using liquid/liquid distribution between cyclohexane and dimethylformamide (DMF). Mix DMF and water in the ratio 9:1 (DMF:water), e.g. 180 ml DMF and 20 ml de-ionised water (MilliQ-plus).
Often samples may be ready for GC/MS at this stage. Dirty samples may require an additional HPLC-clean-up step.
Cleaned cyclohexane extracts, except low level samples from background areas, are cleaned using a silica-column (Lichrosorb SI-60-7, 250 x 4.6 mm). More polar compounds than unsubstituted PAHs will adsorb to the column, whereas the PAH-fraction elutes rapidly to be collected. The UV-detector is operated at 254 mm. The column is flushed with chloroform after each sample.
Between the pump and the eluent reservoirs a solvent switch makes it easy to change from one eluent to another. Cyclohexane, which is saturated with water, and chloroform are used as eluents. Water saturated cyclohexane is made by adding a few millilitres of water (MilliQ plus) to a bottle of cyclohexane. Sonicate the bottle for 30 min. Leave to separate overnight and decant the cyclohexane phase into another bottle the next day.
If there is, disconnect column, vent the air from the system and connect the column again.
With time the columns separating performance will decrease and a cleaning is necessary. Make mixes of methanol/water 1:1 (50% vol. water) and methanol/ water 19:1 (5% water) and hexane/acetic acid/dimethoxypropane 44:5:1 (88% vol. hexane, 10% vol. acetic acid, 2% vol. dimethoxypropane). Use the following solvents at a flow of 2 ml/min.:
The standards should have the highest possible purity and, if available, be certified.
Certified standards from Community Bureau of Reference (BCR): | Purity (%) |
Fluoranthene Pyrene Benzo[ghi]fluoranthene Benz[a]anthracene Chrysene Triphenylene Benzo[a]fluoranthene Benzo[b]fluoranthene Benzo[j]fluoranthene Benzo[e]pyrene Benzo[a]pyrene Indeno[1,2,3-cd]pyrene Dibenz[a,c]anthracene Dibenz[a,h]anthracene Benzo[ghi]perylene Coronene |
99.49 99.75 99.4 99.78 99.20 99.77 99.5 99.5 99.5 99.0 99.3 99.8 99.5 99.8 99.0 99.83 |
Standards from Tokyo Kasei Kogyo, Ltd., Japan: | Purity (%) |
Biphenyl Acenaphthene Phenanthrene Anthracene Fluorene |
>99 >99 Zone Refined, 30 passes Zone Refined, 70 passes Zone Refined, 70 passes |
Standard from Dr. Ehrensdorfer GmbH, Germany: | Purity (%) |
Dibenzothiophene | 99.7 |
Standards from Promochem GmbH, Wesel, Germany: | Purity (%) |
Naphtalene 1-Methylnaphtalene 2-Methylnaphtalene Acenaphtylene 3-Methylphenanthrene 2-Metyhlphenanthrene 2-Metyhlanthracene 9-Methylphenanthrene Benzo[b]fluorene Cyclopenta[cd]pyrene Anthanthrene Perylene Dibenzo[a,e]pyrene Dibenzo[a,i]pyrene Dibenzo[a,h]pyrene |
99.8 97 98 99.8 99.8 99 >99 99.9 99.5 99 >99 99.6 99.8 99.9 99.8 |
Certified standards from Chem Service, Inc., West Chester, USA: | Purity (%) |
1-Methylphenanthrene Dibenzofuran Benzo[a]fluorene Retene Benzo[k]fluoranthene |
99.5 98 99 85 99.0 |
Labelled standards from C/D/N Isotopes Inc., Canada: | Purity (%) |
2-Methylnaphtalene-D10 Acenaphthene-D10 Anthracene-D10 Fluoranthene-D10 Pyrene-D10 Benz[a]anthracene-D10 Benzo[e]pyrene-D12 Benzo[ghi]perylene-D12 |
99.3 99.7 99.3 98.8 99.9 99.1 99.6 99.1 |
The following compounds may be included in the main standard:
Naphtalene 2-Methylnaphtalene 1-Methylnaphtalene Biphenyl Acenaphthylene Acenaphthene Dibenzofuran Fluorene Dibenzothiophene Phenanthrene Anthracene 3-Methylphenanthrene 2-Methylphenanthrene 2-Methylanthracene 9-Methylphenanthrene 1-Methylphenanthrene Fluoranthene Pyrene Benzo[a]fluoren Retene Benzo[b]fluoren |
Benzo[ghi]fluoranthene Syklopenta[cd]pyrene Benz[a]anthracene Chrysene Triphenylene Benzo[b]fluoranthene Benzo[j]fluoranthene Benzo[k]fluoranthene Benzo[a]fluoranthene Benzo[e]pyrene Benzo[a]pyrene Perylene Indeno[1,2,3-cd]pyrene Dibenz[a,c]anthracene Dibenz[a,h]anthracene Benzo[ghi]perylene Anthranthene Coronene Dibenzo[ae]pyrene Dibenzo[ai]pyrene Dibenzo[ah]pyrene |
Perform the weighing using the micro balance. Use gloves and a dust mask. Spatulas and other equipment should be rinsed with toluene before use. When all compounds are weighed, transfer them to a 25 ml volumetric flask using a Pasteur pipette and toluene. Fill the flask to the mark and sonicate until all PAH is dissolved. Compute the exact concentration for each compound (ng/ml). The individual concentration should be in the range 15±10 ng/ml. This corresponds to 300±100 mg of each single compound. Transfer mix to well labelled vial with Teflon lined screw cap. Weigh the vial and store it in a freezer.
The internal standard includes the following compounds: 2-methylnaphtalene-D10 (ISTD I), acenaphthene-D10 (ISTD II), anthracene-D10 (ISTD III), pyrene-D10 (ISTD IV), benz[a]anthracene-D12 (ISTD V), benzo[e]pyrene-D12 (ISTD VI), benzo[ghi]perylene-D112 (ISTD VII).
Ca. 1 mg of ISTD I, II, III, IV and ca 0.5 mg of ISTD V, VI, VII are weighed as described under 6.1.1 and transferred to a 25 ml volumetric flask filled to the mark with cyclohexane. The concentration range is ca 20-40 ng/µl. Transfer mix to a well-labelled vial with Teflon lined screw cap. Weigh the vial and store it in a freezer.
Transfer ca. 1 ml to a sample vial. Label the vial and use as working standard. Weighing is not necessary. Store in a refrigerator.
As recovery standard fluoranthene-D10 may be used. Weigh in ca 1 mg, transfer to a 25 ml volumetric flask and fill to the mark with cyclohexane as described in 6.1.1. Transfer mix to well labelled vial with Teflon lined screw cap. Weigh the vial and store it in a freezer.
Transfer ca 1 ml to a sample vial. Mark the vial and use as working standard. Weighing is not necessary. Store in a refrigerator.
Remove the flasks with main standard, internal standard and recovery standard from the freezer and leave to melt at room temperature. Use aluminium foil to protect the flasks against sunlight. Remove the foil and sonicate flasks for 5 minutes. Check that all PAH is dissolved. If crystals are visible, repeat sonication. Check the flasks for weight loss and compensate an eventual loss with solvent. 2 ml main standard is transferred to a 50 ml volumetric flask using a 2 ml pipette. To the same flask further 1 ml internal standard and 2 ml recovery standard are transferred. Fill the flask to the mark with cyclohexane. Transfer mix a well labelled vial with Teflon lined screw cap. Weigh the vial and store it in a freezer.
Transfer ca. 1 ml to a sample vial. Label the vial and use as working standard. Weighing is not necessary. Store in a refrigerator.
This standard is used to check the GC/MS quantification performance (chapter 6.2.4). The standard may contain the following compounds:
Biphenyl
Phenanthrene
Fluoranthene
Benzo[a]anthracene
Benzo[e]pyrene
Benzo[ghi]perylene
First a mother standard is made as described under 6.1.1. 300±100 mg of each compound is weighed and dissolved in cyclohexane in a 10 ml volumetric flask. Label the flask, weigh and store in a freezer.
Working solution is made as described under 6.1.2. 1 ml. The mother standard is diluted to 50 ml with cyclohexane in a 50 ml volumetric flask. Store the flask in a freezer.
Transfer ca. 1 ml to a 2 ml sample vial. Label the vial and use as working standard. Weighing is not necessary. Store in a refrigerator.
This standard is used to check retention times before HPLC clean-up.
Ca. 5 mg naphthalene and ca. 10 mg coronene is dissolved in 100 ml cyclohexane. The concentrations are ca. 50 mg naphthalene and ca. 100 mg coronene pr. ml. Store in a refrigerator.
The cleaned sample extracts are analysed using gas chromatography combined with mass spectrometry (GC/MS). The compounds are identified according to their retention time and molecular weight and quantified using internal standards.
GC conditions: | ||
- Column | : | CP-Sil 8CB, 25 m x 0.25 mm x 0.12 µm film thickness or similar. |
- Injector temperature | : | 300°C |
- Temperature program | : | 50-100°C with 20°C/min., 100-300°C with 10°C/min., 300°C for 5-10 min. |
- Carrier, helium | : | 85 kPa |
- Split gasflow | : | 40 +10 ml/min. |
- Sample volume | : | 1 ml splitless, (Autosampler or hot-needle injection) |
Autosampler conditions: | ||
- Solvent A | : | toluene |
- Solvent B | : | cyclohexane |
- Sample wash | : | 0 |
- Sample pumps | : | 6 |
- Sample volume | : | 1 µl |
- Solvent A, washes | : | 6 |
- Solvent B, washes | : | 6 |
Solvent A and B for syringe cleaning must be exchanged each day.
Electron impact ionisation (EI) is used. Inject the quantification standard before each sample series and after each 10 samples.
MS conditions for EI (MSD 5970 and MSD 5973): | ||
- GC/MS-interface | : | 290°C |
- Electron energy | : | 70 eV |
- Calibration compound | : | Perfluorotributylamine (PFTBA) |
Automatic tuning (Autotune), or
manual optimisation of mass scale and transmission of the mass filter
(quadrupole) with PFTBA using mass fragments m/z 69.0, 219.0 and 502.0. Mass
resolution, signal width at half height: 0.55±0.03. Calibration of mass
scale at ±0.05
amu.
The following SIM-program may be used for quantification:
SIM-function |
Compound group |
Mass |
1
|
Naphtalene |
128.1 |
2-Methylnaphtalene |
142.1 |
|
1-Methylnaphtalene |
142.1 |
|
d10 z-Methylnaphtalene |
152.1 |
|
Biphenyl |
154.1 |
|
2
|
Acenaphthylene |
152.1 |
Acenaphthene |
154.1 |
|
d10 Acenaphthene |
164.1 |
|
Dibenzofuran |
168.1 |
|
Fluorene |
166.1 |
|
3
|
Dibenzothiophene |
184.1 |
Phenanthrene |
178.1 |
|
Anthracene |
178.1 |
|
d10 Anthracene |
188.1 |
|
3-Metylphenanthrene |
192.1 |
|
2-Methylphenanthrene |
192.1 |
|
2-Methylanthracene |
192.1 |
|
9-Methylphenanthrene |
192.1 |
|
1-Methylphenanthrene |
192.1 |
|
4
|
Fluoranthene |
202.1 |
d10 Fluoranthene |
212.1 |
|
Pyrene |
202.1 |
|
d10Pyrene |
212.1 |
|
Benzo[a]fluorene |
216.1 |
|
Retene |
234.1 |
|
Benzo[b]fluorene |
216.1 |
|
5
|
Benzo[ghi]fluoranthene |
226.1 |
Cyclopenta[cd]pyrene |
226.1 |
|
Benz[a]anthracene |
228.1 |
|
d12 Benz[a]anthracene |
240.1 |
|
Chrysene/triphenylene |
228.1 |
|
6
|
Benzo[b/j/k]fluoranthenes |
252.1 |
Benzo[a]fluoranthene |
252.1 |
|
Benzo[e]pyrene |
252.1 |
|
d10 Benzo[e]pyrene |
264.1 |
|
Benzo[a]pyrene |
252.1 |
|
Perylene |
252.1 |
|
7
|
Inden[1,2,3-cd]pyrene |
276.1 |
Dibenz[a,c/a,h]anthracenes |
278.1 |
|
Benzo[ghi]perylene |
276.1 |
|
d10 Benzo[ghi]perylene |
288.1 |
|
Anthanthrene |
276.1 |
|
Coronene |
300.1 |
|
Dibenzo[a,e]pyrene |
302.1 |
|
Dibenzo[a,i]pyrene |
302.1 |
|
Dibenzo[a,h]pyrene |
302.1 |
If compounds occur at concentrations which saturate the detector these compounds may be quantified using the signal from the 13C-isotope in the compound, detected at a mass one amu higher than the number indicated in the table.
1. Relative response factors, RRFi, are computed for the single compounds relative to the internal standard (ISTD) after analysing the quantification standard with known concentrations.
RRFi : Relative
response factor of compound i
Amt.ISTD : Amount
of internal standard injected
Amt.i : Amount
of compound i injected
Areai : Peak area of
compound i
AreaISTD : Peak area of internal standard
2. Quantification of samples is based on relative response factor, added amount internal standard and the peak area of each compound i.
Amt.i :
Amount
of compound i in the sample
Amt.ISTD :
Amount
of internal standard added
Areai :
Peak area of
compound i
RRFi :
Relative
response factor for compound i
AreaISTD :
Peak
area of internal standard
Check the chromatogram for eventual interference and correct retention times before quantification.
3. Recovery of internal standard (added before sample clean-up) is computed relative to amount of recovery standard added before the quantification. Relative response factors based on the recovery standard (RRFg) is calculated for each ISTD-compound from the quantification standard analysis.
Amt.ISTD : Amount internal standard added before extraction
Amt.GSTD :
Amount of recovery standard added before quantification
AreaISTD : Peak area of internal standard
AreaGSTD : Peak area of recovery standard
The detection limit is defined as 3 times the noise level (compound signal/noise = 3:1) as shown in the following figure.
For outdoor air the detection limit is normally reported in the unit pg/m3. The detection limit varies according to the concentration level in the sample, the sample volume, the purity of the sample extract, sample loss during clean-up and the pre-concentration factor.
Incoming samples must be registered in a sample journal with date, a sample number and an analysis form which follows the sample during the whole analysis.
A cleaned sample extract ready for GC/MS may be stored up to 6 months in a freezer before analysis.
PAHs dissolved in cyclohexane and stored in a freezer (dark) are stable for years. The main standard, quantification standard, internal standard, control standard and recovery standard must be stored in a freezer (<18°C). Control of weight and corrections for weight loss must be recorded. Max. storage time is 5 years.
The working solutions are stored in a refrigerator (4-6°C) and weight loss control is not necessary. Max. storage time is 6 months.
All new standards must be checked against the old standard and concentration deviations should be within ±10%.
To check the GC/MS quantification a control standard must be analysed. The standard covers the whole volatility range of the PAHs to be quantified.
The standard must be analysed after every 20th sample or every 14th day when less then 20 samples are analysed. For long periods without any activity (months), the standard must be analysed once a month.
Plots showing the single results for each compound and an average of the last 10 analyses must be available.
Before GC/MS-analysis a recovery standard is added to the sample in an amount according to the amount of internal standard added.
The recovery of the internal standards should be within the following limits:
ISTD II >10 to 100%
ISTD III,
IV >20 to 100%
ISTD V,
IV, VII >30 to 120%
Since the results for bicyclic PAHs is uncertain and the interest for these compounds is low, no limit is given for ISTD I.
If recovery is outside limits a note about this must be given in the analysis report. Low recovery is caused by losses during clean-up and pre-concentration. The result will be less reliable than with normal recovery.
Too high recovery may be caused by interference on the ISTD-signal. In such cases the quantification should be based on another ISTD with normal recovery.
Blank tests must be performed on clean filters and PUF plugs using the complete method. 2 unexposed plugs and 1 unexposed filter are extracted and analysed as if it was a normal sample. This must be done after each 20th sample.
The following criteria must be fulfilled for a satisfactory identification and quantification: