SIST ISO 7981-1:2007
(Main)Water quality -- Determination of polycyclic aromatic hydrocarbons (PAH) -- Part 1: Determination of six PAH by high-performance thin-layer chromatography with fluorescence detection after liquid-liquid extraction
Water quality -- Determination of polycyclic aromatic hydrocarbons (PAH) -- Part 1: Determination of six PAH by high-performance thin-layer chromatography with fluorescence detection after liquid-liquid extraction
This part of ISO 7981 specifies the determination of six selected PAH in drinking water by high-performance thin-layer chromatography with fluorescence detection after liquid-liquid extraction. The six PAH are: fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene (see Table 1). A screening method (method A) is described to exclude those samples containing less than 20 % of the limit values given in References [1], [2], [3] and [4]. A quantitative method (method B) is also described, with a working range of 40 ng/l to 240 ng/l (sum of 6 PAH). Higher concentrations can be determined by using a smaller aliquot of the sample. With some modifications, this method is also applicable for the analysis of ground waters and moderately polluted surface waters.
Qualité de l'eau -- Détermination des hydrocarbures aromatiques polycycliques (HAP) -- Partie 1: Dosage de six HAP par chromatographie de haute performance sur couche mince avec détection fluorimétrique à la suite d'une extraction liquide-liquide
Kakovost vode - Določevanje policikličnih aromatskih ogljikovodikov (PAH) - 1. del: Določevanje šestih policikličnih aromatskih ogljikovodikov s tankoplastno kromatografijo visoke ločljivosti s fluorescenčno detekcijo po tekočinsko-tekočinski ekstrakciji
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INTERNATIONAL ISO
STANDARD 7981-1
First edition
2005-06-15
Water quality — Determination of
polycyclic aromatic hydrocarbons
(PAH) —
Part 1:
Determination of six PAH by high-
performance thin-layer chromatography
with fluorescence detection after
liquid-liquid extraction
Qualité de l'eau — Détermination des hydrocarbures aromatiques
polycycliques (HAP) —
Partie 1: Dosage de six HAP par chromatographie de haute
performance sur couche mince avec détection fluorimétrique à la suite
d'une extraction liquide-liquide
Reference number
ISO 7981-1:2005(E)
©
ISO 2005
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ISO 7981-1:2005(E)
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ii © ISO 2005 – All rights reserved
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ISO 7981-1:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Principle. 1
3 Interferences . 2
4 Reagents. 3
5 Apparatus . 4
6 Sampling. 5
7 Procedure . 6
8 Evaluation. 8
9 Calibration . 11
10 Determination of the recovery. 13
11 Blank measurements. 13
12 Calculation. 13
13 Expression of results . 14
14 Test report . 15
15 Accuracy. 15
Annex A (informative) R values of the six PAH under various chromatographic conditions . 16
f
Annex B (informative) Spectroscopic identification. 17
Annex C (informative) Examples for the construction of special apparatus . 18
Annex D (informative) Accuracy . 21
Bibliography . 22
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ISO 7981-1:2005(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 7981-1 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
ISO 7981 consists of the following parts, under the general title Water quality — Determination of polycyclic
aromatic hydrocarbons (PAH):
Part 1: Determination of six PAH by high-performance thin-layer chromatography with fluorescence
detection after liquid-liquid extraction
Part 2: Determination of six PAH by high-performance liquid chromatography with fluorescence detection
after liquid-liquid extraction
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ISO 7981-1:2005(E)
Introduction
Polycyclic aromatic hydrocarbons (PAH) are present in nearly all types of waters. These substances are
adsorbed on solids (sediments, suspended matter) as well as dissolved in the liquid phase.
Some PAH are known or suspected to cause cancer. The maximum acceptable levels of PAH in waters
[1] [2] [3] [4]
intended for human consumption are given in European Legislation .
The sum of the mass concentrations of the six PAH specified in this part of ISO 7981 normally is about
0,01 µg/l to 0,05 µg/l in ground water and up to 1 µg/l in surface water.
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INTERNATIONAL STANDARD ISO 7981-1:2005(E)
Water quality — Determination of polycyclic aromatic
hydrocarbons (PAH) —
Part 1:
Determination of six PAH by high-performance thin-layer
chromatography with fluorescence detection after liquid-liquid
extraction
WARNING — Some substances being measured are presumed to be carcinogenic. Acetonitrile and
hexane are harmful.
Persons using this part of ISO 7981 should be familiar with normal laboratory practise. This standard
does not purport to address all of the safety problems, if any, associated with its use. It is the
responsibility of the user of this part of ISO 7981 to establish appropriate safety and health practices
and to ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this part of ISO 7981 be
carried out by suitably trained staff.
1 Scope
This part of ISO 7981 specifies the determination of six selected PAH in drinking water by high-performance
thin-layer chromatography with fluorescence detection after liquid-liquid extraction. The six PAH are:
fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, and
benzo[ghi]perylene (see Table 1).
A screening method (method A) is described to exclude those samples containing less than 20 % of the limit
values given in References [1], [2], [3] and [4].
A quantitative method (method B) is also described, with a working range of 40 ng/l to 240 ng/l (sum of
6 PAH). Higher concentrations can be determined by using a smaller aliquot of the sample.
With some modifications, this method is also applicable for the analysis of ground waters and moderately
polluted surface waters.
2 Principle
Since PAH can to a large extent be adsorbed on particulate matter, the whole test sample is analysed.
NOTE For the analysis of surface water, a differentiation between dissolved and undissolved PAH may be desirable,
but this is not relevant for drinking water.
PAH are extracted from the water sample by liquid-liquid extraction. The extract is evaporated to dryness and
the residue is taken up in a solvent and analysed.
Extracts of surface waters and other contaminated water samples should be cleaned prior to analysis (7.4).
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ISO 7981-1:2005(E)
PAH are then separated by high-performance thin-layer chromatography (HPTLC) on appropriate stationary
phases and detected either visually or by in situ fluorescence measurement at constant or differing
wavelength combinations.
Table 1 — Polycyclic aromatic hydrocarbons determinable by this method
Chemical Carbon
Name Molar mass CAS-number Structure
formula fraction
g/mol
C H
Fluoranthene 202,26 95,0 206-44-0
16 10
C H
Benzo[b]fluoranthene 252,32 95,2 205-99-2
20 12
C H
Benzo[a]pyrene 252,32 95,2 50-32-8
20 12
C H
Benzo[k]fluoranthene 252,32 95,2 207-08-9
20 12
C H
Indeno[1,2,3-cd]pyrene 276,34 95,6 193-39-5
22 12
C H
Benzo[ghi]perylene 276,34 95,6 191-24-2
22 12
3 Interferences
3.1 Interferences with screening method (method A)
Other compounds and/or impurities can interfere in the screening method, thus the use of spectroscopic
identification is recommended in order to lower the occurrence of false positives in screening test samples.
It may be necessary to clean coloured extracts or test samples known to contain other organic substances on
silica prior to analysis (7.4).
3.2 Interferences with sampling and extraction
Use sampling containers made of materials (preferably of glass or steel) that do not affect the test sample
during the contact time. Avoid plastics and other organic materials during sampling, sample storage or
extraction.
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ISO 7981-1:2005(E)
If automatic samplers are used, avoid the use of silicone or rubber material for the tubes. If present, make
sure that the tubes are as short as possible. Rinse the sampling line with the water to be sampled before the
test sample is taken. ISO 5667-2 and ISO 5667-3 can be used for guidance.
Keep the samples from direct sunlight and prolonged exposure to light.
During storage of the test sample, losses of PAH can occur due to adsorption on the walls of the containers.
The extent of the losses depends on the storage time.
3.3 Interferences with HPTLC
Substances that exhibit either fluorescence or quenching and co-elute with the PAH to be determined can
interfere with the determination. These interferences can lead to incompletely resolved signals and can,
depending on their magnitude, affect the accuracy and precision of the analytical results. Band overlap will
make an interpretation of the result impossible. Unsymmetrical bands and bands broader than the
corresponding bands of the reference substance suggest interferences.
The identity and purity of the bands can be checked by recording the excitation and emission spectra.
4 Reagents
Use only reagents of recognized analytical grade (e.g. “for residue analysis” or “for HPLC analysis”) as far as
available, and only distilled water or water of equivalent purity showing the lowest possible fluorescence.
Monitor the blank to guarantee that the reagents do not contain PAH in detectable concentrations (see
Clause 11).
4.1 Solvents
4.1.1 Extraction and clean-up solvents
4.1.1.1 Cyclohexane, C H
6 12
4.1.1.2 Hexane, C H
6 14
4.1.1.3 Dichloromethane, CH Cl
2 2
Other volatile solvents may be used as well, if it is proved that the recovery is equivalent or better.
NOTE Dichloromethane often contains stabilizers, e.g. ethanol or amylene. Stabilizers can influence the elution
strength of the eluent. Without stabilizer, free radicals might develop. This can lead to degradation of PAH. The presence
of hydrogen chloride indicates the presence of radicals. Hydrogen chloride can be determined by extracting
dichloromethane with water and measuring the pH value.
4.1.2 HPTLC solvents
4.1.2.1 Methanol, CH OH
3
4.1.2.2 Acetonitrile, CH CN
3
4.1.2.3 2,4-dimethyl-3-oxypentane (diisopropyl ether), C H O
6 14
4.1.2.4 2-propanol (isopropanol), C H OH
3 7
4.2 Sodium thiosulfate pentahydrate, Na S O ·5H O
2 2 3 2
4.3 Sodium chloride, NaCl
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ISO 7981-1:2005(E)
4.4 Sodium sulfate, Na SO , anhydrous, precleaned by heating to 500 °C.
2 4
4.5 Nitrogen, having a purity (volume fraction) of at least 99,999 %.
4.6 Silica, with an average particle size of approximately 40 µm and stored in a desiccator to ensure
maximum activity.
NOTE Prepacked silica cartridges are commercially available.
4.7 Molecular sieve beads, pore size 0,4 nm.
4.8 Caffeine, C H N O
8 10 4 2
4.9 Liquid paraffin
4.10 Reference substances (see Table 1)
Because of the dangerous nature of the substances to be used, it is highly recommended to use commercially
available, preferably certified, standard solutions. Avoid skin contact.
4.11 Single-substance stock solutions, of those listed in Table 1, diluted in cyclohexane (4.1.1.1) or
methanol (4.1.2.1) to a mass concentration of, for example, 10 µg/ml.
4.12 Multiple-substance stock solution, preferably certified, diluted in cyclohexane (4.1.1.1) or methanol
(4.1.2.1) to a mass concentration of, for example, 10 µg/ml for fluoranthene and 2 µg/ml for the other
reference substances (4.10).
4.13 Calibration solutions
Transfer 20 µl, 40 µl, 60 µl, 80 µl, 100 µl and 120 µl of the stock solution (4.12) into a graduated 10 ml flask
(5.14) and make up to volume with cyclohexane (4.1.1.1) or methanol (4.1.2.1).
1 ml of this reference solution contains 20 ng, 40 ng, 60 ng, 80 ng, 100 ng and 120 ng of fluoranthene and
4 ng, 8 ng, 12 ng, 16 ng, 20 ng and 24 ng of the other reference substances (4.10).
NOTE The solutions 4.11 to 4.13 are stable for at least one year when stored in the dark at room temperature and
protected from evaporation.
5 Apparatus
Use standard laboratory apparatus, cleaned to eliminate all interferences.
Clean all glassware, for example by rinsing with detergent and hot water, and dry for about 15 min to 30 min
at about 120 °C. After cooling, rinse with acetone, seal the glassware and store in a clean environment.
Glassware that has been in contact with waste water samples or samples with high PAH concentrations shall
not be re-used for drinking water analysis.
5.1 Brown glass bottles, narrow-necked, flat-bottomed, nominal capacity 1 000 ml, with solid glass
stopper.
5.2 Magnetic stirrer with stirring rods, PTFE-coated, kept under cyclohexane, with a maximum rotational
–1
frequency of 1 000 min .
5.3 Measuring cylinders, nominal capacities 10 ml, 25 ml and 1 000 ml.
5.4 Separating funnel, nominal capacity 1 000 ml, with PTFE stopcock, kept under cyclohexane, and glass
stopper, e.g. a Squibb funnel.
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ISO 7981-1:2005(E)
5.5 Conical flask, nominal capacity 100 ml, with glass stopper.
5.6 Reduction flask, nominal capacity 50 ml (see Figure C.1).
5.7 Centrifuge with rotor and centrifuge tubes, with tapered bottom, nominal capacity 50 ml (see
–1
Figure C.2) and with a rotational frequency of about 3 000 min .
5.8 Pasteur pipettes
5.9 Evaporation assembly, such as a rotary evaporator with vacuum stabilizer and water bath.
5.10 Shaking apparatus, with adjustable rotational speed, suitable for test tubes.
5.11 Blow-down assembly, nitrogen pressure cylinder with pressure-reducing valve and needle valve for
fine adjustment.
5.12 Polypropene or glass cartridges, filled with at least 0,5 g silica (4.6).
5.13 Glass vials, e.g. centrifuge tubes, nominal capacity 10 ml, with glass stoppers.
5.14 Graduated flasks, nominal capacity 10 ml, 20 ml, 100 ml and 250 ml.
5.15 High-performance thin-layer precoated plates, e.g. silica 60, preferably with fluorescence indicator
without concentrating zone.
For caffeine impregnation of the silica plates, dip the precoated plates by means of a mechanical dipping
device during 4 s into a solution containing 4 g caffeine (4.8) in 96 g dichloromethane (4.1.1.3). Dry the plates
for 30 min at 110 °C, and store in a desiccator until use.
Prior to use, clean the pretreated plate by blank chromatography to the upper edge, dry for 30 min at 110 °C,
and store in a desiccator until use.
NOTE These plates are commercially available.
5.16 High-performance thin-layer precoated plates, e.g. RP-18, preferably with fluorescence indicator
and without concentrating zone.
5.17 Development chamber, for low consumption of mobile phase, suitable for trace analysis.
5.18 Automatic dosing and application device, suitable for spot and band applications, or microlitre
syringes.
5.19 UV lamp, operating at 366 nm.
5.20 TLC scanner, for the direct fluorimetric measurement.
6 Sampling
When sampling drinking water from a tap of the water supply, collect the test sample prior to sterilizing the tap
for bacteriological sampling.
Plastics materials – with the exception of polytetrafluoroethene (PTFE) – may not be used during sampling
and sample treatment, as losses may occur due to adsorption of PAH on the material. Take care during
handling of the samples to keep them from direct sunlight, as PAH may decompose.
Collect the test sample in brown glass bottles (5.1) of known mass. Dechlorinate water samples containing
chlorine by immediately adding approximately 50 mg of sodium thiosulfate (4.2).
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ISO 7981-1:2005(E)
Fill the bottle to the shoulder (approximately 1 000 ml) and store the test sample at about + 4 °C and protected
from light until the extraction is carried out. Ensure that the extraction is carried out within 24 h after sampling
in order to avoid losses due to adsorption. If the complete analysis cannot be performed within 24 h, the
following procedure shall be performed within this time limit. If necessary remove part of the homogenized
sample from the sampling bottle until a sample volume of about 1 000 ml ± 10 ml remains, and determine the
volume of the test sample by weighing the bottle. Add 25 ml of cyclohexane (4.1.1.1) and shake well. The
pretreated test sample may be stored for 72 h at about + 4 °C, protected from light.
7 Procedure
7.1 Extraction
Take care during the handling of the samples to keep them from direct sunlight, as PAH can decompose.
Homogenize the test sample, e.g. with a magnetic stirrer. Remove a part of the test sample from the sampling
bottle until a test sample volume of about 1 000 ml ± 10 ml remains, and determine the volume of the test
sample by weighing the bottle.
Add 20 g of sodium chloride (4.3) to improve the extraction efficiency. Add 25 ml of cyclohexane (4.1.1.1) and
mix. Keep the test sample in a cool and dark place until the extraction is carried out.
Add a stirring rod and put the lid on the bottle. Then thoroughly mix the test sample using the magnetic stirrer
–1
(5.2) at maximum setting (1 000 min ) for 60 min. Transfer the test sample to a separating funnel (5.4) and
allow the phases to separate for at least 5 min.
For the extraction of waste water and other water samples with high concentrations of PAH, only 10 ml to
100 ml of the homogeneous test sample should be transferred to a 250 ml graduated flask (5.14) and diluted
with water to 200 ml. After adding 20 g of sodium chloride (4.3) and 25 ml of cyclohexane (4.1.1.1), proceed
as described above.
The extraction procedure may also be carried out using a microseparator (see Figure C.3).
If a stable emulsion is formed during the extraction process, collect it in a centrifuge tube (5.7) and centrifuge
–1
it for 10 min at about 3 000 min .
Transfer the aqueous phase into the sample bottle (5.1) and collect the cyclohexane extract in an conical flask
(5.5). Dry the extract in accordance with 7.2.
7.2 Drying of the extract
Rinse the separating funnel with 10 ml of cyclohexane (4.1.1.1) and add the cyclohexane to the total extract.
Dry the extract with sodium sulfate (4.4) for at least 30 min, swirling the vessel frequently.
Decant the dry extract into a reduction flask (5.6). Rinse the conical flask (5.5) twice with 5 ml of cyclohexane
(4.1.1.1) and add to the same reduction flask.
7.3 Enrichment
Evaporate the filtered cyclohexane extract until it fills only the tapered tip of the reduction flask (5.6)
(approximately 500 µl), with the evaporation assembly (5.9), e.g. the rotary evaporator, at 120 hPa and 30 °C.
Dissolve any residues that might have been deposited on the glass wall by shaking the extract using the
shaking apparatus (5.10).
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ISO 7981-1:2005(E)
If the extract is colourless or precleaned according to 7.4, evaporate the remaining cyclohexane with the blow-
down assembly (5.11) using nitrogen (4.5) until incipient dryness. Dissolve the dry residue in 40 µl (for spot
application) to 120 µl (for band application) of cyclohexane (4.1.1.1) or methanol (4.1.2.1).
If necessary, pre-clean the extract in accordance with 7.4.
7.4 Clean-up of the extract
For clean-up of the extract, use columns or cartridges (5.12) containing at least 0,5 g of silica (4.6). Clean the
silica in the column or in the cartridge by rinsing with five bed volumes of dichloromethane, followed by
conditioning with the same volume of hexane.
Dry the solvents used for cleaning the extract by applying molecular sieve (4.7).
Transfer the concentrated extract (7.3) with a Pasteur pipette (5.8) onto the hexane-covered silica and allow to
soak almost completely into the silica. Collect the eluate in a glass vial (5.13).
Rinse the reduction flask with 500 µl of hexane (4.1.1.2), add this solution to the column and allow to soak
almost completely into the silica.
Elute the PAH with a mixture of dichloromethane (4.1.1.3)/hexane (4.1.1.2) 1:1 volume fraction, and evaporate
the eluate until it fills only the tapered tip of the reduction flask (5.6) (approximately 500 µl), with the
evaporation assembly (5.9), followed by the blow-down assembly (5.11) using nitrogen (4.5) until incipient
dryness. Dissolve the residue in 40 µl or 120 µl of cyclohexane (4.1.1.1) or methanol (4.1.2.1).
NOTE Commercially available cartridges containing 0,5 g of silica require a volume of at least 3 ml of the mixture of
dichloromethane/hexane (1:1) for the elution of the PAH.
7.5 High-performance thin-layer chromatography
7.5.1 Application of the extracts
On one HPTLC plate, several samples may be analysed simultaneously, together with two or more reference
solutions of varying concentration. If a considerable number of samples has to be analysed, apply the extracts
on both ends of the HPTLC plate, provided the plate is developed in a horizontal development chamber.
Apply an aliquot of the total extract (7.3) either by means of the automated volume dosing device (5.18) or by
hand as spots or bands. For screening purposes, apply about half of the extract as a spot. If using band
application, apply 7 mm bands with 3 mm intervals.
7.5.2 Screening method (method A)
7.5.2.1 General
The screening method is a preliminary examination which is meant to exclude samples containing less than
20 % of the maximum acceptable levels (total concentration 30 ng/l to 40 ng/l).
Choose one of the following procedures for the screening test:
a) separation on caffeine impregnated silica plates (7.5.2.2);
b) separation at room temperature on HPTLC-RP-18 material (7.5.2.3).
Procedure a), if performed at –20 °C, is also recommended as a quantitative procedure. This method shows a
linear correlation between peak height and mass applied in the range of 2 ng to 12 ng for fluoranthene and
0,4 ng to 2,4 ng for the other PAH. If an aliquot of 10 % of the concentrated extract of a 1 000 ml water sample
is applied on the HPTLC plate, the concentration of fluoranthene can be reliably determined in the range of
20 ng/l to 120 ng/l and the other PAH in the range of 4 ng/l to 24 ng/l.
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ISO 7981-1:2005(E)
7.5.2.2 Separation on caffeine-impregnated silica plates at room temperature
Condition the plates after application of the extracts for 30 min at room temperature over water in a
conditioning chamber. Immediately develop the chromatogram vertically in a trough chamber or horizontally in
a horizontal development chamber.
As mobile phase, use 2,4-dimethyl-3-oxypentane (4.1.2.3)/hexane (4.1.1.2), 4:1 volume fraction.
Using a trough chamber, the run time for a migration distance of 6,5 cm is about 25 min. In a horizontal
development chamber, the run time for a migration distance of 6,5 cm is about 15 min, and for 4,5 cm about
10 min.
Dry the plate for 2 min in a stream of air at ambient temperature, then dip it for 2 s into a solution of liquid
paraffin (4.9)/hexane (4.1.1.2) 1:2 volume fraction to stabilize and increase the fluorescence intensity (by a
factor of 5 for benzo[a]pyrene and benzo[ghi]perylene and by a factor of 2 for the other substances), then dry
again for 2 min.
7.5.2.3 Separation on HPTLC-RP-18 plates
Develop the chromatogram vertically in a trough chamber or horizontally in a horizontal development chamber
(5.17).
As mobile phase, use acetonitrile (4.1.2.2)/2-propanol (4.1.2.4)/methanol (4.1.2.1), 1:2:1 volume fraction. Dry
the chromatogram during 2 min in a stream of air at ambient temperature.
Using a trough chamber, the run time for a migration distance of 6,5 cm is 20 min to 25 min. In a horizontal
development chamber, the run time for a migration distance of 6,5 cm is about 20 min, and for 4,5 cm about
15 min (without chamber saturation).
NOTE A better separation can be achieved by working at a lower temperature.
7.5.3 Separation on caffeine-impregnated silica plates at –20 °C in the freezer (method B)
Precool the plates at –20 °C for 20 min, then develop them using dichloromethane (4.1.1.3) as mobile phase
at –20 °C.
Using a trough chamber, the run time for a migration distance of 6,5 cm is about 20 min. In a horizontal
development chamber, the run time for a migration distance of 6,5 cm is about 17 min, and for 4,5 cm about
12 min.
Dry the plate for 2 min in a stream of air at ambient temperature, then dip it for 2 s into a solution of liquid
paraffin (4.9)/hexane (4.1.1.2) 1:2 volume fraction to stabilize and increase the fluorescence intensity (by a
factor of 5 for benzo[a]pyrene and benzo[ghi]perylene and by a factor of 2 for the other substances), then dry
again for 2 min.
8 Evaluation
8.1 Visual evaluation of the screening method
For R values for the PAH under various chromatographic conditions see Annex A.
f
Dry the plate for 2 min in a stream of air at ambient temperature, then place the plate under a UV lamp (5.19)
(λ = 366 nm) and evaluate the fluorescence intensity of the sample by comparison with the standard solutions
by allocating the zones by colour, R value and relative position to the reference chromatogram.
f
NOTE On RP-18-plates, only 3 zones can be recognized (see Figure 2).
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ISO 7981-1:2005(E)
It is not necessary to determine the substance-specific spectra of the individual spots.
If
...
SLOVENSKI STANDARD
SIST ISO 7981-1:2007
01-februar-2007
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Water quality -- Determination of polycyclic aromatic hydrocarbons (PAH) -- Part 1:
Determination of six PAH by high-performance thin-layer chromatography with
fluorescence detection after liquid-liquid extraction
Qualité de l'eau -- Détermination des hydrocarbures aromatiques polycycliques (HAP) --
Partie 1: Dosage de six HAP par chromatographie de haute performance sur couche
mince avec détection fluorimétrique à la suite d'une extraction liquide-liquide
Ta slovenski standard je istoveten z: ISO 7981-1:2005
ICS:
13.060.50 3UHLVNDYDYRGHQDNHPLþQH Examination of water for
VQRYL chemical substances
SIST ISO 7981-1:2007 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST ISO 7981-1:2007
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SIST ISO 7981-1:2007
INTERNATIONAL ISO
STANDARD 7981-1
First edition
2005-06-15
Water quality — Determination of
polycyclic aromatic hydrocarbons
(PAH) —
Part 1:
Determination of six PAH by high-
performance thin-layer chromatography
with fluorescence detection after
liquid-liquid extraction
Qualité de l'eau — Détermination des hydrocarbures aromatiques
polycycliques (HAP) —
Partie 1: Dosage de six HAP par chromatographie de haute
performance sur couche mince avec détection fluorimétrique à la suite
d'une extraction liquide-liquide
Reference number
ISO 7981-1:2005(E)
©
ISO 2005
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SIST ISO 7981-1:2007
ISO 7981-1:2005(E)
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ii © ISO 2005 – All rights reserved
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SIST ISO 7981-1:2007
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Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Principle. 1
3 Interferences . 2
4 Reagents. 3
5 Apparatus . 4
6 Sampling. 5
7 Procedure . 6
8 Evaluation. 8
9 Calibration . 11
10 Determination of the recovery. 13
11 Blank measurements. 13
12 Calculation. 13
13 Expression of results . 14
14 Test report . 15
15 Accuracy. 15
Annex A (informative) R values of the six PAH under various chromatographic conditions . 16
f
Annex B (informative) Spectroscopic identification. 17
Annex C (informative) Examples for the construction of special apparatus . 18
Annex D (informative) Accuracy . 21
Bibliography . 22
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SIST ISO 7981-1:2007
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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 7981-1 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
ISO 7981 consists of the following parts, under the general title Water quality — Determination of polycyclic
aromatic hydrocarbons (PAH):
Part 1: Determination of six PAH by high-performance thin-layer chromatography with fluorescence
detection after liquid-liquid extraction
Part 2: Determination of six PAH by high-performance liquid chromatography with fluorescence detection
after liquid-liquid extraction
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Introduction
Polycyclic aromatic hydrocarbons (PAH) are present in nearly all types of waters. These substances are
adsorbed on solids (sediments, suspended matter) as well as dissolved in the liquid phase.
Some PAH are known or suspected to cause cancer. The maximum acceptable levels of PAH in waters
[1] [2] [3] [4]
intended for human consumption are given in European Legislation .
The sum of the mass concentrations of the six PAH specified in this part of ISO 7981 normally is about
0,01 µg/l to 0,05 µg/l in ground water and up to 1 µg/l in surface water.
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SIST ISO 7981-1:2007
INTERNATIONAL STANDARD ISO 7981-1:2005(E)
Water quality — Determination of polycyclic aromatic
hydrocarbons (PAH) —
Part 1:
Determination of six PAH by high-performance thin-layer
chromatography with fluorescence detection after liquid-liquid
extraction
WARNING — Some substances being measured are presumed to be carcinogenic. Acetonitrile and
hexane are harmful.
Persons using this part of ISO 7981 should be familiar with normal laboratory practise. This standard
does not purport to address all of the safety problems, if any, associated with its use. It is the
responsibility of the user of this part of ISO 7981 to establish appropriate safety and health practices
and to ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this part of ISO 7981 be
carried out by suitably trained staff.
1 Scope
This part of ISO 7981 specifies the determination of six selected PAH in drinking water by high-performance
thin-layer chromatography with fluorescence detection after liquid-liquid extraction. The six PAH are:
fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, and
benzo[ghi]perylene (see Table 1).
A screening method (method A) is described to exclude those samples containing less than 20 % of the limit
values given in References [1], [2], [3] and [4].
A quantitative method (method B) is also described, with a working range of 40 ng/l to 240 ng/l (sum of
6 PAH). Higher concentrations can be determined by using a smaller aliquot of the sample.
With some modifications, this method is also applicable for the analysis of ground waters and moderately
polluted surface waters.
2 Principle
Since PAH can to a large extent be adsorbed on particulate matter, the whole test sample is analysed.
NOTE For the analysis of surface water, a differentiation between dissolved and undissolved PAH may be desirable,
but this is not relevant for drinking water.
PAH are extracted from the water sample by liquid-liquid extraction. The extract is evaporated to dryness and
the residue is taken up in a solvent and analysed.
Extracts of surface waters and other contaminated water samples should be cleaned prior to analysis (7.4).
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PAH are then separated by high-performance thin-layer chromatography (HPTLC) on appropriate stationary
phases and detected either visually or by in situ fluorescence measurement at constant or differing
wavelength combinations.
Table 1 — Polycyclic aromatic hydrocarbons determinable by this method
Chemical Carbon
Name Molar mass CAS-number Structure
formula fraction
g/mol
C H
Fluoranthene 202,26 95,0 206-44-0
16 10
C H
Benzo[b]fluoranthene 252,32 95,2 205-99-2
20 12
C H
Benzo[a]pyrene 252,32 95,2 50-32-8
20 12
C H
Benzo[k]fluoranthene 252,32 95,2 207-08-9
20 12
C H
Indeno[1,2,3-cd]pyrene 276,34 95,6 193-39-5
22 12
C H
Benzo[ghi]perylene 276,34 95,6 191-24-2
22 12
3 Interferences
3.1 Interferences with screening method (method A)
Other compounds and/or impurities can interfere in the screening method, thus the use of spectroscopic
identification is recommended in order to lower the occurrence of false positives in screening test samples.
It may be necessary to clean coloured extracts or test samples known to contain other organic substances on
silica prior to analysis (7.4).
3.2 Interferences with sampling and extraction
Use sampling containers made of materials (preferably of glass or steel) that do not affect the test sample
during the contact time. Avoid plastics and other organic materials during sampling, sample storage or
extraction.
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If automatic samplers are used, avoid the use of silicone or rubber material for the tubes. If present, make
sure that the tubes are as short as possible. Rinse the sampling line with the water to be sampled before the
test sample is taken. ISO 5667-2 and ISO 5667-3 can be used for guidance.
Keep the samples from direct sunlight and prolonged exposure to light.
During storage of the test sample, losses of PAH can occur due to adsorption on the walls of the containers.
The extent of the losses depends on the storage time.
3.3 Interferences with HPTLC
Substances that exhibit either fluorescence or quenching and co-elute with the PAH to be determined can
interfere with the determination. These interferences can lead to incompletely resolved signals and can,
depending on their magnitude, affect the accuracy and precision of the analytical results. Band overlap will
make an interpretation of the result impossible. Unsymmetrical bands and bands broader than the
corresponding bands of the reference substance suggest interferences.
The identity and purity of the bands can be checked by recording the excitation and emission spectra.
4 Reagents
Use only reagents of recognized analytical grade (e.g. “for residue analysis” or “for HPLC analysis”) as far as
available, and only distilled water or water of equivalent purity showing the lowest possible fluorescence.
Monitor the blank to guarantee that the reagents do not contain PAH in detectable concentrations (see
Clause 11).
4.1 Solvents
4.1.1 Extraction and clean-up solvents
4.1.1.1 Cyclohexane, C H
6 12
4.1.1.2 Hexane, C H
6 14
4.1.1.3 Dichloromethane, CH Cl
2 2
Other volatile solvents may be used as well, if it is proved that the recovery is equivalent or better.
NOTE Dichloromethane often contains stabilizers, e.g. ethanol or amylene. Stabilizers can influence the elution
strength of the eluent. Without stabilizer, free radicals might develop. This can lead to degradation of PAH. The presence
of hydrogen chloride indicates the presence of radicals. Hydrogen chloride can be determined by extracting
dichloromethane with water and measuring the pH value.
4.1.2 HPTLC solvents
4.1.2.1 Methanol, CH OH
3
4.1.2.2 Acetonitrile, CH CN
3
4.1.2.3 2,4-dimethyl-3-oxypentane (diisopropyl ether), C H O
6 14
4.1.2.4 2-propanol (isopropanol), C H OH
3 7
4.2 Sodium thiosulfate pentahydrate, Na S O ·5H O
2 2 3 2
4.3 Sodium chloride, NaCl
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4.4 Sodium sulfate, Na SO , anhydrous, precleaned by heating to 500 °C.
2 4
4.5 Nitrogen, having a purity (volume fraction) of at least 99,999 %.
4.6 Silica, with an average particle size of approximately 40 µm and stored in a desiccator to ensure
maximum activity.
NOTE Prepacked silica cartridges are commercially available.
4.7 Molecular sieve beads, pore size 0,4 nm.
4.8 Caffeine, C H N O
8 10 4 2
4.9 Liquid paraffin
4.10 Reference substances (see Table 1)
Because of the dangerous nature of the substances to be used, it is highly recommended to use commercially
available, preferably certified, standard solutions. Avoid skin contact.
4.11 Single-substance stock solutions, of those listed in Table 1, diluted in cyclohexane (4.1.1.1) or
methanol (4.1.2.1) to a mass concentration of, for example, 10 µg/ml.
4.12 Multiple-substance stock solution, preferably certified, diluted in cyclohexane (4.1.1.1) or methanol
(4.1.2.1) to a mass concentration of, for example, 10 µg/ml for fluoranthene and 2 µg/ml for the other
reference substances (4.10).
4.13 Calibration solutions
Transfer 20 µl, 40 µl, 60 µl, 80 µl, 100 µl and 120 µl of the stock solution (4.12) into a graduated 10 ml flask
(5.14) and make up to volume with cyclohexane (4.1.1.1) or methanol (4.1.2.1).
1 ml of this reference solution contains 20 ng, 40 ng, 60 ng, 80 ng, 100 ng and 120 ng of fluoranthene and
4 ng, 8 ng, 12 ng, 16 ng, 20 ng and 24 ng of the other reference substances (4.10).
NOTE The solutions 4.11 to 4.13 are stable for at least one year when stored in the dark at room temperature and
protected from evaporation.
5 Apparatus
Use standard laboratory apparatus, cleaned to eliminate all interferences.
Clean all glassware, for example by rinsing with detergent and hot water, and dry for about 15 min to 30 min
at about 120 °C. After cooling, rinse with acetone, seal the glassware and store in a clean environment.
Glassware that has been in contact with waste water samples or samples with high PAH concentrations shall
not be re-used for drinking water analysis.
5.1 Brown glass bottles, narrow-necked, flat-bottomed, nominal capacity 1 000 ml, with solid glass
stopper.
5.2 Magnetic stirrer with stirring rods, PTFE-coated, kept under cyclohexane, with a maximum rotational
–1
frequency of 1 000 min .
5.3 Measuring cylinders, nominal capacities 10 ml, 25 ml and 1 000 ml.
5.4 Separating funnel, nominal capacity 1 000 ml, with PTFE stopcock, kept under cyclohexane, and glass
stopper, e.g. a Squibb funnel.
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5.5 Conical flask, nominal capacity 100 ml, with glass stopper.
5.6 Reduction flask, nominal capacity 50 ml (see Figure C.1).
5.7 Centrifuge with rotor and centrifuge tubes, with tapered bottom, nominal capacity 50 ml (see
–1
Figure C.2) and with a rotational frequency of about 3 000 min .
5.8 Pasteur pipettes
5.9 Evaporation assembly, such as a rotary evaporator with vacuum stabilizer and water bath.
5.10 Shaking apparatus, with adjustable rotational speed, suitable for test tubes.
5.11 Blow-down assembly, nitrogen pressure cylinder with pressure-reducing valve and needle valve for
fine adjustment.
5.12 Polypropene or glass cartridges, filled with at least 0,5 g silica (4.6).
5.13 Glass vials, e.g. centrifuge tubes, nominal capacity 10 ml, with glass stoppers.
5.14 Graduated flasks, nominal capacity 10 ml, 20 ml, 100 ml and 250 ml.
5.15 High-performance thin-layer precoated plates, e.g. silica 60, preferably with fluorescence indicator
without concentrating zone.
For caffeine impregnation of the silica plates, dip the precoated plates by means of a mechanical dipping
device during 4 s into a solution containing 4 g caffeine (4.8) in 96 g dichloromethane (4.1.1.3). Dry the plates
for 30 min at 110 °C, and store in a desiccator until use.
Prior to use, clean the pretreated plate by blank chromatography to the upper edge, dry for 30 min at 110 °C,
and store in a desiccator until use.
NOTE These plates are commercially available.
5.16 High-performance thin-layer precoated plates, e.g. RP-18, preferably with fluorescence indicator
and without concentrating zone.
5.17 Development chamber, for low consumption of mobile phase, suitable for trace analysis.
5.18 Automatic dosing and application device, suitable for spot and band applications, or microlitre
syringes.
5.19 UV lamp, operating at 366 nm.
5.20 TLC scanner, for the direct fluorimetric measurement.
6 Sampling
When sampling drinking water from a tap of the water supply, collect the test sample prior to sterilizing the tap
for bacteriological sampling.
Plastics materials – with the exception of polytetrafluoroethene (PTFE) – may not be used during sampling
and sample treatment, as losses may occur due to adsorption of PAH on the material. Take care during
handling of the samples to keep them from direct sunlight, as PAH may decompose.
Collect the test sample in brown glass bottles (5.1) of known mass. Dechlorinate water samples containing
chlorine by immediately adding approximately 50 mg of sodium thiosulfate (4.2).
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Fill the bottle to the shoulder (approximately 1 000 ml) and store the test sample at about + 4 °C and protected
from light until the extraction is carried out. Ensure that the extraction is carried out within 24 h after sampling
in order to avoid losses due to adsorption. If the complete analysis cannot be performed within 24 h, the
following procedure shall be performed within this time limit. If necessary remove part of the homogenized
sample from the sampling bottle until a sample volume of about 1 000 ml ± 10 ml remains, and determine the
volume of the test sample by weighing the bottle. Add 25 ml of cyclohexane (4.1.1.1) and shake well. The
pretreated test sample may be stored for 72 h at about + 4 °C, protected from light.
7 Procedure
7.1 Extraction
Take care during the handling of the samples to keep them from direct sunlight, as PAH can decompose.
Homogenize the test sample, e.g. with a magnetic stirrer. Remove a part of the test sample from the sampling
bottle until a test sample volume of about 1 000 ml ± 10 ml remains, and determine the volume of the test
sample by weighing the bottle.
Add 20 g of sodium chloride (4.3) to improve the extraction efficiency. Add 25 ml of cyclohexane (4.1.1.1) and
mix. Keep the test sample in a cool and dark place until the extraction is carried out.
Add a stirring rod and put the lid on the bottle. Then thoroughly mix the test sample using the magnetic stirrer
–1
(5.2) at maximum setting (1 000 min ) for 60 min. Transfer the test sample to a separating funnel (5.4) and
allow the phases to separate for at least 5 min.
For the extraction of waste water and other water samples with high concentrations of PAH, only 10 ml to
100 ml of the homogeneous test sample should be transferred to a 250 ml graduated flask (5.14) and diluted
with water to 200 ml. After adding 20 g of sodium chloride (4.3) and 25 ml of cyclohexane (4.1.1.1), proceed
as described above.
The extraction procedure may also be carried out using a microseparator (see Figure C.3).
If a stable emulsion is formed during the extraction process, collect it in a centrifuge tube (5.7) and centrifuge
–1
it for 10 min at about 3 000 min .
Transfer the aqueous phase into the sample bottle (5.1) and collect the cyclohexane extract in an conical flask
(5.5). Dry the extract in accordance with 7.2.
7.2 Drying of the extract
Rinse the separating funnel with 10 ml of cyclohexane (4.1.1.1) and add the cyclohexane to the total extract.
Dry the extract with sodium sulfate (4.4) for at least 30 min, swirling the vessel frequently.
Decant the dry extract into a reduction flask (5.6). Rinse the conical flask (5.5) twice with 5 ml of cyclohexane
(4.1.1.1) and add to the same reduction flask.
7.3 Enrichment
Evaporate the filtered cyclohexane extract until it fills only the tapered tip of the reduction flask (5.6)
(approximately 500 µl), with the evaporation assembly (5.9), e.g. the rotary evaporator, at 120 hPa and 30 °C.
Dissolve any residues that might have been deposited on the glass wall by shaking the extract using the
shaking apparatus (5.10).
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If the extract is colourless or precleaned according to 7.4, evaporate the remaining cyclohexane with the blow-
down assembly (5.11) using nitrogen (4.5) until incipient dryness. Dissolve the dry residue in 40 µl (for spot
application) to 120 µl (for band application) of cyclohexane (4.1.1.1) or methanol (4.1.2.1).
If necessary, pre-clean the extract in accordance with 7.4.
7.4 Clean-up of the extract
For clean-up of the extract, use columns or cartridges (5.12) containing at least 0,5 g of silica (4.6). Clean the
silica in the column or in the cartridge by rinsing with five bed volumes of dichloromethane, followed by
conditioning with the same volume of hexane.
Dry the solvents used for cleaning the extract by applying molecular sieve (4.7).
Transfer the concentrated extract (7.3) with a Pasteur pipette (5.8) onto the hexane-covered silica and allow to
soak almost completely into the silica. Collect the eluate in a glass vial (5.13).
Rinse the reduction flask with 500 µl of hexane (4.1.1.2), add this solution to the column and allow to soak
almost completely into the silica.
Elute the PAH with a mixture of dichloromethane (4.1.1.3)/hexane (4.1.1.2) 1:1 volume fraction, and evaporate
the eluate until it fills only the tapered tip of the reduction flask (5.6) (approximately 500 µl), with the
evaporation assembly (5.9), followed by the blow-down assembly (5.11) using nitrogen (4.5) until incipient
dryness. Dissolve the residue in 40 µl or 120 µl of cyclohexane (4.1.1.1) or methanol (4.1.2.1).
NOTE Commercially available cartridges containing 0,5 g of silica require a volume of at least 3 ml of the mixture of
dichloromethane/hexane (1:1) for the elution of the PAH.
7.5 High-performance thin-layer chromatography
7.5.1 Application of the extracts
On one HPTLC plate, several samples may be analysed simultaneously, together with two or more reference
solutions of varying concentration. If a considerable number of samples has to be analysed, apply the extracts
on both ends of the HPTLC plate, provided the plate is developed in a horizontal development chamber.
Apply an aliquot of the total extract (7.3) either by means of the automated volume dosing device (5.18) or by
hand as spots or bands. For screening purposes, apply about half of the extract as a spot. If using band
application, apply 7 mm bands with 3 mm intervals.
7.5.2 Screening method (method A)
7.5.2.1 General
The screening method is a preliminary examination which is meant to exclude samples containing less than
20 % of the maximum acceptable levels (total concentration 30 ng/l to 40 ng/l).
Choose one of the following procedures for the screening test:
a) separation on caffeine impregnated silica plates (7.5.2.2);
b) separation at room temperature on HPTLC-RP-18 material (7.5.2.3).
Procedure a), if performed at –20 °C, is also recommended as a quantitative procedure. This method shows a
linear correlation between peak height and mass applied in the range of 2 ng to 12 ng for fluoranthene and
0,4 ng to 2,4 ng for the other PAH. If an aliquot of 10 % of the concentrated extract of a 1 000 ml water sample
is applied on the HPTLC plate, the concentration of fluoranthene can be reliably determined in the range of
20 ng/l to 120 ng/l and the other PAH in the range of 4 ng/l to 24 ng/l.
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7.5.2.2 Separation on caffeine-impregnated silica plates at room temperature
Condition the plates after application of the extracts for 30 min at room temperature over water in a
conditioning chamber. Immediately develop the chromatogram vertically in a trough chamber or horizontally in
a horizontal development chamber.
As mobile phase, use 2,4-dimethyl-3-oxypentane (4.1.2.3)/hexane (4.1.1.2), 4:1 volume fraction.
Using a trough chamber, the run time for a migration distance of 6,5 cm is about 25 min. In a horizontal
development chamber, the run time for a migration distance of 6,5 cm is about 15 min, and for 4,5 cm about
10 min.
Dry the plate for 2 min in a stream of air at ambient temperature, then dip it for 2 s into a solution of liquid
paraffin (4.9)/hexane (4.1.1.2) 1:2 volume fraction to stabilize and increase the fluorescence intensity (by a
factor of 5 for benzo[a]pyrene and benzo[ghi]perylene and by a factor of 2 for the other substances), then dry
again for 2 min.
7.5.2.3 Separation on HPTLC-RP-18 plates
Develop the chromatogram vertically in a trough chamber or horizontally in a horizontal development chamber
(5.17).
As mobile phase, use acetonitrile (4.1.2.2)/2-propanol (4.1.2.4)/methanol (4.1.2.1), 1:2:1 volume fraction. Dry
the chromatogram during 2 min in a stream of air at ambient temperature.
Using a trough chamber, the run time for a migration distance of 6,5 cm is 20 min to 25 min. In a horizontal
development chamber, the run time for a migration distance of 6,5 cm is about 20 min, and for 4,5 cm about
15 min (without chamber saturation).
NOTE A better separation can
...
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