SIST ISO 7981-2:2007
(Main)Water quality -- Determination of polycyclic aromatic hydrocarbons (PAH) -- Part 2: Determination of six PAH by high-performance liquid chromatography with fluorescence detection after liquid-liquid extraction
Water quality -- Determination of polycyclic aromatic hydrocarbons (PAH) -- Part 2: Determination of six PAH by high-performance liquid chromatography with fluorescence detection after liquid-liquid extraction
This part of ISO 7981 specifies the determination of six selected PAH in drinking, mineral and table waters and ground and surface waters in mass concentrations above 0,005 µg/l, by high-performance liquid 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). With some modification, this method is also applicable for the analysis of moderately polluted waste waters.
Qualité de l'eau -- Détermination des hydrocarbures aromatiques polycycliques (HAP) -- Partie 2: Dosage de six HAP par chromatographie de haute performance en phase liquide avec détection fluorimétrique à la suite d'une extraction liquide-liquide
Kakovost vode - Določevanje policikličnih aromatskih ogljikovodikov (PAH) - 2. del: Določevanje šestih policikličnih aromatskih ogljikovodikov s tekočinsko kromatografijo visoke ločljivosti s fluorescenčno detekcijo po tekočinsko-tekočinski ekstrakciji
General Information
Buy Standard
Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 7981-2
First edition
2005-06-15
Water quality — Determination of
polycyclic aromatic hydrocarbons
(PAH) —
Part 2:
Determination of six PAH by high-
performance liquid chromatography with
fluorescence detection after liquid-liquid
extraction
Qualité de l'eau — Détermination des hydrocarbures aromatiques
polycycliques (HAP) —
Partie 2: Dosage de six HAP par chromatographie de haute
performance en phase liquide avec détection fluorimétrique à la suite
d'une extraction liquide-liquide
Reference number
ISO 7981-2:2005(E)
©
ISO 2005
---------------------- Page: 1 ----------------------
ISO 7981-2:2005(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2005
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2005 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 7981-2:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Principle. 1
4 Interferences . 2
5 Reagents. 3
6 Apparatus . 4
7 Sampling. 5
8 Procedure . 6
9 Calibration . 8
10 Measurement of samples. 9
11 Determination of the recovery. 9
12 Blank measurement. 10
13 Calculation. 10
14 Expression of results . 10
15 Test report . 11
16 Accuracy. 11
Annex A (informative) Examples of chromatographic conditions and columns. 12
Annex B (informative) Examples for the construction of special apparatus . 14
Annex C (informative) Accuracy . 17
Bibliography . 18
© ISO 2005 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 7981-2:2005(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 7981-2 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
iv © ISO 2005 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 7981-2:2005(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 usually is about
0,01 µg/l to 0,05 µg/l in ground water, up to 1 µg/l in surface water, and up to 1 000 µg/l in waste water.
© ISO 2005 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 7981-2:2005(E)
Water quality — Determination of polycyclic aromatic
hydrocarbons (PAH) —
Part 2:
Determination of six PAH by high-performance liquid
chromatography with fluorescence detection after liquid-liquid
extraction
WARNING — Some compounds 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, mineral and table waters
and ground and surface waters in mass concentrations above 0,005 µg/l, by high-performance liquid
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).
With some modification, this method is also applicable for the analysis of moderately polluted waste waters.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 8466-1, Water quality — Calibration and evaluation of analytical methods and estimation of performance
characteristics — Part 1: Statistical evaluation of the linear calibration function
3 Principle
Since PAH can to a large extent be adsorbed on particulate matter, the whole sample is analysed.
NOTE For the analysis of surface water, a differentiation between dissolved and undissolved PAH can be desirable,
but this is not relevant for drinking water.
© ISO 2005 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 7981-2:2005(E)
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 on silica (8.4) prior to
analysis.
PAH are then separated by high performance liquid chromatography (HPLC) on suitable stationary phases
under isocratic conditions, identified and quantified by means of fluorescence detection at a constant
combination of excitation and emission wavelengths.
Table 1 — Polycyclic aromatic hydrocarbons determinable by this method
Chemical Carbon
Name Molar mass CAS-number Structure
formula fraction
g/mol
Fluoranthene C H 202,26 95,0 206-44-0
16 10
Benzo[b]fluoranthene C H 252,32 95,2 205-99-2
20 12
Benzo[a]pyrene C H 252,32 95,2 50-32-8
20 12
Benzo[k]fluoranthene C H 252,32 95,2 207-08-9
20 12
Indeno[1,2,3-cd]pyrene C H 276,34 95,6 193-39-5
22 12
Benzo[ghi]perylene C H 276,34 95,6 191-24-2
22 12
4 Interferences
4.1 Interferences with sampling and extraction
Use sampling containers made of materials (preferably of glass or steel) that do not affect the sample during
the contact time. Avoid plastics and other organic materials during sampling, sample storage or extraction.
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.
2 © ISO 2005 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 7981-2:2005(E)
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.
4.2 Interferences with the HPLC
Substances that show 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 accuracy and precision of the analytical results. Peak overlaps will
prevent the measurement of peak height and/or area. Unsymmetrical peaks and peaks broader than the
respective peaks of the reference substance suggest interferences.
5 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 12).
5.1 Solvents
5.1.1 Solvents for extraction and clean-up of the extract
5.1.1.1 Cyclohexane, C H
6 12
5.1.1.2 Hexane, C H
6 14
5.1.1.3 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.
5.1.2 HPLC solvents
5.1.2.1 Methanol, CH OH
3
5.1.2.2 Acetonitrile, CH CN
3
5.1.2.3 Tetrahydrofuran, C H O, without stabilizer
4 8
NOTE Tetrahydrofuran can contain peroxides. Although peroxides have not yet shown to cause any interference with
the HPLC determination, it is preferred to use batches with low peroxide content (regularly checked using test rods). It is of
advantage to use small packages.
5.2 Sodium thiosulfate pentahydrate, Na S O ·5H O
2 2 3 2
5.3 Sodium chloride, NaCl
5.4 Sodium sulfate, Na SO , anhydrous, precleaned by heating to 500 °C.
2 4
5.5 Nitrogen, having a purity (volume fraction) of at least 99,999 %.
© ISO 2005 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 7981-2:2005(E)
5.6 Helium, having a purity (volume fraction) of at least 99,999 %.
5.7 Silica, with an average particle size approximately 40 µm and stored in a desiccator to ensure
maximum activity.
NOTE Prepacked silica cartridges are commercially available.
5.8 Molecular sieve beads, pore size 0,4 nm.
5.9 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.
5.10 Single-substance stock solutions, of those listed in Table 1, diluted in acetonitrile (5.1.2.2) to a mass
concentration of, for example, 10 µg/ml.
5.11 Multiple-substance stock solution, preferably certified, diluted in acetonitrile (5.1.2.2) to a mass
concentration of, for example, 10 µg/ml for each individual compound.
5.12 Calibration solutions
Prepare at least five calibration solutions by appropriate dilution of the stock solution (5.11), using methanol
(5.1.2.1) or acetonitrile (5.1.2.2) as solvent. The choice of solvent depends on the composition of the mobile
phase.
For example, using 50 µl of the stock solution (5.11) in a graduated 10 ml flask (6.16), make up to volume with
acetonitrile (5.1.2.2) or methanol (5.1.2.1). 1 µl of this reference solution contains 50 pg of the respective
individual substance.
NOTE The solutions 5.10 to 5.12 are stable for at least one year when stored in the dark at room temperature and
protected from evaporation.
6 Apparatus
Standard laboratory equipment cleaned to eliminate all interferences.
Clean all glassware, for example, by rinsing with detergent and hot water, and drying 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.
6.1 Brown glass bottles, narrow-necked, flat-bottomed, nominal capacity 1 000 ml, with solid glass
stopper.
6.2 Magnetic stirrer with stirring rods, PTFE coated, kept under cyclohexane, with a maximal rotational
–1
frequency of 1 000 min .
6.3 Measuring cylinder, nominal capacity 10 ml, 25 ml and 1 000 ml.
6.4 Separating funnel, nominal capacity 1 000 ml, with PTFE stopcock, kept under cyclohexane, and glass
stopper, e.g. a Squibb funnel.
6.5 Conical flask, nominal capacity 100 ml, with glass stopper.
6.6 Reduction flask, nominal capacity 100 ml (see Figure B.1).
4 © ISO 2005 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 7981-2:2005(E)
–1
6.7 Centrifuge with rotor, with a rotational frequency of about 3 000 min and with centrifuge tubes with
tapered bottom, nominal capacity 50 ml (see Figure B.2).
6.8 Pasteur pipettes
6.9 Evaporation assembly, such as a rotary evaporator with vacuum stabilizer and water bath.
6.10 Shaking apparatus, with adjustable rotational speed, suitable for test tubes.
6.11 Blow-down assembly, nitrogen pressure cylinder with pressure-reducing valve and needle valve for
fine adjustment.
6.12 Microfilter, with solvent-resistant membrane, pore size 0,45 µm.
6.13 Autosampler vials, capacity approximately 2 ml, with inert filler cap, e.g. PTFE coated septum.
6.14 Polypropene or glass cartridges, filled with at least 0,5 g silica (5.7).
6.15 Glass vials, e.g. centrifuge tubes, nominal capacity 10 ml, with glass stoppers.
6.16 Graduated flasks, nominal capacities 10 ml, 100 ml and 250 ml.
6.17 High-performance liquid chromatograph, with fluorescence detector and data evaluation system,
including:
degassing assembly, e.g. for degassing with vacuum or helium;
low pulsating analytical pump;
manual or automatic sample applicator;
column thermostat, capable of keeping the temperature constant to within ± 0,5 °C;
fluorescence detector, preferably equipped with a monochromator on either the excitation and
emission sides, or with a filter (8.5.2);
analytical separation column, e.g. a column with length up to 250 mm, internal diameter 2 mm to
4,6 mm, packed with particle size 3 µm to 5 µm material, capable of near baseline separation (at
least as good as in Figure A.1) of the PAH to a large extent.
7 Sampling
When sampling drinking water from a tap of the water supply, collect the test sample before the tap is
sterilized for bacteriological sampling.
Plastics materials – with the exception of polytetrafluorethene (PTFE) – should not be used during sampling
and sample treatment, as losses can occur due to adsorption of PAH on the material. Take care during
handling of the test samples to keep them from direct sunlight, as PAH can decompose.
Collect the test sample in brown glass bottles (6.1) of known mass. Dechlorinate water samples containing
chlorine by immediately adding approximately 50 mg of sodium thiosulfate (5.2).
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. When the complete analysis cannot be performed within 24 h, the
following procedure shall be performed within this time limit. If necessary remove a part of the sample from the
sampling bottle until a sample volume of about 1 000 ml ± 10 ml remains, and determine the volume of the
sample by weighing the bottle, add 25 ml of cyclohexane (5.1.1.1) and shake well. The pretreated sample can
be stored for 72 h at about + 4 °C, protected from light.
© ISO 2005 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 7981-2:2005(E)
8 Procedure
8.1 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 sample from the sampling
bottle until a test sample volume of about 1 000 ml ± 10 ml remains, and determine the volume of the
...
SLOVENSKI STANDARD
SIST ISO 7981-2:2007
01-februar-2007
.DNRYRVWYRGH'RORþHYDQMHSROLFLNOLþQLKDURPDWVNLKRJOMLNRYRGLNRY 3$+
GHO 'RORþHYDQMHãHVWLKSROLFLNOLþQLKDURPDWVNLKRJOMLNRYRGLNRYVWHNRþLQVNR
NURPDWRJUDILMRYLVRNHORþOMLYRVWLVIOXRUHVFHQþQRGHWHNFLMRSRWHNRþLQVNR
WHNRþLQVNLHNVWUDNFLML
Water quality -- Determination of polycyclic aromatic hydrocarbons (PAH) -- Part 2:
Determination of six PAH by high-performance liquid chromatography with fluorescence
detection after liquid-liquid extraction
Qualité de l'eau -- Détermination des hydrocarbures aromatiques polycycliques (HAP) --
Partie 2: Dosage de six HAP par chromatographie de haute performance en phase
liquide avec détection fluorimétrique à la suite d'une extraction liquide-liquide
Ta slovenski standard je istoveten z: ISO 7981-2:2005
ICS:
13.060.50 3UHLVNDYDYRGHQDNHPLþQH Examination of water for
VQRYL chemical substances
SIST ISO 7981-2:2007 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
INTERNATIONAL ISO
STANDARD 7981-2
First edition
2005-06-15
Water quality — Determination of
polycyclic aromatic hydrocarbons
(PAH) —
Part 2:
Determination of six PAH by high-
performance liquid chromatography with
fluorescence detection after liquid-liquid
extraction
Qualité de l'eau — Détermination des hydrocarbures aromatiques
polycycliques (HAP) —
Partie 2: Dosage de six HAP par chromatographie de haute
performance en phase liquide avec détection fluorimétrique à la suite
d'une extraction liquide-liquide
Reference number
ISO 7981-2:2005(E)
©
ISO 2005
---------------------- Page: 2 ----------------------
ISO 7981-2:2005(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
© ISO 2005
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2005 – All rights reserved
---------------------- Page: 3 ----------------------
ISO 7981-2:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Principle. 1
4 Interferences . 2
5 Reagents. 3
6 Apparatus . 4
7 Sampling. 5
8 Procedure . 6
9 Calibration . 8
10 Measurement of samples. 9
11 Determination of the recovery. 9
12 Blank measurement. 10
13 Calculation. 10
14 Expression of results . 10
15 Test report . 11
16 Accuracy. 11
Annex A (informative) Examples of chromatographic conditions and columns. 12
Annex B (informative) Examples for the construction of special apparatus . 14
Annex C (informative) Accuracy . 17
Bibliography . 18
© ISO 2005 – All rights reserved iii
---------------------- Page: 4 ----------------------
ISO 7981-2:2005(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 7981-2 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
iv © ISO 2005 – All rights reserved
---------------------- Page: 5 ----------------------
ISO 7981-2:2005(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 usually is about
0,01 µg/l to 0,05 µg/l in ground water, up to 1 µg/l in surface water, and up to 1 000 µg/l in waste water.
© ISO 2005 – All rights reserved v
---------------------- Page: 6 ----------------------
INTERNATIONAL STANDARD ISO 7981-2:2005(E)
Water quality — Determination of polycyclic aromatic
hydrocarbons (PAH) —
Part 2:
Determination of six PAH by high-performance liquid
chromatography with fluorescence detection after liquid-liquid
extraction
WARNING — Some compounds 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, mineral and table waters
and ground and surface waters in mass concentrations above 0,005 µg/l, by high-performance liquid
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).
With some modification, this method is also applicable for the analysis of moderately polluted waste waters.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 8466-1, Water quality — Calibration and evaluation of analytical methods and estimation of performance
characteristics — Part 1: Statistical evaluation of the linear calibration function
3 Principle
Since PAH can to a large extent be adsorbed on particulate matter, the whole sample is analysed.
NOTE For the analysis of surface water, a differentiation between dissolved and undissolved PAH can be desirable,
but this is not relevant for drinking water.
© ISO 2005 – All rights reserved 1
---------------------- Page: 7 ----------------------
ISO 7981-2:2005(E)
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 on silica (8.4) prior to
analysis.
PAH are then separated by high performance liquid chromatography (HPLC) on suitable stationary phases
under isocratic conditions, identified and quantified by means of fluorescence detection at a constant
combination of excitation and emission wavelengths.
Table 1 — Polycyclic aromatic hydrocarbons determinable by this method
Chemical Carbon
Name Molar mass CAS-number Structure
formula fraction
g/mol
Fluoranthene C H 202,26 95,0 206-44-0
16 10
Benzo[b]fluoranthene C H 252,32 95,2 205-99-2
20 12
Benzo[a]pyrene C H 252,32 95,2 50-32-8
20 12
Benzo[k]fluoranthene C H 252,32 95,2 207-08-9
20 12
Indeno[1,2,3-cd]pyrene C H 276,34 95,6 193-39-5
22 12
Benzo[ghi]perylene C H 276,34 95,6 191-24-2
22 12
4 Interferences
4.1 Interferences with sampling and extraction
Use sampling containers made of materials (preferably of glass or steel) that do not affect the sample during
the contact time. Avoid plastics and other organic materials during sampling, sample storage or extraction.
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.
2 © ISO 2005 – All rights reserved
---------------------- Page: 8 ----------------------
ISO 7981-2:2005(E)
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.
4.2 Interferences with the HPLC
Substances that show 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 accuracy and precision of the analytical results. Peak overlaps will
prevent the measurement of peak height and/or area. Unsymmetrical peaks and peaks broader than the
respective peaks of the reference substance suggest interferences.
5 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 12).
5.1 Solvents
5.1.1 Solvents for extraction and clean-up of the extract
5.1.1.1 Cyclohexane, C H
6 12
5.1.1.2 Hexane, C H
6 14
5.1.1.3 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.
5.1.2 HPLC solvents
5.1.2.1 Methanol, CH OH
3
5.1.2.2 Acetonitrile, CH CN
3
5.1.2.3 Tetrahydrofuran, C H O, without stabilizer
4 8
NOTE Tetrahydrofuran can contain peroxides. Although peroxides have not yet shown to cause any interference with
the HPLC determination, it is preferred to use batches with low peroxide content (regularly checked using test rods). It is of
advantage to use small packages.
5.2 Sodium thiosulfate pentahydrate, Na S O ·5H O
2 2 3 2
5.3 Sodium chloride, NaCl
5.4 Sodium sulfate, Na SO , anhydrous, precleaned by heating to 500 °C.
2 4
5.5 Nitrogen, having a purity (volume fraction) of at least 99,999 %.
© ISO 2005 – All rights reserved 3
---------------------- Page: 9 ----------------------
ISO 7981-2:2005(E)
5.6 Helium, having a purity (volume fraction) of at least 99,999 %.
5.7 Silica, with an average particle size approximately 40 µm and stored in a desiccator to ensure
maximum activity.
NOTE Prepacked silica cartridges are commercially available.
5.8 Molecular sieve beads, pore size 0,4 nm.
5.9 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.
5.10 Single-substance stock solutions, of those listed in Table 1, diluted in acetonitrile (5.1.2.2) to a mass
concentration of, for example, 10 µg/ml.
5.11 Multiple-substance stock solution, preferably certified, diluted in acetonitrile (5.1.2.2) to a mass
concentration of, for example, 10 µg/ml for each individual compound.
5.12 Calibration solutions
Prepare at least five calibration solutions by appropriate dilution of the stock solution (5.11), using methanol
(5.1.2.1) or acetonitrile (5.1.2.2) as solvent. The choice of solvent depends on the composition of the mobile
phase.
For example, using 50 µl of the stock solution (5.11) in a graduated 10 ml flask (6.16), make up to volume with
acetonitrile (5.1.2.2) or methanol (5.1.2.1). 1 µl of this reference solution contains 50 pg of the respective
individual substance.
NOTE The solutions 5.10 to 5.12 are stable for at least one year when stored in the dark at room temperature and
protected from evaporation.
6 Apparatus
Standard laboratory equipment cleaned to eliminate all interferences.
Clean all glassware, for example, by rinsing with detergent and hot water, and drying 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.
6.1 Brown glass bottles, narrow-necked, flat-bottomed, nominal capacity 1 000 ml, with solid glass
stopper.
6.2 Magnetic stirrer with stirring rods, PTFE coated, kept under cyclohexane, with a maximal rotational
–1
frequency of 1 000 min .
6.3 Measuring cylinder, nominal capacity 10 ml, 25 ml and 1 000 ml.
6.4 Separating funnel, nominal capacity 1 000 ml, with PTFE stopcock, kept under cyclohexane, and glass
stopper, e.g. a Squibb funnel.
6.5 Conical flask, nominal capacity 100 ml, with glass stopper.
6.6 Reduction flask, nominal capacity 100 ml (see Figure B.1).
4 © ISO 2005 – All rights reserved
---------------------- Page: 10 ----------------------
ISO 7981-2:2005(E)
–1
6.7 Centrifuge with rotor, with a rotational frequency of about 3 000 min and with centrifuge tubes with
tapered bottom, nominal capacity 50 ml (see Figure B.2).
6.8 Pasteur pipettes
6.9 Evaporation assembly, such as a rotary evaporator with vacuum stabilizer and water bath.
6.10 Shaking apparatus, with adjustable rotational speed, suitable for test tubes.
6.11 Blow-down assembly, nitrogen pressure cylinder with pressure-reducing valve and needle valve for
fine adjustment.
6.12 Microfilter, with solvent-resistant membrane, pore size 0,45 µm.
6.13 Autosampler vials, capacity approximately 2 ml, with inert filler cap, e.g. PTFE coated septum.
6.14 Polypropene or glass cartridges, filled with at least 0,5 g silica (5.7).
6.15 Glass vials, e.g. centrifuge tubes, nominal capacity 10 ml, with glass stoppers.
6.16 Graduated flasks, nominal capacities 10 ml, 100 ml and 250 ml.
6.17 High-performance liquid chromatograph, with fluorescence detector and data evaluation system,
including:
degassing assembly, e.g. for degassing with vacuum or helium;
low pulsating analytical pump;
manual or automatic sample applicator;
column thermostat, capable of keeping the temperature constant to within ± 0,5 °C;
fluorescence detector, preferably equipped with a monochromator on either the excitation and
emission sides, or with a filter (8.5.2);
analytical separation column, e.g. a column with length up to 250 mm, internal diameter 2 mm to
4,6 mm, packed with particle size 3 µm to 5 µm material, capable of near baseline separation (at
least as good as in Figure A.1) of the PAH to a large extent.
7 Sampling
When sampling drinking water from a tap of the water supply, collect the test sample before the tap is
sterilized for bacteriological sampling.
Plastics materials – with the exception of polytetrafluorethene (PTFE) – should not be used during sampling
and sample treatment, as losses can occur due to adsorption of PAH on the material. Take care during
handling of the test samples to keep them from direct sunlight, as PAH can decompose.
Collect the test sample in brown glass bottles (6.1) of known mass. Dechlorinate water samples containing
chlorine by immediately adding approximately 50 mg of sodium thiosulfate (5.2).
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. When the complete analysis cannot be performed within 24 h, the
following procedure shall be performed within this time limit. If necessary remove a part of the sample from the
sampling bottle until a sample volume of about 1 000 ml ± 10 ml remains, and determine the volume
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.