SIST EN ISO 17993:2004

Water quality - Determination of 15 polycyclic aromatic hydrocarbons (PAH) in water by HPLC with fluorescence detection after liquid-liquid extraction (ISO 17993:2002)

With a scope of describing a manual method of making measurements for the determination of volume fraction of major phases in advanced technical ceramics using micrographs of polished and etched sections, overlaying a square grid of lines, and counting the number of intersection lying other each phase.

Wasserbeschaffenheit - Bestimmung von 15 polycyclischen aromatischen Kohlenwasserstoffen (PAK) in Wasser durch HPLC mit Fluoreszenzdetektion nach Flüssig-Flüssig-Extraktion (ISO 17993:2002)

Diese Internationale Norm legt ein Verfahren zur Bestimmung von 15 ausgewählten PAK (siehe Tabelle 1) mit Hilfe der hochauflösenden Dampfraumnanalyse (HPLC) nach flüssig-flüssig-Extraktion und Fluoreszenzs-pektrometrie fest. Das Verfahren wird angewendet auf Trink- und Grundwasser in Massenkonzentrationen oberhalb 0,005 µg/l (Einzelsubstanz) und auf Oberflächenwasser in Massenkonzentrationen oberhalb 0,01 µg/l fest.
Das Verfahren ist nach Anpassung auch geeignet für die Untersuchung von Abwasser. Das Verfahren darf auch auf andere PAK angewendet werden, vorausgesetzt, es wird in jedem Einzelfall validiert.

Qualité de l'eau - Dosage de 15 hydrocarbures aromatiques polycycliques (HAP) dans l'eau par HPLC avec détection par fluorescence apres extraction liquide-liquide (ISO 17993:2002)

La présente Norme internationale spécifie une méthode pour le dosage, par chromatographie en phase liquide à haute performance (HPLC) avec détection par fluorescence, de 15 HAP sélectionnés (voir Tableau 1) dans l'eau potable et dans les eaux souterraines à des concentrations en masse supérieures à 0,005 microgrammes par litre (pour chaque composé individuel), et dans les eaux de surface à des concentrations en masse supérieures à 0,01 microgrammes par litre.
Cette méthode est également appropriée, avec certaines modifications, pour l'analyse des eaux usées. Elle peut être appliquée à d'autres HAP, sous réserve de valider la méthode dans chaque cas.

Kakovost vode – Določevanje 15 policikličnih aromatskih ogljikovodikov (PAH) v vodi s tehniko HPLC s fluorescenčno detekcijo po ekstrakciji tekoče-tekoče (ISO 17993:2002)

General Information

• Status: Published
• Publication Date: 31-Jan-2004
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: KAV - Water quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 01-Feb-2004
• Due Date: 01-Feb-2004
• Completion Date: 01-Feb-2004
• Directive: 01/990 - Proposal for a EP and Council Decision establishing the list of priority substances in the field of water policy COM(2000)47 New version of 01/990: PE-CONS 3645/01 (2001-09-04) : PE and Council Decision on List of Priority Substances for WFD
• Directive: 2000/60/EC - Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy (substitutes Directive Reference 01/991 - Common Position on WFD)
• Directive: 98/83/EC - Quality of water intended for human consumption
• Directive: TP128 - Pravilnik o prvih meritvah in obratovalnem monitoringu odpadnih voda
• Directive: TRRTP064 - Rules on initial measurements and operational monitoring of wastewater
• Ref Project: EN ISO 17993:2003 - Water quality - Determination of 15 polycyclic aromatic hydrocarbons (PAH) in water by HPLC with fluorescence detection after liquid-liquid extraction (ISO 17993:2002)

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 17993:2004
01-februar-2004
.DNRYRVWYRGH±'RORþHYDQMHSROLFLNOLþQLKDURPDWVNLKRJOMLNRYRGLNRY3$+Y
YRGLVWHKQLNR+3/&VIOXRUHVFHQþQRGHWHNFLMRSRHNVWUDNFLMLWHNRþHWHNRþH,62

Water quality - Determination of 15 polycyclic aromatic hydrocarbons (PAH) in water by
HPLC with fluorescence detection after liquid-liquid extraction (ISO 17993:2002)
Wasserbeschaffenheit - Bestimmung von 15 polycyclischen aromatischen
Kohlenwasserstoffen (PAK) in Wasser durch HPLC mit Fluoreszenzdetektion nach
Flüssig-Flüssig-Extraktion (ISO 17993:2002)
Qualité de l'eau - Dosage de 15 hydrocarbures aromatiques polycycliques (HAP) dans
l'eau par HPLC avec détection par fluorescence apres extraction liquide-liquide (ISO
17993:2002)
Ta slovenski standard je istoveten z: EN ISO 17993:2003
ICS:
13.060.50 3UHLVNDYDYRGHQDNHPLþQH Examination of water for
VQRYL chemical substances
SIST EN ISO 17993:2004 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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EUROPEAN STANDARD
EN ISO 17993
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2003
ICS 13.060.50
English version
Water quality - Determination of 15 polycyclic aromatic
hydrocarbons (PAH) in water by HPLC with fluorescence
detection after liquid-liquid extraction (ISO 17993:2002)
Qualité de l'eau - Dosage de 15 hydrocarbures aromatiques Wasserbeschaffenheit - Bestimmung von 15 polycyclischen
polycycliques (HAP) dans l'eau par HPLC avec détection aromatischen Kohlenwasserstoffen (PAK) in Wasser durch
par fluorescence après extraction liquide-liquide (ISO HPLC mit Fluoreszenzdetektion nach Flüssig-Flüssig-
17993:2002) Extraktion (ISO 17993:2002)
This European Standard was approved by CEN on 3 November 2003.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2003 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17993:2003 E
worldwide for CEN national Members.

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EN ISO 17993:2003 (E)
Foreword
The text of ISO 17993:2002 has been prepared by Technical Committee ISO/TC 147 "Water
quality” of the International Organization for Standardization (ISO) and has been taken over as
EN ISO 17993:2003 by Technical Committee CEN/TC 230 "Water analysis", the secretariat of
which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of
an identical text or by endorsement, at the latest by May 2004, and conflicting national standards
shall be withdrawn at the latest by May 2004.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium, Czech
Republic, Denmark, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and
the United Kingdom.
Endorsement notice
The text of ISO 17993:2002 has been approved by CEN as EN ISO 17993:2003 without any
modifications.
NOTE Normative references to International Standards are listed in Annex ZA (normative).
2

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EN ISO 17993:2003 (E)
Annex ZA
(normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions of
any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications, indicated
by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 5667-2 1991 Water quality - Sampling - Part 2: EN 25667-2 1993
Guidance on sampling techniques
ISO 5667-3 1994 Water quality - Sampling - Part 3: EN ISO 5667-3 1995
Guidance on the preservation and
handling of samples
3

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INTERNATIONAL ISO
STANDARD 17993
First edition
2002-08-15


Water quality — Determination of 15
polycyclic aromatic hydrocarbons (PAH) in
water by HPLC with fluorescence detection
after liquid-liquid extraction
Qualité de l'eau — Dosage de 15 hydrocarbures aromatiques polycycliques
(HAP) dans l'eau par HPLC avec détection par fluorescence après
extraction liquide-liquide




Reference number
ISO 17993:2002(E)
©
 ISO 2002

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ISO 17993:2002(E)
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ii © ISO 2002 – All rights reserved

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ISO 17993:2002(E)
Contents Page
Foreword . iv
Introduction. v
1 Scope. 1
2 Normative references. 1
3 Principle . 1
4 Interferences. 3
5 Reagents . 4
6 Apparatus. 5
7 Sampling . 6
8 Procedure. 6
9 Calculation . 11
10 Precision . 11
11 Test report. 13
Annex A (informative) Examples of chromatographic conditions and columns . 14
Annex B (informative) Examples for the construction of special apparatus. 18


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ISO 17993:2002(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 3.
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 International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 17993 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
Annexes A and B of this International Standard are for information only.
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ISO 17993:2002(E)
Introduction
Polycyclic aromatic hydrocarbons (PAH) occur in nearly all types of waters. These compounds 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 Council Directive 98/83/EC on the quality of water
intended for human consumption set the maximum acceptable level for benzo(a)pyrene at 0,010 µg/l, and for the
sum of four specified PAH [benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(ghi)perylene, indeno(1,2,3-cd)-
pyrene] at 0,100 µg/l.

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INTERNATIONAL STANDARD ISO 17993:2002(E)

Water quality — Determination of 15 polycyclic aromatic
hydrocarbons (PAH) in water by HPLC with fluorescence detection
after liquid-liquid extraction
WARNING — Some compounds being measured are presumed to be carcinogenic. Acetonitrile and hexane
are toxic.
Persons using this International Standard should be familiar with normal laboratory practice. This
International Standard does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure
compliance with any national regulatory conditions.
1 Scope
This International Standard specifies a method using high performance liquid chromatography (HPLC) with
fluorescence detection after liquid-liquid extraction for the determination of 15 selected PAH (see Table 1) in
drinking and ground water in mass concentrations greater than 0,005 µg/l (for each single compound) and surface
waters in mass concentrations above 0,01 µg/l.
This method is, with some modification, also suitable for the analysis of wastewater. This method may be
applicable to other PAH, provided the method is validated for each case.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 5667-2, Water quality — Sampling — Part 2: Guidance on sampling techniques
ISO 5667-3, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples
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
The PAH present in the aqueous sample are extracted from the water sample with hexane. The extract is
concentrated by evaporation and the residue taken up in a solvent appropriate for HPLC analysis.
If necessary, extracts of surface water or more contaminated water samples are cleaned by chromatography over
silica prior to analysis.
PAH are separated by HPLC on a suitable stationary phase using gradient elution. Identification and quantification
is performed by means of fluorescence detection with wavelength programming for both the excitation and the
emission wavelength.
NOTE If only a limited number of PAH are to be determined, separation can also be performed under isocratic conditions.
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ISO 17993:2002(E)
Table 1 — Polycyclic aromatic hydrocarbons determinable by this method
Chemical Percentage
Name Molar mass CAS-number Structure
formula carbon
C H
Naphthalene 128,17 g/mol 93,75 % C 091-20-3
10 8

C H
Acenaphthene 154,21 g/mol 93,05 % C 083-32-9
12 10

C H
Phenanthrene 178,23 g/mol 94,05 % C 085-01-8
14 10

C H
Fluoranthene 202,26 g/mol 95,0 % C 206-44-0
16 10

C H
Benzo(a)anthracene 228,29 g/mol 94,45 % C 056-55-3
18 12

a
C H
252,32 g/mol 95,2 % C 205-99-2
Benzo(b)fluoranthene
20 12

a
C H
Benzo(a)pyrene 252,32 g/mol 95,2 % C 050-32-8
20 12

C H
Dibenzo(a,h)anthracene 278,35 g/mol 94,7 % C 053-70-3
22 14
C H
Fluorene 166,22 g/mol 93,59 % C 086-73-7
13 10

C H
Anthracene 178,23 g/mol 94,05 % C 120-12-7
14 10

C H
Pyrene 202,26 g/mol 95,0 % C 129-00-0
16 10

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ISO 17993:2002(E)
Table 1 (continued)
Chemical Percentage
Name Molar mass CAS-number Structure
formula carbon
C H
Chrysene 228,29 g/mol 94,45 % C 218-01-9
18 12

a
C H
252,32 g/mol 95,2 % C 207-08-9
Benzo(k)fluoranthene
20 12

a
C H
276,34 g/mol 95,6 % C 193-39-5
Indeno(1,2,3-cd)pyrene
22 12

a
C H
Benzo(ghi)perylene 276,34 g/mol 95,6 % C 191-24-2
22 12

NOTE The 15 PAH selected for determination by this method correspond to those of the US EPA list with the exception of
acenaphthylene. Acenaphthylene cannot be determined by this method because it is not fluorescent.
a
Compounds specified in the Council Directive 98/83/EC.

4 Interferences
4.1 Sampling and extraction
Use sampling containers of materials (preferably of steel or glass) 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 these materials are
present, make sure that they are as short as possible. Rinse the sampling line with the water to be sampled before
taking the test sample. Refer to ISO 5667-2 and ISO 5667-3 for guidance.
Keep the test samples from direct sunlight and prolonged exposure to light.
During storage of the test samples, losses of PAH may occur due to adsorption on the walls of the containers. The
extent of the losses depends on the storage time.
4.2 HPLC
Compounds that show either fluorescence or quenching and co-elute with the analyte PAH may interfere with the
determination. These interferences may lead to incompletely resolved signals resulting in peak overlap and may,
depending on their magnitude, affect accuracy and precision of the analytical results. Unsymmetrical peaks and
peaks being broader than the corresponding peaks of the reference compound suggest interferences. This problem
may arise for naphthalene and phenanthrene depending on the selectivity of the phases used.
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ISO 17993:2002(E)
Incomplete removal of the solvents used for sample pretreatment (hexane, acetone, dichloromethane) may lead to
poor reproducibility of the retention times and peak broadening or double peaks especially for the 2- and 3-ring
PAH.
Separation between dibenzo(ah)anthracene and indeno(1,2,3-cd)pyrene can be critical. When incomplete
resolution is encountered, peak integration shall be checked and, when necessary, corrected.
Usually perylene is incompletely resolved from benzo(b)fluoranthene, but by choosing a selective wavelength (see
Table A.1) the perylene peak can be suppressed.
As perylene can be detected under the conditions given in the isocratic method for the PAH, which are relevant for
drinking water (see Figure A.3), it should be included in the calibration step.
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 fluorescence possible.
Monitor the blank to guarantee that the reagents do not contain PAH in detectable concentrations (see 8.9).
5.1 Solvents.
5.1.1 Extraction solvents, as follows:
 hexane, C H ;
6 14
 other volatile solvents may be used as well, if it is proved that there is equivalent or better recovery.
5.1.2 Extraction clean-up solvents, as follows:
 dichloromethane, CH Cl (see note);
2 2
 hexane, C H ;
6 14
 N,N-dimethylformamide, (CH ) NCHO;
3 2
 acetone, C H O.
3 6
NOTE Dichloromethane often contains stabilizers, e.g. ethanol or amylene, which may influence the elution strength of the
eluent. Without stabilizer, radicals may develop which may lead to degradation of PAH. The presence of hydrogen chloride
indicates radicals. It can be determined by extracting dichloromethane with water and measuring the pH value.
5.1.3 HPLC solvents, as follows:
 acetonitrile, CH CN;
3
 methanol, CH OH.
3
5.2 Sodium thiosulfate pentahydrate, Na S O ·5H O.
2 2 3 2
5.3 Sodium sulfate, Na SO , anhydrous, precleaned by heating to 500 °C.
2 4
5.4 Nitrogen, having a volume fraction of at minimum 99,999 %.
5.5 Silica, having an average particle size of approximately 40 µm and stored in a desiccator to ensure
maximum activity.
NOTE Prepacked silica cartridges are commercially available.
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ISO 17993:2002(E)
5.6 Molecular sieve beads, having a pore diameter of 0,4 nm and having been completely activated.
5.7 Reference compounds, listed in Table 1.
Because of the dangerous nature of these compounds, it is highly recommended to use commercially available,
preferably certified, standard solutions. Avoid skin contact.
5.8 Stock solutions.
The solutions 5.8.1 and 5.8.2 are stable for at least a year when stored in the dark at room temperature and
protected from evaporation.
5.8.1 Single compound stock solutions, of those listed in Table 1, diluted in acetonitrile (5.1.3) to a mass
concentration of, for example, 10 µg/ml.
These solutions are used for confirmation and identification of single PAH in the chromatogram.
5.8.2 Multiple compound stock solution, certified, diluted in acetonitrile (5.1.3) to a mass concentration of, for
example, 10 µg/ml for each individual compound.
5.9 Reference solutions.
Prepare at least five calibration solutions by appropriate dilution of the stock solution (5.8.2), using methanol (5.1.3)
or acetonitrile (5.1.3) as the solvent. The choice of the solvent depends on the composition of the HPLC mobile
phase.
Transfer, for example, 50 µl of the stock solution into a 5 ml volumetric flask and make up to the mark with
acetonitrile. One microlitre of this reference solution contains 100 pg of the respective individual compounds.
These solutions remain stable for at least a year when stored in the dark at room temperature and protected from
evaporation. To ensure their stability, run a quality control check regularly on the reference solutions.
Checking the mass concentration of the PAH in the stock solution is only possible by comparison with an
independent, preferably certified, standard solution.
6 Apparatus
Standard laboratory glassware cleaned to eliminate all interferences. All glassware can be cleaned, 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.
Do not use glassware that has been in contact with wastewater samples or samples with high PAH concentrations
for drinking water analysis.
6.1 Brown glass bottles, narrow-necked, flat-bottomed, 1 000 ml, with glass stopper, preferably of known
mass.
6.2 Magnetic stirrer, with stirring bars, glass or polytetrafluoroethene (PTFE) coated, for stirring the solvent
used for extraction.
6.3 Separating funnel, of 1 000 ml capacity, with PTFE stopcock and glass stopper.
6.4 Conical flasks, of 100 ml and 250 ml capacity, with glass stopper.
6.5 Microlitre syringes, of 500 µl and 1 000 µl capacity.
6.6 Reduction flask, of 100 ml capacity (see Figure B.1).
6.7 Centrifuge with rotor, for centrifuge tubes with tapered bottoms of 50 ml capacity (see Figure B.2).
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ISO 17993:2002(E)
6.8 Pasteur pipettes.
6.9 Evaporation assembly, for example a rotary evaporator with a vacuum stabilizer and a water bath.
6.10 Shaking apparatus, with adjustable rotational speed.
6.11 Microfilter, with a solvent-resistant hydrophilic membrane and a pore size of 0,45 µm.
6.12 Glass autosampler vials, of approximately 2 ml capacity, with an inert cap, e.g. PTFE coated septum.
6.13 Polypropene or glass cartridges, filled with at least 0,5 g of silica (see 5.5).
NOTE These cartridges are commercially available.
6.14 Glass vials, e.g. centrifuge tubes, graduated (scale division 0,1 ml), nominal capacity 10 ml, with glass
stoppers.
6.15 High performance liquid chromatograph (HPLC), with fluorescence detector and data evaluation system,
including:
6.15.1 Degassing assembly, e.g. for degassing with vacuum or helium.
6.15.2 Analytical pumps, capable of binary gradient elution.
6.15.3 Column thermostat, capable of keeping the temperature constant to within ± 0,5 °C.
6.15.4 Fluorescence detector, capable of programming at least six pairs of wavelengths, including
damping/amplification, preferably equipped with monochromator(s).
6.16 Analytical separation column, meeting the separation requirements given in 8.5.2 (for examples see
annex A).
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.
Collect the test sample in a brown glass bottle (6.1). Dechlorinate water test 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 protect it 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, perform the following
procedure within this time limit. 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 test sample by weighing the bottle, add 25 ml of
hexane (5.1.2) and shake well. The pretreated test sample may be stored for 72 h at about + 4 °C, protected from
light.
8 Procedure
8.1 Extraction
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 25 ml of hexane (5.1.2) and mix. Other volatile solvents may be used for extraction,
provided they give equal or better recovery.
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ISO 17993:2002(E)
Add a stirring bar and put the lid on the bottle. Then thoroughly mix the test sample using the magnetic stirrer (6.2)
−1
at maximum setting (1 000 min ) for 60 min. Transfer the test sample to a separating funnel (6.3) and allow the
phases to separate for at least 5 min. The separation of hexane from water can also be carried out using a
microseparator (an example is given in Figure B.3).
If a stable emulsion forms during the extraction process, collect it in a centrifuge tube (6.14) and centrifuge it for
−1
10 min at about 3 000 min . Remove the separated water with a Pasteur pipette (6.8), transfer the extract to a
100 ml conical flask (6.4). Dry extract according to 8.2.
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 are transferred to a 250 ml conical flask (6.4) with a pipette and diluted with water to
200 ml. After adding 25 ml of hexane (5.1.2) proceed as described above.
8.2 Drying of the extract
Transfer the hexane layer obtained according to 8.1 into a 100 ml conical flask (6.4). Rinse the funnel or centrifuge
tube with 5 ml of hexane (5.1.2) and add it to the total extract.
Dry the extract with sodium sulfate (5.3) for at least 30 min. Swirl the vessel frequently.
Decant the dry extract into a reduction flask (6.6). Rinse the conical flask twice with 5 ml of hexane (5.1.2) and add
to the same reduction flask.
8.3 Enrichment
Evaporate the dried hexane extract obtained according to 8.2 until it fills only the tapered tip of the reduction flask
(approximately 2 ml), with e.g. a rotary evaporator (6.9), with a bath temperature of 30 °C and slowly decreasing
the pressure to 200 hPa.
Do not evaporate the extracts to dryness, as losses of the 2- or 3-ring compounds may occur.
Dissolve any residues that may be deposited on the glass wall by shaking the extract using the shaking apparatus
(6.10).
Clean extracts of wastewater samples and other samples of unknown origin using the method given in 8.4.
Add 250 µl of N,N-dimethylformamide (5.1.2) to the concentrated extract and homogenize the mixture with 500 µl
of acetone (5.1.2). Dichloromethane (5.1.2) may also be used instead of acetone for homogenization after the
addition of dimethylformamide.
Remove the hexane and the acetone completely by a gentle stream of nitrogen (5.4), so as to reduce the volume of
the extract to between 200 µl and 250 µl. The enriched extract should not contain residues of hexane or acetone,
because the presence of these solvents in the measuring solution leads to interferences with the HPLC (see 4.2).
Dilute the extract to a known volume (e.g. 2 ml) with the same solvent that has been used for the preparation of the
reference solutions (5.9). The volume fraction of dimethylformamide should not exceed 20 % in order to avoid peak
broadening in the chromatogram.
Transfer the enriched test sample, if necessary after filtration through a microfilter (6.11), into a glass sample vial
(6.12). Keep the extract in a cool and dark place until the analysis is carried out.
8.4 Clean-up of the extract
To clean the extract, use columns [Pasteur pipettes (6.8)] or cartridges (6.13) containing at least 0,5 g of silica
(5.5). Wash the silica in the column or in the cartrid
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