oSIST prEN 17503:2020

Environmental solid matrices - Determination of polycyclic aromatic hydrocarbons (PAH) by gas chromatography (GC) and high performance liquid chromatography (HPLC)

8. Scope
This European Standard specifies a method for quantitative determination of 16 polycyclic aromatic hydrocarbons (PAH) (see Table ) in soil, sludge, sediment, treated biowaste, and waste, using GC-MS and HPLC-UV-DAD/FLD covering a wide range of PAH contamination levels (see Table 2).
When using fluorescence detection, acenaphthylene cannot be measured.
Table  —Target analytes of this European Standard
Target analyte   CAS-RNa
Naphthalene   91–20–3
Acenaphthene   83–32–9
Acenaphthylene   208–96–8
Fluorene   86–73–7
Anthracene   120–12–7
Phenanthrene   85–01–8
Fluoranthene   206–44–0
Pyrene   129–00–0
Benz[a]anthracene   56–55–3
Chrysene   218–01–9
Benzo[b]fluoranthene   205–99–2
Benzo[k]fluoranthene   207–08–9
Benzo[a]pyrene   50–32–8
Indeno[1,2,3-cd]pyrene   193–39–5
Dibenz[a,h]anthracene   53–70–3
Benzo[ghi]perylene   191–24–2
a   CAS-RN Chemical Abstracts Service Registry Number.
The limit of detection depends on the determinants, the equipment used, the quality of chemicals used for the extraction of the sample and the clean-up of the extract
Under the conditions specified in this European Standard, lower limit of application from 10 μg/kg (expressed as dry matter) for soils, sludge and biowaste to 100 μg/kg (expressed as dry matter) for solid waste can be achieved. For some specific samples (e.g. bitumen) the limit of 100 μg/kg cannot be reached.
Sludge, waste and treated biowaste may differ in properties as well as in the expected contamination levels of PAH and presence of interfering substances. These differences make it impossible to describe one general procedure. This European Standard contains decision tables based on the properties of the sample and the extraction and clean-up procedure to be used.
The method may be applied to the analysis of other PAH not specified in the scope, provided suitability is proven by proper in-house validation experiments.

Feststoffe in der Umwelt - Bestimmung von polycyclischen aromatischen Kohlenwasserstoffen (PAK) mittels Gaschromatographie (GC) und Hochleistungs-Flüssigkeitschromatographie (HPLC)

Dieses Dokument legt verschiedene Verfahren für die quantitative Bestimmung von 16 polycyclischen aromatischen Kohlenwasserstoffen (PAK) (siehe Tabelle 2) in Boden, Schlamm, Sediment, behandeltem Bioabfall und Abfall mittels GC-MS oder HPLC-UV-DAD/FLD fest, wobei ein breites Spektrum an PAK-Kontaminationsniveaus (siehe Tabelle 2) abgedeckt wird.
Bei Anwendung der Fluoreszenzdetektion kann Acenaphthylen nicht gemessen werden.
(tabelle 2)
Die Nachweisgrenze hängt von den zu bestimmenden Substanzen, der verwendeten technischen Ausrüstung, der Qualität der für die Extraktion der Probe verwendeten Chemikalien und der Reinigung des Extrakts ab.
Unter den in diesem Dokument festgelegten Bedingungen kann eine untere Anwendungsgrenze zwischen 10 μg/kg (angegeben als Trockenmasse) für Böden, Schlamm und Bioabfall und 100 μg/kg (angegeben als Trockenmasse) für Feststoffabfall erreicht werden. Bei einigen bestimmten Proben (z. B. Bitumen) kann die Grenze von 100 μg/kg nicht erreicht werden.
Schlamm, Abfall und behandelter Bioabfall können sich in Bezug auf ihre Eigenschaften sowie auf die erwarteten PAK-Kontaminationsniveaus und das Vorhandensein von Störsubstanzen unterscheiden. Aufgrund dieser Unterschiede ist es unmöglich, ein allgemeines Verfahren darzulegen. Dieses Dokument enthält Entscheidungstabellen, die auf den Eigenschaften der Probe und den anzuwendenden Extraktions- und Reinigungsverfahren basieren.
Das Verfahren kann für die Analyse anderer PAK angewendet werden, die im Anwendungsbereich nicht genannt sind, sofern die Eignung des Verfahrens durch ordnungsgemäße interne Validierungsuntersuchungen bewiesen wurde.

Matrices solides environnementales - Dosage des hydrocarbures aromatiques polycycliques (HAP) par chromatographie en phase gazeuse et chromatographie liquide à haute performance

Le présent document spécifie différentes méthodes de dosage de 16 hydrocarbures aromatiques polycycliques (HAP) (voir Tableau 2) dans les sols, les boues, les sédiments, les biodéchets traités et les déchets faisant appel à des méthodes CG-SM ou HPLC-UV-DAD/FLD couvrant une large gamme de niveaux de contamination par les HAP (voir Tableau 2).
L’utilisation d’une méthode de détection fluorimétrique ne permet pas le mesurage de l’acénaphtylène.
(tableau 2)
La limite de détection dépend des composés à analyser, de l’équipement utilisé, de la qualité des réactifs chimiques utilisés pour l’extraction de l’échantillon et la purification de l’extrait.
Dans les conditions spécifiées dans le présent document, une limite inférieure d’application allant de 10 μg/kg (exprimée par rapport à la matière sèche) pour les sols, boues et biodéchets jusqu’à 100 μg/kg (exprimée par rapport à la matière sèche) pour les déchets solides peut être atteinte. Pour certains échantillons particuliers (bitume, par exemple), la limite de 100 μg/kg ne peut pas être atteinte.
Les boues, les déchets et les biodéchets traités peuvent présenter des propriétés, des niveaux de contamination différents par les HAP attendus et des substances interférentes. En raison de ces différences, il est impossible de décrire un mode opératoire général. Le présent document contient des tables de décision basées sur les propriétés de l’échantillon ainsi que sur le mode opératoire d’extraction et de purification à utiliser.
La méthode peut être appliquée à l’analyse d’autres HAP non spécifiés dans le domaine d’application, à condition que son adéquation ait été prouvée par des expériences de validation interne.

Trdni matriksi z vidika okolja - Določevanje policikličnih aromatskih ogljikovodikov (PAH) s plinsko kromatografijo (GC) in s tekočinsko kromatografijo visoke ločljivosti (HPLC)

General Information

• Status: Not Published
• Public Enquiry End Date: 31-May-2020
• ICS: 13.030.01 - Wastes in general
• ICS: 13.080.10 - Chemical characteristics of soils
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: KAT - Soil quality
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 05-Mar-2020
• Due Date: 23-Jul-2020
• Completion Date: 08-Jun-2020
• Ref Project: prEN 17503 - Environmental solid matrices – Determination of polycyclic aromatic hydrocarbons (PAH) by gas chromatography (GC) and high performance liquid chromatography (HPLC)

RELATIONS

• Revised: SIST EN 15527:2009 - Characterization of waste - Determination of polycyclic aromatic hydrocarbons (PAH) in waste using gas chromatography mass spectrometry (GC/MS)
• Effective Date: 01-Apr-2020
• Revised: SIST EN 16181:2018 - Soil, treated biowaste and sludge - Determination of polycyclic aromatic hydrocarbons (PAH) by gas chromatography (GC) and high performance liquid chromatography (HPLC)
• Effective Date: 01-Apr-2020
• Revised: SIST ISO 13877:1999 - Soil quality -- Determination of polynuclear aromatic hydrocarbons -- Method using high -performance liquid chromatography
• Effective Date: 01-Apr-2020

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN 17503:2020
01-maj-2020
Trdni matriksi z vidika okolja - Določevanje policikličnih aromatskih

ogljikovodikov (PAH) s plinsko kromatografijo (GC) in s tekočinsko kromatografijo

visoke ločljivosti (HPLC)

Environmental solid matrices - Determination of polycyclic aromatic hydrocarbons (PAH)

by gas chromatography (GC) and high performance liquid chromatography (HPLC)
Umweltrelevante feste Matrizes - Bestimmung von polycyclischen aromatischen
Kohlenwasserstoffen (PAK) mittels Gaschromatograpie (GC) und Hochleistungs-
Flüssigkeitschromatographie (HPLC)

Matrices solides environnnementales - Determination des hydrocarbures aromatiques

polycycliques (HAP)par chromatographie en phase gazeuse (GC) et chromatographie
liquid a haute performance (HPLC)
Ta slovenski standard je istoveten z: prEN 17503
ICS:
13.030.01 Odpadki na splošno Wastes in general
13.080.10 Kemijske značilnosti tal Chemical characteristics of
soils
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
oSIST prEN 17503:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17503:2020
DRAFT
EUROPEAN STANDARD
prEN 17503
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2020
ICS 13.030.01; 13.080.10 Will supersede EN 15527:2008, EN 16181:2018
English Version
Environmental solid matrices - Determination of polycyclic
aromatic hydrocarbons (PAH) by gas chromatography
(GC) and high performance liquid chromatography (HPLC)

Matrices solides environnnementales ¿ Determination Umweltrelevante feste Matrizes ¿ Bestimmung von

des hydrocarbures aromatiques polycycliques polycyclischen aromatischen Kohlenwasserstoffen

(HAP)par chromatographie en phase gazeuse (GC) et (PAK) mittels Gaschromatograpie (GC) und

chromatographie liquid a haute performance (HPLC) Hochleistungs-Flüssigkeitschromatographie (HPLC)

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 444.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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 CEN-CENELEC

Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17503:2020 E

worldwide for CEN national Members.
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Contents Page

European foreword ............................................................................................................................................ 3

Introduction .......................................................................................................................................................... 4

1 Scope .......................................................................................................................................................... 5

2 Normative references .......................................................................................................................... 6

3 Terms and definitions ......................................................................................................................... 6

4 Principle ................................................................................................................................................... 8

5 Interferences .......................................................................................................................................... 8

6 Safety remarks ....................................................................................................................................... 9

7 Reagents ................................................................................................................................................... 9

8 Apparatus .............................................................................................................................................. 13

9 Sample storage and preservation ................................................................................................. 15

10 Procedure .............................................................................................................................................. 16

11 Performance characteristics........................................................................................................... 29

12 Precision ................................................................................................................................................ 29

13 Test report ............................................................................................................................................. 30

Annex A (informative) Repeatability and reproducibility data ...................................................... 31

A.1 Materials used in the interlaboratory comparison study .................................................... 31

A.2 Interlaboratory comparison results ............................................................................................ 31

Annex B (informative) Examples of instrumental conditions and chromatograms ................ 41

B.1 Measurement of PAH with GC-MS ................................................................................................. 41

B.2 Measurement of PAH with HPLC fluorescence ......................................................................... 47

B.3 Example for measurement conditions of PAH with GC-MS/MS .......................................... 53

Bibliography ....................................................................................................................................................... 55

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European foreword

This document (prEN 17503:2020) has been prepared by Technical Committee CEN/TC 444 “Test

methods for environmental characterization of solid matrices”, the secretariat of which is held by NEN.

This document is currently submitted to the CEN Enquiry.
This document will supersede EN 15527:2008 and EN 16181:2018.
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Introduction

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous because they are released in appreciable

quantities every year into the environment through the combustion of organic matters such as coal, fuel

oils, petrol, wood, refuse and plant materials. Since some of these PAH compounds are carcinogenic or

mutagenic, their presence in the environment (air, water, soil, sediment and waste) is regularly

monitored and controlled. At present determination of PAH is carried out in these matrices in most of the

routine laboratories following the preceding steps for sampling, pre-treatment, extraction, clean-up by

measurement of specific PAH by means of gas chromatography in combination with mass spectrometric

detection (GC-MS) or by high performance liquid chromatography (HPLC) in combination with UV-DAD-

or fluorescence-detection (HPLC-UV-DAD/FLD). Both the GC-MS and the HPLC methods are included in

this horizontal standard.

This document was developed by merging of EN 16181:2018 [1], initially elaborated as a CEN Technical

Specification in the European project 'HORIZONTAL' and validated by CEN/TC400 with the support of

BAM, with EN 15527 [2], published by CEN/TC292.

Considered the different matrices and possible interfering compounds, this document does not contain

one single possible way of working. Several choices are possible, in particular relating to clean-up.

Quantification with both GC-MS-detection and HPLC-DAD-UV/FLD is possible. Four different extraction

procedures are described and three clean-up procedures. The use of internal and injection standards is

described in order to have an internal check on choice of the extraction and clean-up procedure. The

method is as far as possible in agreement with the method described for PCB (EN17322). It has been

tested for ruggedness.

This document is applicable and validated for several types of matrices as indicated in Table 1 (see also

Annex A for the results of the validation).
Table 1 — Matrices for which this document is applicable and validated
Matrix Materials used for validation
Soil Sandy soil
Mix of soil from an industrial area in Brandenburg, Germany and
PCB-free German reference soil
Sludge Mix of municipal waste water treatment plant sludge from the
vicinity of Berlin, Germany
Biowaste Mix of compost from the vicinity of Berlin, Germany
Contaminated soil, building debris, waste wood, roofing tar,
Waste
shredder light fraction

WARNING — Persons using this document should be familiar with usual laboratory practice. This

document 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.

WARNING — It is absolutely essential that tests conducted according to this document be carried out by

suitably trained staff.
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1 Scope

This document specifies different methods for quantitative determination of 16 polycyclic aromatic

hydrocarbons (PAH) (see Table 2) in soil, sludge, sediment, treated biowaste, and waste, using GC-MS or

HPLC-UV-DAD/FLD covering a wide range of PAH contamination levels (see Table 2).
When using fluorescence detection, acenaphthylene cannot be measured.
Table 2 —Target analytes of this document
Target analyte a
CAS-RN
Naphthalene 91–20–3
Acenaphthene 83–32–9
Acenaphthylene 208–96–8
Fluorene 86–73–7
Anthracene 120–12–7
Phenanthrene 85–01–8
Fluoranthene 206–44–0
Pyrene 129–00–0
Benz[a]anthracene 56–55–3
Chrysene 218–01–9
Benzo[b]fluoranthene 205–99–2
Benzo[k]fluoranthene 207–08–9
Benzo[a]pyrene 50–32–8
Indeno[1,2,3-cd]pyrene 193–39–5
Dibenz[a,h]anthracene 53–70–3
Benzo[ghi]perylene 191–24–2
CAS-RN Chemical Abstracts Service Registry Number.

The limit of detection depends on the determinants, the equipment used, the quality of chemicals used

for the extraction of the sample and the clean-up of the extract.

Under the conditions specified in this document, lower limit of application from 10 μg/kg (expressed as

dry matter) for soils, sludge and biowaste to 100 μg/kg (expressed as dry matter) for solid waste can be

achieved. For some specific samples (e.g. bitumen) the limit of 100 μg/kg cannot be reached.

Sludge, waste and treated biowaste may differ in properties as well as in the expected contamination

levels of PAH and presence of interfering substances. These differences make it impossible to describe

one general procedure. This document contains decision tables based on the properties of the sample and

the extraction and clean-up procedure to be used.

The method can be applied to the analysis of other PAH not specified in the scope, provided suitability is

proven by proper in-house validation experiments.
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2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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.

EN ISO 5667-15, Water quality - Sampling - Part 15: Guidance on the preservation and handling of sludge

and sediment samples (ISO 5667-15)

EN ISO 16720, Soil quality - Pretreatment of samples by freeze-drying for subsequent analysis (ISO 16720)

EN ISO 22892, Soil quality - Guidelines for the identification of target compounds by gas chromatography

and mass spectrometry (ISO 22892)

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

ISO 18512, Soil quality — Guidance on long and short term storage of soil samples

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
critical pair

pair of PAH that shall be separated to a predefined degree (e.g. R = 0,5) to ensure chromatographic

separation meets minimum quality criteria
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  t
R2     (1)
YY  
Where:
R peak separation
Δt difference in retention times of the two peaks a and b in seconds (s)
Y peak width at the base of peak a in seconds (s)
Y peak width at the base of peak b in seconds (s)
Figure 1 — Example of a chromatogram of a critical pair
3.2
internal standard

isotopically labelled mostly deuterated PAH or native PAH unlikely to be present in the sample, added to

samples before extraction and used for quantification of the PAH content
3.3
injection standard

PAH that is unlikely to be present in samples, added to the sample extract before injection into the gas

chromatograph, to monitor variability of instrument response and the recovery of the internal standards

3.4
extraction standard

PAH that is unlikely to be present in samples added to the sample prior to extraction. Used for checking

the extraction efficiency and not used for quantification purposes
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3.5
sediment

solid material, both mineral and organic, deposited in the bottom of a water body

[SOURCE: ISO 5667-12:2017 definition 3.5]
4 Principle

Due to the multi matrix character of this document, different procedures for different steps (modules)

are allowed. Which modules should be used depends on the sample. A recommendation is given in this

European Standard. Performance criteria are described and it is the responsibility of the laboratories

applying this European Standard to show that these criteria are met. Using of spiking standards (internal

standards) allows an overall check on the efficiency of a specific combination of modules for a specific

sample. But it does not necessarily give the information regarding the extensive extraction efficiency of

the native PAH bonded to the matrix.
After pre-treatment, the sample is extracted with a suitable solvent.

The extract is concentrated by evaporation. If necessary, interfering compounds are removed by a clean-

up method suitable for the specific matrix, before the concentration step.

If a solvent exchange for HPLC analysis is necessary, the concentrated extract is taken up in an

appropriate less volatile water miscible polar solvent and the non-polar extract residue is removed.

The extract is analysed by GC-MS using a capillary column with a stationary phase of low polarity or by

HPLC-UV-DAD/FLD with an appropriate reversed phase column.

PAH are identified and quantified with GC-MS by comparison of relative retention times and relative peak

heights (or peak areas) with respect to internal standards added, and with HPLC by using the

corresponding variables of the extraction standard solutions. The efficiency of the procedure depends on

the composition of the matrix that is investigated
5 Interferences
5.1 Interference with sampling and extraction

Use sampling containers of materials (preferably of steel, aluminium or glass) that do not affect the

sample during the contact time. Avoid plastics and other organic materials during sampling, sample

storage or extraction. Keep the samples from direct sunlight and prolonged exposure to light.

During storage of the 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.
5.2 Interference with GC-MS

Substances that co-elute with the target PAH may interfere with the determination. These interferences

may lead to incompletely resolved signals and may, depending on their magnitude, affect accuracy and

precision of the analytical results. Peak overlap does not allow an interpretation of the result.

Asymmetrical peaks and peaks broader than the corresponding peaks of the reference substance suggest

interferences.

Chromatographic separation between dibenz[a,h]anthracene and indeno[1,2,3-cd]pyrene are mostly

critical. Due to their molecular mass differences, quantification can be made by mass selective detection.

When incomplete resolution is encountered, peak integration shall be checked and, if necessary,

corrected.
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Sufficient resolution (e.g. 0,8) between the peaks of benzo[b]fluoranthene and benzo[k]fluoranthene as

well as of benzo[a]pyrene and benzo[e]pyrene shall be set as quality criteria for the capillary column.

Benzo[b]fluoranthene and benzo[j]fluoranthene cannot be separated. Triphenylene may not be

completely separated from benz[a]anthracene and chrysene. In this case it shall be stated in the report.

5.3 Interference with the HPLC

Substances that show either fluorescence or quenching and co-elute with the PAHs to be determined may

interfere with the determination. These interferences may lead to incompletely resolved signals and may,

depending on their magnitude, affect accuracy and precision of the analytical results. Peak overlap does

not allow an interpretation of the result. Asymmetrical peaks and peaks being broader than the

corresponding peaks of the reference substance suggest interferences. This problem may arise for

naphthalene and phenanthrene depending on the selectivity of the phases used.

Incomplete removal of the solvents used for sample extraction and clean-up may lead to poor

reproducibility of the retention times and wider peaks or double peaks especially for the 2- and 3-ring

PAHs. Extracts shall be diluted sufficiently with acetonitrile for the HPLC-analysis, otherwise the

detection of naphthalene and 3-ring PAH can be interfered with by a broad toluene peak.

Usually perylene is incompletely resolved from benzo[b]fluoranthene, but by choosing a selective

wavelength, the perylene peak can be suppressed.
6 Safety remarks

Certain PAH are highly carcinogenic and shall be handled with extreme care. Avoid contact with solid

materials, solvent extracts and solutions of standard PAH. Contact of solutions of standard with the body

should be prevented. It is strongly advised that standard solutions are prepared centrally in suitably

equipped laboratories or are purchased from suppliers specialized in their preparation.

PAH contamination of vessels may be detected by irradiation with 366 nm UV-light.

Solvent solutions containing PAH shall be disposed of in a manner approved for disposal of toxic wastes.

For the handling of hexane precautions shall be taken because of its neurotoxic properties.

National regulations should be followed with respect to all hazards associated with this method.

7 Reagents
7.1 General

All reagents shall be of recognized analytical grade. The purity of the reagents used shall be checked by

running a blank test as described in 10.1. The blank shall be less than 50 % of the lowest reporting limit.

7.2 Reagents for extraction
7.2.1 Acetone, (2-propanone), (CH ) CO.
3 2
7.2.2 Toluene, C H .
7 8
7.2.3 Petroleum ether, boiling range 40 °C to 60 °C
Hexane-like solvents with a boiling range between 40 °C and 98 °C are allowed.
7.2.4 Sodium sulfate, Na SO .
2 4
The anhydrous sodium sulfate shall be kept carefully sealed.
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7.2.5 Distilled water or water of equivalent quality, H O.
7.2.6 Sodium chloride, NaCl
7.2.7 Keeper substance. High boiling compound, i.e. octane, nonane
7.3 Reagents for clean-up
7.3.1 Clean-up A using aluminium oxide
7.3.1.1 Aluminium oxide, Al O
2 3
Basic or neutral, specific surface 200 m /g, activity Super I [11].
7.3.1.2 Deactivated aluminium oxide
Deactivated with approximately 10 % water.

Add approximately 10 g of water (7.2.5) to 90 g of aluminium oxide (7.3.1.1). Shake until all lumps have

disappeared. Allow the aluminium oxide to condition before use for some 16 h, sealed from the air, use it

for maximum of two weeks.

NOTE 1 The activity depends on the water content. It might be necessary to adjust the water content.

NOTE 2 Commercially available aluminium oxides with 10 % mass fraction water can also be used.

7.3.2 Clean-up B using silica gel 60 for column chromatography
7.3.2.1 Silica gel 60, particle size 63 µm to 200 µm
7.3.2.2 Silica gel 60, water content: mass fraction w(H O) = 10 %.

Silica gel 60 (7.3.2.1), heated for at least 3 h at 450 °C, cooled down and stored in a desiccator containing

magnesium perchlorate or a suitable drying agent. Before use, heat at least for 5 h at 130 °C in a drying

oven. Then allow cooling in a desiccator and add 10 % water (7.2.5) (mass fraction) in a flask. Shake for

5 min intensively until all lumps have disappeared and then for 2 h in a shaking device (8.1.3). Store the

deactivated silica gel in the absence of air, use it for a maximum of two weeks.
7.3.3 Clean-up C using gel permeation chromatography (GPC)
7.3.3.1 Bio-Beads® S-X3

NOTE Bio-Beads is an example of a suitable product available commercially. This information is given for

convenience of users of this European Standard and does not constitute an endorsement by CEN of this product.

Equivalent products can be used if they can be shown to lead to the same results.

7.3.3.2 Ethyl acetate, C4H8O2
7.3.3.3 Cyclohexane, C H
6 12

Preparation of GPC, for example: Put 50 g Bio-Beads S-X3 (7.3.3.1) into a 500 ml Erlenmeyer flask and

add 300 ml elution mixture made up of cyclohexane (7.3.3.3) and ethyl acetate (7.3.3.2) 1:1 (volume

fraction) in order to allow the beads to swell. After swirling for a short time until no lumps are left,

maintain the flask closed for 24 h. Drain the slurry into the chromatography tube for GPC. After

approximately three days, push in the plungers of the column so that a filling level of

approximately 35 cm is obtained. To further compress the gel, pump approximately 2 l of elution mixture

through the column at a flow rate of 5 ml · min and push in the plungers to obtain a filling level of

approximately 33 cm.
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7.4 Reagents for chromatographic analysis
7.4.1 GC-Analysis
7.4.1.1 Carrier gas for GC-MS

Operating gases of high purity and in accordance with the manufacturer’s specifications.

7.4.2 HPLC-analysis
7.4.2.1 Acetonitrile (CH CN) or methanol (CH OH), HPLC purity grade
3 3
7.4.2.2 Ultra-pure water, HPLC purity grade.
7.4.2.3 Helium, He, of suitable purity for degasification of solvents
7.5 Standards
7.5.1 General

Choose the internal and/or extraction standards whose physical and chemical properties (such as

extraction behaviour, retention time) are similar to those of the compounds to be analysed. For GC-MS a

minimum of four deuterated internal standards shall be used as internal standards for evaluation of

results.
7.5.2 Calibration substances and internal standards

Table 3 contains native and deuterated PAH to be used for calibration. Verify the stability of the internal

standards regularly.
Table 3 — Native PAH and deuterated PAH
PAH reference substances Internal standard substances deuterated PAHs
Naphthalene (CAS-RN 91-20-3) Naphthalene-d8 (CAS-RN 1146-65-2)
Acenaphthene (CAS-RN 83-32-9) Acenaphthene-d10 (CAS-RN 15067-26-2)
Acenaphthylene (CAS-RN 208-96-8) Acenaphthylene-d8 (CAS-RN 93951-97-4)
Fluorene (CAS-RN 86-73-7) Fluorene-d10 (CAS-RN 81103-79-9)
Anthracene (CAS-RN 120-12-7) Anthracene-d10 (CAS-RN 1719-06-8)
Phenanthrene (CAS-RN 85-01-8) Phenanthrene-d10 (CAS-RN 1517-22-2)
Fluoranthene (CAS-RN 206-44-0) Fluoranthene-d10 (CAS-RN 93951-69-0)
Pyrene (CAS-RN 129-00-0) Pyrene-d10 (CAS-RN 1718- 52–1)
Benz[a]anthracene (CAS-RN 56-55-3) Benz[a]anthracene-d12 (CAS-RN 1718-53-2)
Chrysene (CAS-RN 218-01-9) Chrysene-d12 (CAS-RN 1719-03-5)

Benzo[b]fluoranthene (CAS-RN 205-99-2) Benzo[b]fluoranthene-d12 (CAS-RN 93951-98-5)

Benzo[k]fluoranthene (CAS-RN 207-08-9) Benzo[k]fluoranthene-d12 (CAS-RN 93952-01-3)

(CAS-RN-205–82–
Benzo[j]fluoranthene
Benzo[a]pyrene (CAS-RN 50-32-8) Benzo[a]pyrene-d12 (CAS-RN 63466-71-7)
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PAH reference substances Internal standard substances deuterated PAHs
(CAS-RN 192-97-2)
Benzo[e]pyrene

Indeno[1,2,3-cd]pyrene (CAS-RN 193-39-5) Indeno[1,2,3-cd]pyrene-d12 (CAS-RN 203578-33-0)

Dibenz[a,h]anthracene (CAS-RN 53-70-3) Dibenz[a,h]anthracene -d14 (CAS-RN 13250-98-1)

Benzo[ghi]perylene (CAS-RN 191-24-2) Benzo[ghi]perylene-d12 (CAS-RN 93951-66-7)

Not part of 16 target analytes, but applicable for resolution check for the separation with benzo[a]pyrene for

GC measurements
NOTE 1 C12-labelled PAH standards can also be used as internal standards.

NOTE 2 Certified solutions of PAH, and single solid PAH substances with certified purity are available from a

limited number of suppliers.

When highly contaminated samples are analysed, an aliquot of the extract is often used for further clean-

up. This makes the costs of analyses caused by the use of deuterated standard very high. In these cases,

it is allowed to add the internal standard in two steps. Step 1 addition of unlabelled internal standards to

the sample. Step 2 addition of deuterated compounds to the aliquot of the extract used for clean-up.

For HPLC 6-methylchrysene, 1-Methylnaphthalene or other alkylated PAH which are not present in the

sample and which are sufficient separated from the target PAH can be used as internal or extraction

standard.
7.5.3 Injection standard

If required for GC-MS a deuterated PAH such as 1-methylnaphthalene-d10, triphenylene-d12 and

perylene-d12 can be added to the final extract before GC-MS injection to verify the recovery of the

deuterated internal standards.
7.6 Preparation of standard solutions
7.6.1 General

The procedure for the preparation of standard solutions for GC-MS and HPLC is the same only different

solvents are used.

Because of the dangerous nature of the substances to be used, commercially available - preferably

certified - standard solutions or mixed standard solutions are preferred. It is recommended to avoid skin

contact.
The working standard solutions shall be in the same solvent as the extract.

Store the primary and diluted standard solutions in a dark place at a temperature of (5 ± 3) °C. The

solutions are stable for at least one year, provided that evaporation of solvent is negligible.

Components present in mixed standard solutions should be separated by the chromatographic columns

used.
7.6.2 Preparation of calibration standard solutions for GC-MS

Prepare individual concentrated primary standard solutions of about 0,4 mg/ml in hexane-like solvent

(7.2.3) by weighing approximately 10 mg of each of the calibration standards (7.5.2 Table 3 left column)

to the nearest 0,1 mg and dissolving them in 25 ml of hexane-like solvent.

Combine small quantities (2 ml to 10 ml) of these individual primary standard solutions into a mixed

standard solution of PAH.
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Because of the dangerous nature of the substances to be used, commercially available - preferably

certified - standard solutions or mixed standard solutions are preferred. It is recommended to avoid skin

contact.
7.6.3 Preparation of internal standard solution for GC-MS
Prepare a concentrated primary internal
...