1、 ISO 2014 Soil quality Determination of polycyclic aromatic hydrocarbons (PAH) by gas chromatography (GC) and high performance liquid chromatography (HPLC) Qualit du sol Dtermination des hydrocarbures aromatiques polycycliques (HAP) par chromatographie en phase gazeuse (CPG) et chromatographie liqui
2、de haute performance (CLHP) INTERNATIONAL STANDARD ISO 13859 First edition 2014-02-01 Reference number ISO 13859:2014(E) ISO 13859:2014(E)ii ISO 2014 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2014 All rights reserved. Unless otherwise specified, no part of this publication may be reproduc
3、ed or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester. I
4、SO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ISO 13859:2014(E) ISO 2014 All rights reserved iii Contents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references 2 3 T erm
5、s and definitions . 2 4 Principle 3 5 Interferences 3 5.1 Interference with sampling and extraction 3 5.2 Interference with GC-MS 4 5.3 Interferences with the HPLC . 4 6 Safety remarks 4 7 Reagents 5 7.1 General . 5 7.2 Reagents for extraction 5 7.3 Reagents for clean-up 5 7.4 Reagents for chromatog
6、raphic analysis 6 7.5 Standards . 7 7.6 Preparation of standard solutions . 8 7.7 Preparation of internal standard solutions 9 7.8 Preparation of injection standard solution 9 8 Apparatus . 9 9 Sample storage and preservation 11 9.1 Sample storage .11 9.2 Sample pretreatment 11 10 Procedure11 10.1 B
7、lank test 11 10.2 Extraction .12 10.3 Concentration or dilution .14 10.4 Clean-up of the extract 15 10.5 Addition of the injection standard 16 10.6 Gas chromatographic analysis (GC) .17 10.7 High performance liquid chromatographic analysis (HPLC) .20 11 Performance characteristics .23 12 Precision 2
8、3 13 Test report 23 Annex A (informative) Repeatability and reproducibility data .24 Annex B (informative) Examples of instrumental conditions and chromatograms .27 Bibliography .37 ISO 13859:2014(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of natio
9、nal 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. Internati
10、onal 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. The procedures used to develop this document and those intende
11、d for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.is
12、o.org/directives). 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. Details of any patent rights identified during the development of the document wil
13、l be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents). Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement. For an explanation on the meaning of ISO specific terms and expres
14、sions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 3, C
15、hemical methods and soil characteristics.iv ISO 2014 All rights reserved ISO 13859:2014(E) 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, fu
16、el 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 m
17、ost of the routine laboratories following the preceding steps for sampling, pretreatment, 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 wit
18、h UV-DAD or fluorescence detection (HPLC-UV-DAD/FLD). Both the GC-MS and the HPLC methods are included in this horizontal standard. It is to be underlined that the target contamination level of PAH can lie in the range of about 0,01 mg/kg per individual PAH (agricultural soil and sediment) to about
19、200 mg/kg and higher (e.g. contaminated soil at coking plant sites or waste). The use of internal and injection standards is described in order to have an internal check on execution of the extraction and clean-up procedure. The method is as far as possible in agreement with the method described for
20、 PCBs (see EN 16167). This International Standard is the result of a desk study “Horizontal International Standard for determination of PAH in sludge, soil, and biowaste” in the project “Horizontal” and aims at evaluating the latest developments in assessing PAH in sludge, soil, treated biowaste, an
21、d neighbouring fields. After an evaluation study, in which the ruggedness of the method was studied, a European-wide validation of the draft standard has taken place. The results of the desk studies as well as the evaluation and validation studies have been subject to discussions with all parties co
22、ncerned in CEN. This International Standard 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 International Standard is applicable and validated Matrix Materials used for validation
23、Sludge Municipal sludge Biowaste Fresh compost ISO 2014 All rights reserved v Soil quality Determination of polycyclic aromatic hydrocarbons (PAH) by gas chromatography (GC) and high performance liquid chromatography (HPLC) WARNING Persons using this International Standard should be familiar with us
24、ual 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. IM
25、PORTANT It is absolutely essential that tests conducted according to this International Standard be carried out by suitably trained staff. 1 Scope This International Standard specifies the quantitative determination of 16 PAH (see Table 2) in sludge, soil, and treated biowaste using GC-MS and HPLC-U
26、V-DAD/FLD covering a wide range of PAH contamination levels (see also Annex B). When using fluorescence detection, acenaphthylene cannot be measured. Table 2 Polycyclic aromatic hydrocarbons which can be analysed using this International Standard Target analyte CAS-RN a Naphthalene 91-20-3 Acenaphth
27、ene 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,
28、h)anthracene 53-70-3 Benzo(ghi)perylene 191-24-2 aChemical 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. Typically, a lower limit of applicati
29、on of 0,01 mg/kg (expressed as dry matter) can be ensured for each individual PAH. This depends on instrument and sample. INTERNATIONAL ST ANDARD ISO 13859:2014(E) ISO 2014 All rights reserved 1 ISO 13859:2014(E) Sludge, soil, and treated biowaste can differ in properties and also in the expected co
30、ntamination levels of PAH and presence of interfering substances. These differences make it impossible to describe one general procedure. This International Standard contains decision tables based on the properties of the sample and the extraction and clean-up procedure to be used. Two general lines
31、 are followed, an agitation procedure (shaking) or use of Soxhlet/pressurized liquid extraction. NOTE Other PAH compounds can also be analysed with this method, provided suitability has been proven. 2 Normative references The following documents, in whole or in part, are normatively referenced in th
32、is document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 5667-15, Water quality Sampling Part 15: Guidance on the preservation and handlin
33、g of sludge and sediment 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 ISO 11465, Soil quality Determination of dry matter and water content on a mass
34、basis Gravimetric method ISO 14507, Soil quality Pretreatment of samples for determination of organic contaminants ISO 18512, Soil quality Guidance on long and short term storage of soil samples ISO 22892, Soil quality Guidelines for the identification of target compounds by gas chromatography and m
35、ass spectrometry 3 T erms a nd definiti ons For the purposes of this document, the following terms and definitions apply. 3.1 critical pair pair of congeners that will be separated to a predefined degree (e. g. R = 0,5) to ensure chromatographic separation meets minimum quality criteria SOURCE: EN 1
36、5308:2008, 3.6 Note 1 to entry: See Figure 1.2 ISO 2014 All rights reserved ISO 13859:2014(E) Key t difference in retention times of the two peaks a and b, in seconds (s) Y a peak width at the base of peak a, in seconds (s) Y b peak width at the base of peak b, in seconds (s) Figure 1 Example of a c
37、hromatogram of a critical pair 4 Principle After pretreatment to reduce the moisture content and to increase the homogeneity (see 9.2), the test sample is extracted with a solvent. The extract is concentrated and interfering compounds are removed by a clean-up method suitable for the specific matrix
38、. The eluate is concentrated. For HPLC analysis, the concentrated eluate is taken up in an appropriate less volatile water miscible polar solvent and the non-polar eluate residue is removed. The extract is analysed by GC-MS using a capillary column with a stationary phase of low polarity or by HPLC-
39、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 external standard
40、 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 change the sample during the contact ti
41、me. 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 can occur due to adsorption on the walls of the containers. The extent of the losses depe
42、nds on the storage time. ISO 2014 All rights reserved 3 ISO 13859:2014(E) 5.2 Interference with GC-MS Substances that co-elute with the target PAH can interfere with the determination. These interferences can lead to incomplete resolved signals and can, depending on their magnitude, affect accuracy
43、and precision of the analytical results. Peak overlap does not allow an interpretation of the result. Unsymmetrical 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)pyre
44、ne 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. Sufficient resolution (e. g. 0,8) between the peaks of benzo(b)fluoranth
45、ene 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 cannot be completely separated from benz(a)anthracene and chrysene. In this case it
46、 shall be stated in the report. 5.3 Interferences with the HPLC Substances that show either fluorescence or quenching and co-elute with the PAH to be determined can interfere the determination. These interferences can lead to incompletely resolved signals and can, depending on their magnitude, affec
47、t 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 can arise for naphthalene and phenanthrene depending
48、 on the selectivity of the phases used. Incomplete removal of the solvents used for sample extraction and clean-up can lead to poor reproducibility of the retention times and wider peaks or double peaks especially for the 2-ring and 3-ring PAH. Extracts shall be diluted sufficiently with acetonitril
49、e for the HPLC analysis, otherwise the detection of naphthalene and 3-ring PAH can be interfered by a broad toluene peak. Separation between dibenz(a,h)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, the perylene peak can be suppressed. 6 Safety remarks Certain PAH are highly carcinogenic and shall b
