SIST EN ISO 22478:2006

SLOVENSKI STANDARD
01-julij-2006
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Water quality - Determination of certain explosives and related compounds - Method
using high-performance liquid chromatography (HPLC) with UV detection (ISO
22478:2006)
Wasserbeschaffenheit - Bestimmung ausgewählter Explosivstoffe und verwandter
Verbindungen - Verfahren mittels Hochleistungs- Flüssigkeitschromatographie (HPLC)
mit UV-Detektion (ISO 22478:2006)
Qualité de l'eau - Dosage de certains explosifs et de composés apparentés - Méthode
utilisant la chromatographie en phase liquide a haute performance (CLHP) avec
détection UV (ISO 22478:2006)
Ta slovenski standard je istoveten z: EN ISO 22478:2006
ICS:
13.060.50
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 22478
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2006
ICS 13.060.50
English Version
Water quality - Determination of certain explosives and related
compounds - Method using high-performance liquid
chromatography (HPLC) with UV detection (ISO 22478:2006)
Qualité de l'eau - Dosage de certains explosifs et de Wasserbeschaffenheit - Bestimmung ausgewählter
composés apparentés - Méthode utilisant la Explosivstoffe und verwandter Verbindungen - Verfahren
chromatographie en phase liquide à haute performance mittels Hochleistungs-Flüssigkeitschromatographie (HPLC)
(CLHP) avec détection UV (ISO 22478:2006) mit UV-Detektion (ISO 22478:2006)
This European Standard was approved by CEN on 6 February 2006.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, 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
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 22478:2006: E
worldwide for CEN national Members.

Foreword
This document (EN ISO 22478:2006) has been prepared by Technical Committee ISO/TC 147
"Water quality" in collaboration with 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 August 2006, and conflicting national
standards shall be withdrawn at the latest by August 2006.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Endorsement notice
The text of ISO 22478:2006 has been approved by CEN as EN ISO 22478:2006 without any
modifications.
INTERNATIONAL ISO
STANDARD 22478
First edition
2006-02-15
Water quality — Determination of certain
explosives and related compounds —
Method using high-performance liquid
chromatography (HPLC) with UV
detection
Qualité de l'eau — Dosage de certains explosifs et de composés
apparentés — Méthode utilisant la chromatographie en phase liquide à
haute performance (CLHP) avec détection UV

Reference number
ISO 22478:2006(E)
©
ISO 2006
ISO 22478:2006(E)
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ii © ISO 2006 – All rights reserved

ISO 22478:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Principle. 1
4 Interference . 4
5 Reagents. 4
6 Apparatus . 5
7 Sampling. 6
8 Procedure . 6
9 Calibration . 8
10 Evaluation. 10
11 Expression of results . 11
12 Test report . 11
Annex A (informative) Examples of HPLC chromatograms. 12
Annex B (informative) UV spectra of compounds from Table 1. 19
Annex C (informative) Interference due to degradation of tetryl (CE) on exposure to daylight . 22
Annex D (informative) Precision data. 24

ISO 22478:2006(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 22478 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
iv © ISO 2006 – All rights reserved

ISO 22478:2006(E)
Introduction
Explosives and related compounds are frequently encountered in groundwater areas near to soil sites
contaminated by armaments waste and may also be found in drinking water taken from nearby catchment
areas. The range of pollutants will depend on the waste concerned, but will not, as a rule, include all the
compounds listed in Table 1. Instead, samples of groundwater containing such pollutants may contain
numerous other substances, such as nitro- and dinitrobenzoic acid, nitrophenols and aromatic amines. The
compounds listed in Table 1 are frequently used for exploratory examinations of armaments waste.
When using this International Standard, it may be necessary in some cases to determine whether and to what
extent particular problems will require the specification of additional conditions.

INTERNATIONAL STANDARD ISO 22478:2006(E)

Water quality — Determination of certain explosives and related
compounds — Method using high-performance liquid
chromatography (HPLC) with UV detection
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. 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 to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this International
Standard be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for determination of certain explosives, in particular
nitrotoluenes, nitroamines and nitrate esters, and related compounds (by-products and degradation products),
such as those listed in Table 1, in drinking water, groundwater and surface water.
Depending on the type of sample and the compound to be analysed, the lower limit of the working range for
nitroaromatics and nitramines can be assumed to be between 0,1 µg/l and 0,5 µg/l (in some cases, the lower
limit may be extended down to 0,05 µg/l). The lower limit of the working range for nitrate esters may be
assumed to be higher (0,5 µg/l or more).
Similar compounds, in particular other nitroaromatics, may also be determined by this method, but its
applicability will have to be checked in each individual case.
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 3696, Water for analytical laboratory use — Specification and test methods
ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes
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 water
samples
3 Principle
The substances in the water sample are concentrated by solid-phase extraction using a
polystyrene/divinylbenzene-based adsorbent. After elution with a solvent mixture, the eluate is concentrated
and the substances are separated by high-performance liquid chromatography (HPLC) and analysed using a
UV photodiode array (UV-PDA) detector.
ISO 22478:2006(E)
Table 1 — Explosives and related compounds determined by this method (the compounds listed here
are particularly likely to be encountered in water samples containing armaments waste)
Molar Reference No./
mass letter in example
a b chromatograms
Name Abbreviation Other name CAS No.
in Clause
g/mol A.1 A.2 A.3 B.1
2,4,6-Trinitrophenol PA Picric acid 88-89-1 229,1 1 2 1 a
1,3,5,7-Tetranitro-octahydro-1,3,5,7- HMX Octogen 2691-41-0 296,2 2 1 2 b
tetrazocine
1,3,5-Trinitro-hexahydro-1,3,5-triazine RDX Hexogen 121-82-4 222,1 3 3 4 d
2,2',4,4',6,6'-Hexanitrodiphenylamine — Hexyl 131-73-7 439,2 4 19 3 c
Ethylene glycol dinitrate EGDN — 628-96-6 152,1 5 4 5 e
Diethylene glycol dinitrate DEGN — 693-21-0 196,1 6 5 6 f
1,3,5-Trinitrobenzene 1,3,5-TNB — 99-35-4 213,1 7 6 7 g
1,3-Dinitrobenzene 1,3-DNB — 99-65-0 168,1 8 7 9 i
N-Methyl-N-2,4,6-tetranitroaniline CE Tetryl 479-45-8 287,2 9 8 8 h
Glycerol trinitrate NG Nitroglycerine 55-63-0 227,1 10 9 10 j
2,4,6-Trinitrotoluene 2,4,6-TNT TNT 118-96-7 227,1 11 10 11 k
4-Amino-2,6-dinitrotoluene 4-A-2,6-DNT — 19406-51-6197,1 12 11 12 l
2-Amino-4,6-dinitrotoluene 2-A-4,6-DNT — 35572-78-2197,1 13 12 13 m
2,6-Dinitrotoluene 2,6-DNT — 606-20-2 182,1 14 13 14 n
2,4-Dinitrotoluene 2,4-DNT — 121-14-2 182,1 15 14 15 o
2-Nitrotoluene 2-NT — 88-72-2 137,1 16 15 17 q
Pentaerythritol tetranitrate PETN Nitropenta 78-11-5 316,2 17 18 16 p
4-Nitrotoluene 4-NT — 99-99-0 137,1 18 16 18 r
3-Nitrotoluene 3-NT — 99-08-1 137,1 19 17 19 s
Diphenylamine DPA — 122-39-4 169,2420 — — —
a
Standard abbreviations, some of Anglo-Saxon origin.
b
Chemical Abstracts Service.
The structural formulae of selected compounds are given in Figure 1.
2 © ISO 2006 – All rights reserved

ISO 22478:2006(E)
Picric acid (PA) Octogen (HMX) Hexogen (RDX)

Hexyl Ethylene glycol dinitrate (EGDN) Diethylene glycol dinitrate (DEGN)

Tetryl (CE) Nitroglycerine (NG) Nitropenta (PETN)

1,3,5-Trinitrobenzene 1,3-Dinitrobenzene 2,4,6-Trinitrotoluene (TNT) 4-Amino-2,6-dinitrotoluene

2-Amino-4,6-dinitrotoluene 2,6-Dinitrotoluene 2,4-Dinitrotoluene 2-Nitrotoluene

4-Nitrotoluene 3-Nitrotoluene Diphenylamine (DPA)
Figure 1 — Structural formulae of selected explosives
ISO 22478:2006(E)
4 Interference
4.1 Sampling
To avoid interference, collect samples as specified in Clause 7, observing the instructions given in ISO 5667-1,
ISO 5667-2 and ISO 5667-3.
4.2 Filtration
If the sample contains suspended matter, first filter it through a borosilicate glass fibre filter to prevent
blockage of the solid phase concentrating cartridge and record this operation in the test report.
4.3 Concentration by evaporation
Mononitrotoluenes may be lost when the eluates are evaporated as specified in 8.2.
4.4 Chromatography
Asymmetrical peaks and peaks that are wider than normal may indicate incompletely resolved peaks or peak
overlapping due to compounds with similar retention times and/or absorption at the same or at a similar
wavelength to those of the compounds being determined. To detect peak overlaps, check the peak purity and
compare the UV spectrum with that of a reference material.
Interference may also be caused by humic matter eluted in the same range as the analytes being determined.
4.5 Exposure to light
Reference solutions, samples and sample extracts may decompose on exposure to light. This is particularly
important in the case of tetryl (see Annex C). Therefore a detailed investigation of the recovery of tetryl should
be carried out.
4.6 Determination of hexogen
When determining small concentrations of hexogen, interference may be caused by high concentrations of
nitroaromatics.
5 Reagents
The reagents shall not have blank values that would interfere with the HPLC analysis.
If available, use reagents of HPLC grade or residue grade. The solvents shall not contain any measurable UV-
absorbing substances that interfere with the method.
5.1 Water, complying with grade 1 as defined in ISO 3696. The water shall not contain any measurable
quantities of UV-absorbing substances that interfere with the determination.
5.2 Nitrogen, purity not less than 99,996 % by volume, for drying the adsorbent and, if necessary, for
concentrating the eluates.
5.3 Helium, purity not less than 99,996 % by volume, for degassing the eluent if a vacuum degasser is not
used.
5.4 Microporous polystyrene/divinylbenzene adsorbent, with a high specific surface area (e.g. greater
than 900 m /g). Other adsorbents may be used provided that the recovery rate is greater than 85 % for
octogen (HMX) at a concentration of 1 µg/l in a 1 l sample.
4 © ISO 2006 – All rights reserved

ISO 22478:2006(E)
5.5 Methanol, CH OH.
5.6 Acetonitrile, CH CN.
5.7 Sodium chloride, NaCl.
5.8 Potassium dihydrogen phosphate solution, c(KH PO ) = 0,025 mol/l (3,40 g/l).
2 4
5.9 Orthophosphoric acid, w(H PO ) = 85 %.
3 4
5.10 Reference compounds, as listed in Table 1, some of which are obtainable only as commercial
solutions with a specified mass concentration.
5.11 Solutions of reference compounds, prepared as follows:
Add methanol (5.5) to 100 mg of the reference compound in a 100 ml volumetric flask, dissolve the reference
compound and make up to volume with methanol.
Prepare solutions of hexyl and octogen using acetonitrile (5.6) because of their poor solubility in methanol.
Store the solutions at a maximum of 6 °C, protected from light; their shelf-life is limited (six months at most).
5.12 Stock solutions, prepared as follows:
Transfer 1 ml of each of the solutions of reference compounds (5.11) to a separate 100 ml volumetric flask
and make up to volume with methanol (5.5).
Store the solutions at a maximum of 6 °C, protected from light; the solutions are stable for up to six months.
5.13 Reference solutions for multipoint calibration, prepared as follows:
Prior to each calibration, prepare at least five dilutions of each of the stock solutions prepared in 5.12,
preferably using a methanol/water mixture (50 + 50 parts by volume) as solvent.
Store the reference solutions at a maximum of 6 °C, protected from light; check their concentrations regularly
as their stability is limited.
5.14 Buffer solution, to improve gradient elution of hexyl and picric acid, prepared by adjusting the pH of a
0,025 mol/l solution of potassium dihydrogen phosphate (5.8) to 3,2 using orthophosphoric acid (5.9).
6 Apparatus
Those parts of the apparatus that come into contact with the sample or the extract shall be free of compounds
that may produce blank values. The apparatus shall be made of materials that do not adsorb the compounds
under investigation and that provide protection from light, e.g. brown glass, stainless steel or perfluorinated
plastics.
6.1 Brown-glass narrow-necked flat-bottomed flasks, 1 000 ml.
6.2 Vacuum or pressure equipment, for sample enrichment.
6.3 Glass fibre filter, diameter of fibres 0,75 µm to 1,5 µm, of borosilicate glass containing an inorganic
binder.
6.4 Solvent-resistant micromembrane filter, e.g. polyamide or cellulose membrane, pore size of 0,2 µm
to 0,45 µm, for filtration of HPLC extracts.
6.5 Glass or polypropene cartridges, packed with a polystyrene/divinylbenzene-based adsorbent (5.4).
ISO 22478:2006(E)
6.6 Glass volumetric flasks, 1 ml, 10 ml and 100 ml.
6.7 Graduated cylinder, 100 ml.
6.8 pH-meter, with electrodes.
6.9 Equipment for concentrating the eluates by evaporation, e.g. a rotary evaporator, regulatable for
constant vacuum and with a temperature-controlled water bath, or stripping equipment using nitrogen gas.
6.10 Glass vessels, for collecting and concentrating the eluates by evaporation (e.g. tapered flask with
cylindrical graduated extension).
6.11 Glass sample bottles, with inert closure (e.g. PTFE-coated septum), for storing the extracts.
6.12 High-performance liquid chromatograph, consisting of:
a) an analytical pump system for isocratic or gradient elution;
b) a manual or automatic sample introduction device;
c) a degassing device;
d) a column thermostat;
e) a photodiode array (PDA) detector with a wavelength range of e.g. 200 nm to 400 nm;
f) a data evaluation system;
g) an analytical column (examples of suitable column types are given in Annex A).
7 Sampling
Fill carefully cleaned brown-glass flat-bottomed flasks with the water to be examined. Store the samples
immediately after sampling and during transport at not more than 4 °C, protected from light (e.g. in a cool box).
Extract the sample, if possible, within three days after collection since the concentration of explosives and
related compounds is more stable in the organic eluates than in the samples. If storage cannot be avoided,
keep the sample at about 4 °C in the dark.
Carry out the determination as soon as possible after extraction.
8 Procedure
8.1 Conditioning of adsorbent
Use not less than 0,2 g of polystyrene/divinylbenzene adsorbent (5.4) in a cartridge (6.5) for each 1 000 ml of
sample. Add 3 ml of methanol (5.5) over a period of 5 min and wash with 3 ml of acetonitrile (5.6) and then
with 10 ml of water. Ensure that the adsorbent does not run dry.
8.2 Concentration and elution
If necessary, filter the sample through a glass fibre filter (6.3) to remove suspended matter.
Take e.g. 1 000 ml of the sample to be examined and dissolve in it about 5 g of sodium chloride (5.7). Pass
the sample through the adsorbent conditioned as in 8.1 at a flow rate of up to 1 000 ml/h and then flush with
1 ml of water (5.1). Dry the adsorbent in a stream of nitrogen (5.2) for not less than 10 min.
6 © ISO 2006 – All rights reserved

ISO 22478:2006(E)
Elute once with 2 ml of a methanol/acetonitrile
...