ISO-TC147-SC2 N1474 SC2WG52 N 76 Proposal New Project Arseni

ISO/TC 147/SC 2
N 1474
ISO/TC 147/SC 2
Physical, chemical and biochemical methods
Email of secretary: gabriela.barz@din.de
Secretariat: DIN (Germany)
SC2WG52 N 76 Proposal New project Arsenic species HPLC HG-AFS

Document type:
Working draft
Date of document:
2015-03-27
Expected action:
INFO
Background:
Dear member,
Enclosed please find a proposal for the new project "Water quality - Determination of four chemical
arsenic species - Method using high performance liquid chromatography (HPLC) with hydride
generation atomic fluorescence spectrometry (HG-AFS)".
This draft will be discussed at the meeting of SC 2/WG 52 "Antimony, arsenic and selenium" in
Conshohocken, Philadelphia, USA 2015-06-01.
Best regards
Gabriela Barz
Committee URL:
http://isotc.iso.org/livelink/livelink/open/tc147sc2
Proposal for a new project

Closing date for voting
Reference number
(to be given by the Secretariat)
Date of circulation
Secretariat
ISO/TC 147/SC 2/WG 52

(replaces Doc WG 52 N 75)
N 76
For discussion at the meeting of SC 2/WG 52 in Conshohocken, Philadelphia, USA, 2015-06-01;

Point 6 of the agenda
IMPORTANT NOTE: Proposals without adequate justification risk rejection or referral to originator.
Guidelines for proposing and justifying a new work item are contained in Annex C of the ISO/IEC Directives, Part 1.
The proposer has considered the guidance given in the Annex C during the preparation of the NWIP.
Proposal (to be completed by the proposer)
Title of the proposed deliverable.
(in the case of an amendment, revision or a new part of an existing document, show the reference number and current title)
English title
Water quality - Determination of four chemical arsenic species - Method using high performance liquid
chromatography (HPLC) with hydride generation atomic fluorescence spectrometry (HG-AFS)
French title
Qualit de l'eau - Dtermination de quatre formes chimiques de larsenic Mthode par chromatographie
liquide haute performance (CLHP) avec spectromtrie de fluorescence atomique gnration d'hydrures
(HG-AFS)
(if available)
Scope of the proposed deliverable.
The proposed standard will specify a method for the determination of four arsenic species dissolved in waters intended for
human consumption, such as surface waters, groundwaters and rainwaters. The working linear dynamic range is dependent
on the operating conditions; under standard conditions, it ranges from 1 g/l to 50 g/l for the four species in question. It
will use a simple relatively low-cost filter atomic fluorescence for detection of the various arsenic hydride species
FORM 4 (ISO) v. 2013
Page 1 of 4
Purpose and justification of the proposal*
In the environment, metals and metalloids are found in the form of various chemical species. Chemical speciation makes it
possible to identify and quantify these different species. For the same metal or metalloid, given that the toxicity of each
compound may vary significantly, it can be very useful to quantify each of the significant toxicological arsenic species
present in a given sample. The proposed method is applicable to the determination of arsenite (As(III)), arsenate (As(V)),
monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA).
In natural water samples, according to the literature, the main arsenic species encountered are arsenite and arsenate.
However, the organic arsenic species MMA and DMA may also be encountered in some surface waters. For arsenic, the
toxicity of these arsenic species varies considerably; inorganic species are recognized as being more toxic than organic
species and, the toxicity of As III is greater than that of As V. The proposed method is applicable to the determination of
As(III), As (V), MM and DMA.
All current ISO and CEN arsenic in waters standards can only determine "total arsenic". The proposed new standard method
will provide results for all of the four arsenic species given above. This information can be very useful for selecting and
optimising the appropriate removal of arsenic technique for treating groundwaters containing unacceptable levels of
naturally occurring arsenic.
The setting up and routine running of proposed robust speciation method should be within the capability of any competent
drinking water analysis laboratory
It is hoped to be able to find an adequate number of laboratories to carry out a method validation trial.
It should be noted that arsenic is one of WHOs 10 chemicals of major public health concern.
See also: http://www.who.int/mediacentre/factsheets/fs372/en/ and
http://www.sustainablefuture.se/arsenic/towards_effective.html
*The reason for requiring justification statements with approval or disapproval votes is primarily to collect input on market or stakeholder
needs, and on market relevance of the proposal, to benefit the development of the proposed ISO standard(s). Any NSB vote in relation to a
proposal for new work may result in significant commitments of resources by all parties (NSBs, committee leaders and delegates/experts) or
may have significant implications for ISO's relevance in the global community. It is especially important that NSBs consider and express
why they vote the way they do. In addition, it is felt that it would be useful for ISO and its committees to have documentation as to why the
NSBs feel a proposal has market need and market relevance. Therefore, please ensure that your justifying statements with your approval or
disapproval vote convey the reason(s) why your national consensus does or does not support the market need and/or global relevance of
the proposal.
If a draft is attached to this proposal,:

Please select from one of the following options (note that if no option is selected, the default will be the first
option):
Draft document will be registered as new project in the committee's work programme (stage 20.00)
Draft document can be registered as a Working Draft (WD stage 20.20)
Draft document can be registered as a Committee Draft (CD stage 30.00)
Draft document can be registered as a Draft International Standard (DIS stage 40.00)
Is this a Management Systems Standard (MSS)?
Yes
No
NOTE: if Yes, the NWIP along with the Justification study (see Annex SL of the Consolidated ISO Supplement) must be sent
to the MSS Task Force secretariat (tmb@iso.org) for approval before the NWIP ballot can be launched.
Indication(s) of the preferred type or types of deliverable(s) to be produced under the proposal.
International Standard
Technical Specification
Proposed development track
1 (24 months)
Publicly Available Specification

2 (36 months - default)
Technical Report
3 (48 months)
Known patented items (see ISO/IEC Directives, Part 1 for important guidance)
Yes
No
If "Yes", provide full information as annex
Page 2 of 4
A statement from the proposer as to how the proposed work may relate to or impact on existing work, especially
existing ISO and IEC deliverables. The proposer should explain how the work differs from apparently similar work,
or explain how duplication and conflict will be minimized.
This will be a lower cost alternative method to the CD Water quality Determination of four chemical arsenic species
Method using high-performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICPMS)
A listing of relevant existing documents at the international, regional and national levels.
Currently there are no ISO or CEN standards relating to arsenic speciation using atomic spectroscopic detection. However
there is a Draft TS 19620 using the much more costly ICP-MS detection technique
A simple and concise statement identifying and describing relevant affected stakeholder categories (including small
and medium sized enterprises) and how they will each benefit from or be impacted by the proposed deliverable(s)
There is an inceasing demand for arsenic speciation analysis as the toxicity of arsenic cannot simply be determined by
carrying out a total arsenic analysis. Almost certainly regulators will be looking to set requirements for specific arsenic
species. Also, the efficiency of arsenic removal processes from groundwater is affected by the arsenic speciation and this
analysis is required for optimisation of this key process
Liaisons:
A listing of relevant external international organizations
or internal parties (other ISO and/or IEC committees) to
be engaged as liaisons in the development of the
deliverable(s).
Joint/parallel work:
Possible joint/parallel work with:
IEC
(please specify committee ID)
CEN
(please specify committee ID) TC 230
Other
(please specify)
A listing of relevant countries which are not already P-members of the committee.
Preparatory work (at a minimum an outline should be included with the proposal)
A draft is attached
An outline is attached
An existing document to serve as initial basis
The proposer or the proposer's organization is prepared to undertake the preparatory work required
Yes
Proposed Project Leader (name and e-mail address)
K. Clive Thompson
(Convenor of ISO/TC 147/SC 2/WG 52)
clive.thompson@alcontrol.com
No
Name of the Proposer

(include contact information)
K. Clive Thompson
clive.thompson@alcontrol.com
(NSB: BSI)
Supplementary information relating to the proposal

This proposal relates to a new ISO document;
This proposal relates to the adoption as an active project of an item currently registered as a Preliminary Work Item;
This proposal relates to the re-establishment of a cancelled project as an active project.
Other:
Annex(es) are included with this proposal (give details)
PNWI ISO/WD Arsenic HPLC HG-AFS
Page 3 of 4
- page intentionally left blank -
Page 4 of 4
ISO 2015 All rights reserved
ISO/TC 147/SC 2 N
1474
Date: 2015-03-26

ISO/TC 147/SC 2/WG 52
N 76
Secretariat: DIN
Water quality Determination of four chemical arsenic species

Method using high performance liquid chromatography (HPLC) with
hydride generation atomic fluorescence spectrometry (HG-AFS)
Qualit de l'eau Dtermination de quatre fromes chimiques de l'arsenic Mthode par chromatographie
liquide haute performance (CLHP) avec spectromtrie de fluorescence atomique gnration d'hydrures
(HG-AFS)
Warning
This document is not an ISO International Standard. It is distributed for review and comment. It is subject to
change without notice and may not be referred to as an International Standard.
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.
Document type: International Standard

Document subtype:
Document stage: (20) Preparatory
Document language: E
Copyright notice
This ISO document is a working draft or committee draft and is copyright-protected by ISO. While the
reproduction of working drafts or committee drafts in any form for use by participants in the ISO standards
development process is permitted without prior permission from ISO, neither this document nor any extract
from it may be reproduced, stored or transmitted in any form for any other purpose without prior written
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Reproduction for sales purposes may be subject to royalty payments or a licensing agreement.
Violators may be prosecuted.
ii
Contents
Page
Foreword ............................................................................................................................................................ iv
Introduction ......................................................................................................................................................... v
1
Scope ...................................................................................................................................................... 1
Normative references ............................................................................................................................ 1
Terms and definitions ........................................................................................................................... 2
Principle.................................................................................................................................................. 3
Interferences .......................................................................................................................................... 3
Apparatus ............................................................................................................................................... 3
Reagents and standards ....................................................................................................................... 4
Sampling, preservation and storage of drinking, surface and ground water samples .................. 6
Procedure ............................................................................................................................................... 7
10
Expression of results ............................................................................................................................ 8
11
Test report .............................................................................................................................................. 8
Annex A (informative) Example - Layout of LC-HG-AFS ................................................................................. 9

Annex B (informative) Example of experimental conditions ........................................................................ 10
Bibliography ...................................................................................................................................................... 15
iii
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 Arsenic HPLC HG-AFS was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee
SC 2, Physical, chemical and biochemical methods.
iv
Introduction
In the environment, metals and metalloids are found in the form of various chemical species. Chemical
speciation makes it possible to identify and quantify these different species. For the same metal or metalloid,
given that the toxicity of each compound may vary significantly, it can be useful to quantify each of the species
present in a given sample. For arsenic, the toxicity of the various species varies considerably; inorganic
species are recognized as being more toxic than organic species and, for example, the toxicity of As(III) is
greater than that of As(V). The proposed method is applicable to the determination of arsenite (As(III)),
arsenate (As(V)), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA).
In natural water samples, according to the literature, the main arsenic species encountered are arsenite and
arsenate. However, the organic species MMA and DMA may also be encountered in surface waters.
Arsenobetaine and other organic arsenic species are not present in natural water samples and are therefore
not included in this method.
WORKING DRAFT
Water quality Determination of four chemical arsenic

species Method using high performance liquid
chromatography (HPLC) with hydride generation atomic
fluorescence spectrometry (HG-AFS)
WARNING Persons using this document should be familiar with normal laboratory practice. This
document is not intended to cover any safety problems associated with its use, if applicable. It is the
responsibility of the user to establish appropriate safety and health methods and to ensure
compliance with any national regulatory conditions.
IMPORTANT It is absolutely essential that tests conducted in accordance with this document be
carried out by suitably qualified staff.
Scope
This International Standard specifies the determination of four arsenic species dissolved in waters intended for
human consumption, surface waters, ground waters and rain waters. The working linear dynamic range is
dependent on the operating conditions; under standard conditions, it ranges from 0,1 g/l to 50 g/l for the
four species in question. Samples containing arsenic at concentrations higher than the linear dynamic range
can be analysed after suitable dilution.
The sensitivity of this method is dependent on the instrument operating conditions selected. The limit of
quantification of the method (LOQ) is also dependent on the operating conditions of the analytical system
used and the extent of the calibration range used. The limits of quantification (LOQ) are given as examples in
Annex B, and are between 0,1 g/l and 0,4 g/l. This information is given as an indicative guide.
Normative references
The following documents, in whole or in part, are normatively referenced in this 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 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 and
sampling techniques
ISO 5667-3, Water quality Sampling Part 3: Preservation and handling of water samples
ISO 17378-1, Water quality Determination of arsenic and antimony Part 1: Method using hydride
generation atomic fluorescence spectrometry (HG-AFS)
ISO 17378-2, Water quality Determination of arsenic and antimony Part 2: Method using hydride
generation atomic absorption spectrometry (HG-AAS)
Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
analyte
element to be determined
[SOURCE: ISO 17294-1:2004, 3.2]
3.2
blank calibration solution
solution prepared in the same way as the calibration solution but leaving out the analyte
[SOURCE: ISO 17294-1:2004, 3.3]
3.3
calibration solution
solution used to calibrate the instrument, prepared from (a) stock solution(s) or from a certified standard
3.4
stock solution
solution with accurately known analyte concentration(s), prepared from suitably pure chemicals
[SOURCE: ISO 17294-1:2004, 3.30]
3.5
test sample
sample prepared from the laboratory sample, for example by grinding or homogenizing
[SOURCE: ISO 17294-1:2004, 3.31]
3.6
determination (Remark from SC 2 secretariat: This term is of very general nature; could be precised?)
entire process from preparing the test sample solution up to and including measurement and calculation of the
final result
3.7
linearity
straight line relationship between the result of measurement (signal) and the quantity (concentration) of the
analyte to be determined
Note 1 to entry:
Or straight line relationship between the (mean) replicate results of measurement (signal) and the
quantity (concentration) of the component to be determined.
(Request from SC 2 secretariat: Kindly check the ISO Online Browsing Platform, whether definition text could
be adapted to already existing definitions)
3.8
limit of quantification (LOQ)
lowest concentration of an analyte that can be determined with a specified degree of accuracy and precision
(Please check ISO OBP)
Principle
The different arsenic species are separated using a specific column in a high-performance liquid
chromatograph (HPLC). The separation of arsenic species in natural water is typically achieved using strong
anion exchange ion chromatography. On elution form the column the arsenic species are acidified and
subsequently reduced using a sodium tetrahydroborate reductant to form the species specific covalent
gaseous hydride. After gas liquid separation the arsenic hydride species are delivered to an atomizer such as
hydrogen diffusion flame or heated quartz cuvette using an argon carrier gas. Moisture is removed from this
TM
stream using a hygroscopic Nafion membrane dryer. After atomisation the arsenic is determined by atomic
fluorescence spectrometry (AFS) equipped with an Arsenic boosted discharge hollow cathode lamp. Atomic
absorption and other detection methods with hydride generation can be used provided that the performance
specification of the method is achieved.
The concentration of arsenic is determined by comparing the quantity of atomic arsenic detected in the
sample in relation to the calibration solutions of each species.
Annex A presents a block diagram of the measurement apparatus.
Interferences
The hydride generation technique is prone to interferences by transition and easily reducible metals. For the
majority of natural water samples, this type of interference should not be significant. The user should carry out
recovery tests on typical waters and also determine the maximum concentrations of potentially interfering
elements, using appropriate methods. If such interferences are indicated, the level of interferences should be
assessed by performing spike recoveries. However, the atomic fluorescence technique has a high linear
dynamic range and a very low detection limit. In most cases interferences can be removed by a simple dilution
step as long as the final arsenic concentrations are above the LOQ. It is important that the excitation source
does not contain any significant amount of other hydride-forming elements (e.g. antimony when analysing for
arsenic or arsenic when analysing for antimony) that emit fluorescent radiation over the band pass of the
interference filter used in the detector, if these elements are present in the sample.
6
6.1
Apparatus
General
Due to significant differences between the various instrument models and brands available, it is not possible
to give detailed instructions on their operation. The operator should thus refer to the instructions provided by
the manufacturer of each instrument.
Usual laboratory glassware and equipment and, in particular, the following:
6.2 Hydride generation, including a peristaltic pump unit to deliver reagents and load sample injection
loop, mixing manifold to produce covalent hydride species, gas-liquid separator to separate hydride species
from liquid phase reagents and moisture removal device.
NOTE
The concentration and flow of reagents are specifically related to the hydride generation apparatus. The user
should use optimal conditions to ensure that best analytical performance is obtained.
((Remark from SC 2 secretariat: "Should" forms a requirement, which is not allowed in Notes; to be rephrased
or to be changed to normal text))
6.3 High-performance liquid chromatograph (HPLC), including a column for analyte separation and
optionally a chromatographic pre-column. The HPLC system is optionally equipped with an in-line degassing
system and injection system for introducing the sample. In most cases an isocratic pump can be used
although the use of gradient pumps to provide optimal separation times is permitted.
6.4
Sample changer
It is recommended that the HPLC system should be coupled with a sample changer.
NOTE
Annex B.
6.5
Various column/eluent pairings can be used for separating arsenic species. Some examples are described in
Vacuum filtration system, for filtering the eluent and reagents prepared.
Reagents and standards
7.1
General requirements
All reagents shall be of known analytical grade. The concentration of the analyte or interfering substances in
the reagents and water should be negligible compared to the lowest concentration to be determined (LOQ for
example).
NOTE
purity.
Standard stock solutions are commercially available or can be prepared using chemicals of known analytical
7.2
Deionized water, grade 1, as defined in ISO 3696.
7.3
HPLC grade water
HPLC grade water is used to prepare the mobile phase, the calibration solutions and produce the sample
dilutions. It can be prepared by suitably purifying deionized water (7.2).
7.4
Nitric acid (HNO3) = 1,4 g/ml
Acid used at a rate of 0,5 % volume fraction to stabilize the samples for the total arsenic concentration
determination.
Nitric acid is available in the following forms: (HNO3) = 1,40 g/ml (w(HNO3) = 650 g/kg)
(HNO3) = 1,42 g/ml (w(HNO3) = 690 g/kg). These two forms are suitable for use with this method.
NOTE
7.5
97 % sodium hydroxide, analytical grade.
7.6
1N sodium hydroxide solution
and
Weigh 4 g of sodium hydroxide pellets (7.5) and pour them into a 100 ml flask. Make up to the mark with
deionized water (7.2) and stir until the pellets have completely dissolved.
7.7
Hydrochloric acid (HCl) = 1,18 g/ml
7.8
10 % Hydrochloric acid hydride generation solution
Measure 100 ml of high purity hydrochloric acid (7.7) and carefully add to 900 ml of deionized water (7.2).
7.9
Sodium tetrahydroborate NaBH4, analytical reagent grade pellets.
7.10 1,4 % m/V sodium tetrahydroborate / 0,4 % m/V sodium hydroxide hydride generation solution
Weigh 14 g of NaBH4 pellets (7.9) and transfer to a 1 l flask. Add 4 g of NaOH pellets (7.5) and make up to the
mark with deionized water (7.2). Filter if necessary and prepare daily.
7.11 Disodium ethylene diamine tetra-acetate (EDTA), C10H14N2Na2O82H2O (CAS No. 6381-92-6).
7.12 0,2 N EDTA solution

In a 100 ml volumetric flask, dissolve 7,445 g of EDTA (7.11) in approximately 80 ml of water (7.2) and make
up to the mark. This solution has a shelf-life of six months.
7.13 Standard substances
As(III): Arsenous oxide As2O3 (CAS No. 1327-53-3)
As(V): Di-sodium hydrogen arsenate Na2HAsO47H2O (CAS No.10048-95-0)
DMA: Dimethylarsinic acid (CH3)2 AsO2Na 3H2O (CAS No. 124-65-2)
MMA: Monomethylarsonic acid CH3AsO(ONa)26H2O (CAS No. 144-21-8)
7.14 Stock solutions
For each of the species, As(III), As(V), DMA and MMA, prepare a stock solution with a concentration
= 1 000 mg/l expressed as As.
These solutions are prepared from the standard substances (7.13).
In 100 ml flasks, dissolve the suitable mass of each substance (7.13) as shown in Table 1.
Commercially available stock solutions of the required concentration can also be used.
Table 1 Preparation guidelines for 1 000mg/l arsenic standards
Species
Masses to weigh
Final volume
ml
As(III)
0,132
4 ml of 1N NaOH (7.6) made up

to 100 ml with water (7.3)
As(V)
0,416
100 ml in water (7.3)
DMA
0,286
MMA
0,390
These solutions, when stored protected from light and at 4 C, are considered to be stable for one year.
The 10 mg/l single-element working solutions are also stable for one year if they are stored protected from
light and at 4 C and stabilized in 4 % m/V NaOH (7.6) for As(III).
7.15 Calibration solutions

The calibration solutions are prepared from the 1 000 mg/l stock solutions (7.14). Tables 2 and 3 are given as
examples.
Table 2 Preparation guidelines for working arsenic standards
Concentration
Sample volume
g/l
Solution for dilution
qs HPLC grade water

(7.3)
1 000
100 l of each solution
1 000 mg/l (7.14)

As(III), As(V), DMA, MMA
100 ml
500 l
1 000 g/l
100 ml
10
1 000 l
1 000 g/l
100 ml
25
2 500 l
1 000 g/l
100 ml
50
5 000 l
1 000 g/l
100 ml
To prepare standard solutions below 5 g/l use the 10 mg/l intermediate stock to produce a mixed standard of
100 g/l. Table 3 is given as an example.
Table 3 Preparation guidelines for working arsenic standards
Concentration
Sample volume
Solution for dilution
qs HPLC grade water

(7.3)
g/l
100
1 000 l of each solution
10 mg/l (7.14)
As(III), As(V), DMA, MMA
100 ml
0.2
200 l
100 g/l
100 ml
0.5
500 l
100 g/l
100 ml
1.0
1 000 l
100 g/l
100 ml
2.0
2 000 l
100 g/l
100 ml
7.16 Eluents
Various eluents can be used and the choice depends on the type of separation column chosen. The eluent
compositions described in Annex B and in the associated tables are given as an example.
7.17 HPLC trace analysis grade methanol
8 Sampling, preservation and storage of drinking, surface and ground water

samples
Sampling shall be performed in accordance with 5667-1 and ISO 5667-3, using suitable sampling containers,
e.g. HDPE or glass.
The preservation technique described in ISO 5667-3 shall only be applied for the total arsenic determination.
In ISO 5667-3 it is suggested to preserve samples with HNO3 or HCl at pH <2.
This document is based on the preservation of the arsenic species contained in the sample and suggests,
depending on the type of samples, filtering at 0,45 m preferably on-site, particularly in the case of ground
waters (piezometers) and surface waters.
The bibliographic study showed that there were a number of methods to restrict the evolvement of the As(III)
and As(V) species. It has been demonstrated that acidifying with 0,1 % m/V HCl, 0,4 % m/V HNO3 or
0,2 % m/V H2SO4 m/V made it possible to keep both species intact at ambient temperature and at 4 C for at
least 30 d.
It has also been shown that natural water samples filtered at 0,45 m and stored at 4 C in bottles filled to the
rim are stable for 30 d.
In the case of samples of water intended for human consumption with turbidity levels <2 NFU, filtering at
0,45 m is not required.
In the case of dissolved iron-enriched ground water, it is recommended to stabilize with the 0,2 N EDTA
solution (7.11), add 0,5 ml per 9,5 ml of sample under analysis; under these conditions, the samples are
stable for 3 months.
NOTE
If filtration and stabilization are not feasible on-site and to minimize variations in terms of speciation, these
procedures shall be carried out on receipt in the laboratory and within not more than 48 h after sampling.
((Remark SC 2 secretariat: to be rephrased or to be changed to normal text))
9
9.1
Procedure
Instrument optimization
Use the instrument under the manufacturer's standard conditions.

The efficiency of hydride generation is highly dependent on the design and operating conditions of the
apparatus and concentration of reagents. The hydride generation and measurement should be optimized to
ensure that that optimal performance is achieved.
Annex A provides some examples for LC-HG-AFS using different separations.
Start up the HPLC system, set the pump flow rate and couple the column outlet to the hydride generation
manifold. A waiting interval is necessary to obtain a stable signal.
Before running the calibration, check that the chromatographic conditions selected enable satisfactory
separation with minimum resolution between 2 peaks for the highest concentration of standard solution.
Independent from the detection system, identify the analytes by comparison of the retention times for samples
and reference standards. Minimum requirement for identification are retention times within 0,1 min,
respectively relative retention times within 0,5 % over the total run of a chromatogram.
Retention time shift may occur for some natural waters especially they are enriched with other metals. These
compete with the active sites on the column. Sample spikes using the each arsenic species should therefore
be used to confirm species identity if retention shift is observed. The sample may also be diluted to overcome
this effect or alternatively a smaller injection volume may be used.
9.2
Calibration
As a general rule, proceed as follows:

Prepare and measure the blank calibration solution and the calibration solutions prepared in 7.15.
Prepare a calibration graph in accordance with the manufacturer's instructions according to the processing
software used for signal acquisition in coupling mode.
9.3
Sample measurement
The water samples can be injected with or without dilution depending on the total arsenic concentrations
measured previously.
Samples which exhibit retention time shift due to sample matrix should be diluted to overcome the effect if
they have sufficiently high arsenic concentrations to fall in the calibration range. Alternatively the retention
time shift should be verified by species specific spike additions at similar concentration found.
NOTE 1
The use of a smaller injection volume, e.g. 50 l, will help to overcome retention time shift.
NOTE 2
The preliminary total arsenic analysis on the filtered sample provides information on any dilution required prior
to injecting the sample and serves to check the consistency of the results. It is important to ensure that the sum of the
species measured remains less than or equal to the total arsenic value measured.
10 Expression of results
The results obtained are expressed as g As/l, applying the dilution factors used for each sample. Give the
results within 2 significant digits.
Example:
Arsenic(III)
1,2 g/l
Arsenic(V)
0,5 g/l
11 Test report
The test report shall contain at least the following information:
a)
the test method used, together with a reference to this International Standard (PNWI ISO/WD);
b)
complete identification of the sample;
c)
expression of the results as indicated in Clause 10;
d)
any details not specified in this document or considered to be optional, and any particular factor liable to
have affected the results.
Annex A
(informative)
Example - Layout of LC-HG-AFS
3
6
10
11
1
AUTOSAMPLER
2 INJECTION VALUE
3 MOBILE PHASE
4 LC PUMP
5 COLUMN
6 HCL
7 NaBH4
8 GLS
9
AUX H2
10 NAFION DRYER
11 AFS
Figure A.1 Layout of LC-HG-AFS
Annex B
(informative)
Example of experimental conditions
Figure B.1 Chromatogram representing four arsenic species containing 5 g/l;

As(III), DMA, MMA, and As(V) (order of elution)
Table B.1 HG-AFS conditions
HCl concentration
25 % volume fraction
HCl flowrate
1,7 ml/min
NaBH4 concentration
0,7 % m/V in 0,4 % m/V NaOH
NaBH4 flowrate
5 ml/min
Argon carrier gas flowrate
250 ml/min
Air dryer gas flowrate
2,5 l/min
Aux. hydrogen flowrate
Optional (not used in example shown)
Primary current
27,5 mA
Boost current
35,0 mA
Acquisition
Peak area
Table B.2 Chromatographic conditions

Flow rate
Elution gradient: fixed flow rate at 1 ml/min
Column
Hamilton PRP-X 100 (250 mm 4,6 mm) 10 m
Eluent
A: 12,5 mM ammonium carbonate at pH 9

B: 60 mM ammonium carbonate at pH 9
T = 0 min: 100 % A, 0 % B
Gradient
T = 2 min: 0 % A, 100 % B
T = 10 min: 100 % A, 0 % B
Injection loop
10
50 l to 250 l
Table B.3 Calibration conditions

Number of standards
Blank + 5 points
Calibration dynamic range
0,5 g/l to 50 g/l

0,2 g/l to 2 g/l
Limit of detection
(3n-1 calculated)
0,05 g/l for As(III), As(V), DMA & MMA
Limit of quantification
0,16 g/l for As(III), As(V), DMA & MMA

Same conditions as in Table B.1

Pump
Isocratic
Flow rate
Isocratic conditions: fixed flow rate at 1 ml/min
Column
Eluent
2,5 mM NaH2PO4 + 0,5 mM 2Na-EDTA + 2,5 % (volume fraction) methanol at pH=6
Injection loop
50 l to 250 l

Number of standards
Blank + 5 points
0,5 g/l to 50 g/l
0,2 g/l to 2 g/l
0,05 g/l for As(III)
Limit of detection (calculated)
0,1 g/l for As(V) and MMA

0,2 g/l for DMA
0,16 g/l for As(III)
0,33 g/l for As(V) and MMA

0,66 g/l for DMA
11

Table B.7 HG-AFS Conditions

Pump
Isocratic
Flow rate
Isocratic conditions: fixed flow rate at 1 ml/min
Column
Eluent
20 mM (NH4)2HPO4 at pH = 6
Injection loop
50 l to 250 l

Number of standards
12
Blank + 5 points
0,5 g/l to 50 g/l
0,2 g/l to 2 g/l
0,05 g/l for As(III) and MMA
0,1 g/l for As(V) and DMA
0,33 g/l for As(V) and DMA


Pump
Binary
Flow rate
Eluent gradient: fixed flow rate at 1 ml/min
Column

A: 10 mM (NH4)2HPO4/(NH4)H2PO4 in 3 % methanol at pH = 7
Eluent
B: 100 mM (NH4)2HPO4 in 3 % methanol at pH = 8,5

T = 0 min: 100 % A, 0 % B
Gradient
T = 3 min: 0 % A, 100 % B
T = 10 min: 100 % A, 0 % B
Injection loop
50 l to 250 l

Number of standards
Blank + 5 points
0,5 g/l to 50 g/l

0,2 g/l to 2 g/l
0,05 g/l for As(III), MMA and As(V)

0,1 g/l for DMA
0,16 g/l for As(III), MMA and As(V)
0,33 g/l for DMA
13


Pump
Isocratic
Flow rate
Eluent gradient: fixed flow rate at 1 ml/min
Column
Eluent
20 mM NaH2PO4/Na2HPO4 at pH = 6,2
Injection loop
50 l to 250 l

Number of standards
14
Blank + 5 points
0,5 g/l to 50 g/l
0,2 g/l to 2 g/l
0,1 g/l for DMA and As(V)
0,33 g/l for DMA and As(V)
Bibliography
[1]
Cheam, V. and Agemian, H. (1980). "Preservation of inorganic arsenic species at microgram levels
in water samples." Analyst 105(1253): pp. 737-743.
[2]
Aggett, J. and Kriegman, M.R. (1987). "Preservation Arsenic (III) and Arsenic (V) in samples of
Sediment Interstitial water." Analyst 112: pp. 153-157.
[3]
E.M. Hall, G., Pelchat, J.C. et al. (1999). "Stability of inorganic arsenic (III) and arsenic (V) in water
samples." Journal of Analytical Atomic Spectrometry 14(2): pp. 205-213.
[4]
Gallagher, P.A., Schwegel, C.A. et al. (2001). "Speciation and preservation of inorganic arsenic in
drinking water sources using EDTA with IC separation and ICP-MS detection." Journal of
Environmental Monitoring 3(4): pp. 371-376.
[5]
Francesconi, K.A. and Kuehnelt, D. (2004). "Determination of arsenic species: A critical review of
methods and applications, 2000-2003." Analyst 129(5): pp. 373-395.
[6]
GomezAriza, J.L., Sanchez-Rodas, D., Beltran, R., Corns, W.T., Stockwell, P.B. (1998) Evaluation of
Atomic Fluorescence Spectrometry as a Sensitive Detection Technique for Arsenic Speciation. Appl.
Organomet. Chem. 12, pp 439-447.
[7]
Sanchez-Rodas, D., Corns, W.T., Chen, B. and Stockwell, P.B. (2010). Critical Review Atomic
Fluorescence Spectrometry: a suitable detection technique in speciation studies for arsenic, selenium,
antimony and mercury J. Anal. At. Spectrom., 25, pp 933946.
[8]
Gomez-Ariza, J.L., Sanchez-Rodas, D., Giraldez, I., and Morales, E. (2000). A comparison between
ICP-MS and AFS detection for arsenic speciation in environmental samples.Talanta.,2000, 51
pp 257268.
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ISO/TC 147/SC 2

SC2WG52 N 76 Proposal New project Arsenic species HPLC HG-AFS

Proposal for a new project

ISO/TC 147/SC 2/WG 52

For discussion at the meeting of SC 2/WG 52 in Conshohocken, Philadelphia, USA, 2015-06-01;

Scope of the proposed deliverable.

FORM 4 (ISO) v. 2013

Proposal for a new project

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If "Yes", provide full information as annex

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(please specify committee ID)

(please specify committee ID) TC 230

Name of the Proposer

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PNWI ISO/WD Arsenic HPLC HG-AFS

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FORM 4 (ISO) v. 2013

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

Water quality Determination of four chemical arsenic species

Document type: International Standard

PNWI ISO/WD Arsenic HPLC HG-AFS

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

Normative references ............................................................................................................................ 1

Terms and definitions ........................................................................................................................... 2

Reagents and standards ....................................................................................................................... 4

Expression of results ............................................................................................................................ 8

Test report .............................................................................................................................................. 8

Annex A (informative) Example - Layout of LC-HG-AFS ................................................................................. 9

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

Water quality Determination of four chemical arsenic

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

Terms and definitions

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

Reagents and standards

Deionized water, grade 1, as defined in ISO 3696.

HPLC grade water

Nitric acid (HNO3) = 1,4 g/ml

97 % sodium hydroxide, analytical grade.

1N sodium hydroxide solution

Hydrochloric acid (HCl) = 1,18 g/ml

10 % Hydrochloric acid hydride generation solution

Sodium tetrahydroborate NaBH4, analytical reagent grade pellets.

ISO 2015 All rights reserved

PNWI ISO/WD Arsenic HPLC HG-AFS

7.12 0,2 N EDTA solution