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Practical Examples on

Traceability,
Measurement Uncertainty
and Validation
in Chemistry
Volume 2
Edited by
Nineta Majcen, Philip Taylor, Tomas Martišius
Guest Editors: Antonio Menditto, Marina Patriarca

Authors:
Ilaria Altieri Mustafa Memić
Sabrina Barbizzi Antonio Menditto
Jelena Bebić Tidža Muhić-Šarac
Maria Belli Marina Patriarca
Elena Amico di Meane Giancarlo Pistone
Gordana Horvat Michela Sega
Nada L. Lazić Antonella Semeraro
Snježana Marinčić Marjana Simonič
Munir Mehović Brigita Tepuš

EUR24688 EN - 2011
Practical Examples on
Traceability,
Measurement Uncertainty
and Validation
in Chemistry
Volume 2

Edited by:
Nineta Majcen, Philip Taylor
Tomas Martišius

Guest Editors:
Antonio Menditto,
Marina Patriarca

Authors:
Ilaria Altieri
Sabrina Barbizzi
Jelena Bebić
Maria Belli
Elena Amico di Meane
Gordana Horvat
Nada L. Lazić Marina Patriarca
Snježana Marinčić Giancarlo Pistone
Munir Mehović Michela Sega
Mustafa Memić Antonella Semeraro
Antonio Menditto Marjana Simonič
Tidža Muhić Šarac Brigita Tepuš
The mission of the JRC-IRMM is to promote a common and reliable European
measurement system in support of EU policies.

European Commission
Joint Research Centre
Institute for Reference Materials and Measurements

Contact information
Address:
Institute for Reference Materials and Measurements, European Commission, Joint
Research Centre, Retieseweg 111, 2440 Geel, BELGIUM

E-mail: jrc irmm trainmic@ec.europa.eu


Tel.: +32 14571608
Fax: +32 14571863

http://irmm.jrc.ec.europa.eu/
http://www.jrc.ec.europa.eu/

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Cataloguing data can be found at the end of this publication.

Luxembourg: Publications Office of the European Union, 2011

JRC 65988

EUR 24688
ISBN 978-92-79-18998-2
ISSN 1018-5593
doi: 10.2787/36024

© European Union, 2011

Reproduction is authorised provided the source is acknowledged

Printed in Belgium
Table oF ConTenTs

InTrodUCTIon ..................................................................................................................5

GUesT edITorIal ...............................................................................................................7

aboUT The aUThors .....................................................................................................11

abbreVIaTIons and aCronyMs ...............................................................................21

ChapTer 1..........................................................................................................................23
simultaneous measurement of the concentration of retinol and α-tocopherol
in human serum by hplC with UV and fluorimetric detection
Antonella Semeraro, Ilaria Altieri, Elena Amico di Meane, Sabrina Barbizzi, Maria
Belli, Antonio Menditto, Marina Patriarca, Giancarlo Pistone

ChapTer 2..........................................................................................................................57
Measurement of the concentration of cyclamate in soft drinks
by a high-performance liquid chromatographic method
Gordana Horvat, Snježana Marinčić

ChapTer 3..........................................................................................................................95
Measurement of the concentration of arsenic in groundwater
by flame atomic absorption spectrometry (hydride technique)
Jelena Bebić, Nada L. Lazić

ChapTer 4....................................................................................................................... 131


Measurement of the mass fraction of sodium chloride in milk products
by Volhard’s method
Tidža Muhić‑Šarac, Munir Mehović, Mustafa Memić

ChapTer 5....................................................................................................................... 167


Measurement of the concentration of total organic carbon (ToC) in waste water
Brigita Tepuš, Marjana Simonič

3
appendIx 1 ..................................................................................................................... 227
how to use this book

appendIx 2 ..................................................................................................................... 233


TrainMiC exercises (white pages)

appendIx 3 ..................................................................................................................... 249


briefing of the trainees on the example session

4
Introduction

的汉语意思
Learning is like rowing upstream: not to advance is to drop back.
(approximate translation of a Chinese proverb)

Chemical and bio‑analytical measurements are omnipresent and often very


important in our society. Just think of the quality of the food we eat, the air
we breathe … the role of these measurements in healthcare, in trade and
in research. In all these cases, people strive to get reliable data. There is an
international standard for assuring the quality of measurement data, namely
ISO/IEC 17025:2005 (General requirements for the competence of testing and
calibration laboratories). The standard contains particular management as well
as technical requirements. These technical requirements are linked to the science
behind these measurements, meaning that metrological issues such as traceability,
uncertainty and validation are at the heart of this.

In order to provide life long learning in this area, the TrainMiC® programme (http://
www.trainmic.org) was conceived in 2001 by the Institute for Reference Materials
and Measurements of the European Commission’s Joint Research Centre. Firstly,
it addressed the need arising in those countries wanting to become members of
the European Union (EU) at that time. Rather than approaching such training
in an anecdotal way and organising ad hoc events, a programme was set up —
TrainMiC® — to create harmonised training material as well as to disseminate
knowledge in the various countries via a network of authorised TrainMiC®
trainers. Afterwards, the TrainMiC® programme spread to the rest of the EU and
Europe’s largest Lifelong learning programme in this area was created. Up to
the present, 20 national TrainMiC® teams have been set up and more than 6 000
experts had been trained all across Europe by the end of 2010. Trainers quickly
realised the importance of having suitable examples for their training events and
they soon realised that creating examples adapted to the various audiences is quite
a labour intensive activity. For this reason, sharing such examples proved to be an
attractive proposition. Today, examples are reviewed and then published primarily
in an e‑collaboration environment only available to the authorised trainers. It was
then decided to publish some of the examples in the format of a series of books: a
first volume in 2010 with this book being the second volume.

5
Practical examples of traceability, measurement uncertainty and validation in chemistry

Interestingly, one of the ways examples are generated is via a competition between the
national teams which meet at the biannual TrainMiC® convention. In June 2006, this
competition was won by the Bulgarian team (see the example in Chapter 1, Volume 1)
and, in January 2009, it was the Italian team who won (this is the example in Chapter 1,
Volume 2).

Volume 1 contained five examples from the


area of clinical, environmental, food, and
material analysis. From the feedback we
received, it seems that the book has inspired
laboratory practitioners as to how to present
their way of working during accreditation
audits. It has also been used by teachers,
for example within the Euromaster
Measurement Science in Chemistry
(http://www.msc‑euromaster.eu). This new
volume contains five examples covering
some new areas and produced by other
authors.

Nineta Majcen and Philip Taylor

6
Guest editorial

Guest editorial
Salute a TrainMiC®!

Most European regulations and directives require, for their implementation, results of
analytical measurements. To ensure the uniform application of EU legislation across
the Member States, the same quality of analytical results must be achieved. To this aim,
education of practitioners is a key issue and, even more, the harmonisation across the
EU of such education.

In 2006, we came up with a novel idea: a harmonised platform across Europe for the
interactive education of practitioners applying the concepts of metrology in analytical
sciences to the tests they carry out every day. The TrainMiC® programme, born out
of a project in support of the new accession countries, today provides the basis for a
harmonised interpretation of the technical requirements of ISO/IEC 17025:2005 across
the 27 EU Member States and beyond. The programme also provides an interactive
platform through the implementation, by the European Commission’s JRC‑IRMM,
of innovative IT (eRooms), allowing an ongoing interactive exchange of experiences,
knowledge and discussion on emerging issues, of which the TrainMiC® authorised
trainers are the key players.

The Italian national team was, therefore, formed in 2006; public bodies, entrusted
with responsibilities in the field of measurements in general (the National Institute
for Research in Metrology, INRIM) joined forces with those in specific sectors, such
as public health (the National Institute of Health, ISS), the environment (the National
Institute for Environmental Research and Protection, ISPRA) and food control (the
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta). At various
levels, these parties were already carrying out activities aiming to improve the reliability
of analytical results produced in Italy, each according to their area of competence.
These activities included the promotion and dissemination of metrological concepts;
production, certification and/or distribution of reference materials; organisation of
inter‑laboratory comparisons; and training of laboratory staff, quality managers and
managers. Coming together with the TrainMiC® idea provided the spark to bring our

7
Practical examples of traceability, measurement uncertainty and validation in chemistry

competence and experience together, to fulfil our shared mission to promote further
education in metrology applied to analytical sciences, within a European‑wide
environment.

Over four years (2006–10), nine TrainMiC® events were organised in Italy, all of them
approved by the Italian Ministry of Health as part of the programme for the continuous
education of staff providing services related to health. One of the key features of the
TrainMiC® programme is to promote the use of national languages for training and,
accordingly, TrainMiC® presentations and two examples were translated into Italian.
Additional material was produced by the team to cover general aspects, such as the
content of normative references for laboratories seeking accreditation to ISO/IEC
17025:2005 (ISO/IEC 17000:2004 (Conformity assessment — Vocabulary and general
principles); ISO/IEC Guide 99:2007 (International vocabulary of metrology — Basic
and general concepts and associated terms (VIM) Third edition)); the principles of
metrological confirmation of equipment; and more specific issues such as the structure
of the metrological system in Italy and alternative approaches to the estimate of
measurement uncertainty. In total, 320 laboratory staff were trained including analysts,
quality managers, metrology function managers and internal auditors, as well as end‑users
of analytical data, spread across various areas of Italy (north, centre, south, islands). The
training, which included learning evaluation on the request of the Italian Ministry of
Health, was well received and successful for the vast majority of the participants. The
main feedback from the participants was: ‘We need more!’

A key aspect of TrainMiC® is its focus on ‘learning by practicing’. The TrainMiC®


examples are developed by the TrainMiC® national teams, based on real‑life experiences
and according to a standardised model. Examples help the trainees, working together,
to practice what they have learned and stimulate discussion and exchange of knowledge
between trainees as well as between trainers and trainees. Our best achievement as a
team is, therefore, the first of the five examples presented in this book, which shows,
in a practical way and using real experimental data, how to plan a single‑laboratory
validation of an analytical procedure in order to obtain all the necessary information
to estimate the measurement uncertainty of the results. A lot of time and effort was put
into this work by our team member Antonella Semeraro and we were pleased to see it
acknowledged by the TrainMiC® community with the award of the TrainMiC® Cup for
the best proposed example during the 2009 TrainMiC® Convention.

The mascot in this book, who will guide you through the methodology and the five
examples presented here, is Pinocchio, a well‑known Italian character, born out of
the pen of the Italian writer Carlo Collodi in 1881. Pinocchio’s best‑known feature is
that his nose grows when he tells lies. Analysts do not lie: but they may be mistaken,
confused or contradicted if they do not have a strong basis to hold on to. So we chose
Pinocchio’s story as a good example of how success is achieved by learning from
one’s mistakes, with a little bit of advice from those who know (the talking crickets

8
Guest editorial

— please don’t kill them straight away!), some help from the fairies (Always look
around for help! Fairies? Well, they may also use the Internet nowadays!) and a lot
of commitment to the professional code of conduct and practice. The pictures were
created, on our suggestion, by Antoine Cesaroni, whose contribution is gratefully
acknowledged. We hope you enjoy them!

In 2011, at the same time as TrainMiC®’s 10th birthday, Italy celebrates 150 years of
the birth of Italy as one country. The unification of the Italian territory under one flag
was not an easy task to accomplish. Great effort was needed to bring together people of
different origins, traditions, social organisations and even languages, but it was all worth
it and, today, we are proud to celebrate the statesmen who led the change as well as the
people that made it possible. In a somewhat similar way, TrainMiC® brings together
people from different backgrounds, culture, history and languages, to develop a common
understanding of metrology in chemistry across Europe. We would like to say: “Buon
compleanno TrainMiC®!”

Marina Patriarca and Antonio Menditto


TrainMiC® National Team Leaders, Italy

Acknowledgment
The graphic illustrations in this book were created by Antoine Cesaroni on suggestions from
the Italian Team Leaders at the Istituto Superiore di Sanità and his contribution is gratefully
acknowledged.

9
About the authors

Introduction

Philip Taylor
Professor Philip Taylor completed his PhD in analytical chemistry at the University of
Gent in 1986. He started his career as a research fellow for the Belgian Science Foundation,
he then moved to the European R&D Centre of Proctor & Gamble in Brussels. Since
1990, he has worked at the Institute for Reference Materials and Measurements (Geel,
Belgium), which is part of the European Commission’s Joint Research Centre.

Professor Taylor has had many interests during his research career ranging from atomic
spectrometry to mass spectrometry and isotopic measurements, with an emphasis on how
to produce reliable results, and has produced some 200 research papers in areas ranging
from fundamental constants to food, environmental and industrial reference materials.
He enjoys making his activities in metrology relevant to the broader outside world, both
within the Commission services as well as externally. He heads a unit specialising in
technical assistance regarding quality infrastructure (metrology, accreditation) in support
of EU legislation. He initiated and established the TrainMiC® programme as well as a
joint university programme, the Euromaster Measurement Science in Chemistry. He also
lectures on these topics at the University of Maribor in Slovenia and is the vice‑chair
of the Eurachem working group on training and education. For his contributions, he got
awarded by the Polish Chemical Society and University of Maribor.

Nineta Majcen
Nineta Majcen started her career as a researcher at the University of Ljubljana (Slovenia)
where she gained her PhD on the validation of newly developed methods and chemometrics.
She continued her analytical work in quality control laboratories in industry before
stepping into metrology activities at the national and European level. In metrology, she
has mainly been involved in topics related to metrology in chemistry, issues related to
metrological infrastructure and knowledge transfer activities. She also collaborates closely
with accreditation and standardisation bodies and lectures as a guest lecturer at universities,
postgraduate summer schools and other knowledge transfer events. She is the author of
more than 200 bibliographic publications in both research and expert areas.

Several international conferences, workshops, seminars and high‑level events have been
organised under Nineta Majcen’s leadership: for example, the Eurachem workshop on
proficiency testing (2006), EURAMET’s European metrology research programme
launch event (2008), Quality for south‑eastern European countries (2008), the TrainMiC®
Convention (2009), and the Measurement science in chemistry summer school (2009).

Proactively contributing to the TrainMiC® programme since the beginning of the initiative
in 2001, Nineta Majcen received special recognition in 2005 from EC JRC‑IRMM for

11
Practical examples of traceability, measurement uncertainty and validation in chemistry

her contribution to the TrainMiC® programme. She is the Slovenian TrainMiC® team
leader, a member of the TrainMiC® Management Board and chairs the TrainMiC®
Editorial Board. She is the Slovenian representative at Eurachem and is a member of
the Eurachem working group on training and education. She is currently working as the
Secretary General of the European Association for Chemical and Molecular Sciences
(EuCheMS), managing also policy issues in these areas.

Guest Editorial

Antonio Menditto
Antonio Menditto gained his degree in medicine and surgery at the University of Rome
La Sapienza (Italy). He is a senior scientist at the Department of Public Veterinary
Health and Food Safety of the Istituto Superiore di Sanità (Italian National Institute of
Health) and is active mainly in the food and medical analysis fields. He is the author
of more than 100 scientific publications and has been involved in the organisation and
administration of more than 100 courses in the fields of laboratory accreditation, quality
assurance, method validation, uncertainty of measurement and metrological verification
of measuring equipment.

Antonio Menditto has been involved in the organisation and development of external
quality assessment schemes in clinical, environmental and occupational laboratory
medicine and is a member of the Eurachem working group on proficiency testing. He
is also a lead assessor for ISS ORL, the body involved in the accreditation of Italian
laboratories in charge of official control of food products.

Since 2006, Antonio Menditto has been an authorised TrainMiC® trainer and, jointly with
Marina Patriarca, coordinated the TrainMiC® activities in Italy as national TrainMiC®
team leader.

Marina Patriarca
Marina Patriarca gained her PhD in chemistry from the University of Rome La
Sapienza (Italy) and her MSc in medical sciences from the University of Glasgow
(United Kingdom). She joined the Italian National Institute of Health (Istituto Superiore
di Sanità) in 1981, where she still currently works as a senior research scientist. Her
research activity has mainly been devoted to the application of atomic spectrometry
and has involved the development and validation of analytical methods including the
estimation of uncertainty of measurement; population surveys for risk factors, including
environmental exposure to metals; studies on the metabolism of copper and nickel in
humans; the development and organisation of external quality assessment schemes
and the assessment and certification of reference materials. She is author of more
than 80 papers and a member of the Atomic Spectrometry Updates Editorial Board.

12
About the authors

Currently, she supports the quality system at her home institution by providing advice
on metrology issues related to the implementation of the technical requirements of ISO/
IEC 17025:2005. Together with Enzo Ferrara, she represents Italy at Eurachem.

Marina Patriarca has gained considerable experience in training practitioners by lecturing


at more than 50 courses and seminars for the staff of public and private laboratories
devoted to aspects of quality assurance, implementation of ISO standards in testing
laboratories and the uncertainty of measurement. Recently, she has been involved in
training activities for laboratory staff in developing countries.

Since 2006, Marina Patriarca has been an authorised TrainMiC® trainer and, jointly with
Antonio Menditto, coordinated the TrainMiC® activities in Italy as TrainMiC® team
leader: she is also a member of the TrainMiC® Editorial Board.

Chapter 1

Antonella Semeraro
Antonella Semeraro gained her masters degree in chemistry at the University of Rome La
Sapienza (Italy). She continued her analytical work on biological markers at the Istituto
Superiore di Sanità (Italian National Institute of Health) for three years. Then she spent
six months at the Isotope Measurement Unit of the Institute for Reference Materials and
Measurements as a member of the International Measurement Evaluation Programme
(IMEP). As a trainee under supervision, she organised the inter‑laboratory comparison
IMEP‑27 (levels of Cd, As and Pb in a mineral feedstuff).

Antonella Semeraro is currently working as a research scientist at the Department


of Public Veterinary Health and Food Safety of the Istituto Superiore di Sanità. Her
scientific work is mainly in the field of analysis of trace elements in biological fluids by
Inductively coupled plasma mass spectrometry (ICP‑MS). She is also involved in the
organisation of programmes for external quality assessment in preventive medicine and
food safety.

The author of several scientific papers, Antonella Semeraro has also been involved in the
organisation and administration of seven courses in the field of laboratory accreditation,
quality assurance, method validation and metrological confirmation for measuring
equipment.

Since 2007, Antonella Semeraro has been an authorised TrainMiC® trainer and member
of the Italian TrainMiC® team.

13
Practical examples of traceability, measurement uncertainty and validation in chemistry

Ilaria Altieri
Ilaria Altieri gained her masters degree in biology at the University of Rome La Sapienza
in 1991: she is a specialist in biotechnology applications.

Since 1990, Ilaria Altieri has worked at the Istituto Superiore di Sanità (Italian
National Institute of Health) as a researcher in charge of developing analytical methods
(chromatographic and radioimmulogical) to determine drugs and their metabolites
in biological fluids to detect and monitor the activity and toxicity of anti‑epileptic,
anti‑tumor and antipsycotic drugs in pharmacokinetic studies.

Ilaria Altieri worked for five years on the batch release of blood derivatives following
the implementation in Italy of Legislative Decree (22/4/1996). Since 2004, she has
been working in the Food Safety and Veterinary Public Health Department where she is
developing and applying new methodologies for the measurement of concentration of
biomarkers (biochemical and molecular), indicators of risk and protective factors for the
study of the effects of dietary components on human health, and effects of contaminants
such as xenobiotics, residues and additives.

The author of more than 40 scientific papers, Ilaria Altieri has been involved in the
organisation of more than 20 courses in the field of laboratory accreditation, quality
assurance, method validation and the metrological verification of measuring equipment
and has also collaborated on the translation of some of the TrainMiC® material in to Italian.

Elena Amico di Meane


Elena Amico di Meane gained her PhD in ‘Metrology: Science and Technique of
Measurements’ at the Politecnico of Turin (Italy) in 2002, with an experimental thesis,
on the ‘Study of methods for measurement of concentration of gaseous substances in
conditions traceable to the measurement units of the International System’. The research
was carried out at the Institute of Metrology ‘G. Colonnetti’ of the Italian National Council
of Research (IMGC‑CNR). Afterwards, she continued to work, as a research scientist, at
IMGC‑CNR and at the National Electrotechnical Institute ‘G. Ferraris’ (IEN). In 2006,
both institutes merged into the National Institute of Metrological Research (INRIM)
where she continues to be employed. Her field of interest is focused on metrology in
chemistry applied in gas analysis. She has expertise in analytical techniques and the
preparation of reference gas mixtures by the gravimetric method.

Elena Amico di Meane is the author of more than 30 scientific papers and has contributed
to the organisation of several training courses: she is also taking part in the activities of
CCQM.

Elena Amico di Meane joined the TrainMiC® programme in 2007 as a member of the
Italian team of authorised TrainMiC® trainers.

14
About the authors

Sabrina Barbizzi
Sabrina Barbizzi gained her PhD in physics at the University of Bologna (Italy) and
started her career at the Italian National Agency for New Technologies, Energy and
the Environment. Since 2000, she has worked at ISPRA — Istituto Superiore per la
Protezione e la Ricerca Ambientale in Rome (Italy) — in the field of environmental
metrology.

Sabrina Barbizzi is the author of some 40 scientific papers and reports. Her research
interests include the sampling and laboratory analysis by energy dispersive X‑ray
fluorescence of environmental matrices; production and characterisation of reference
materials; evaluation of data of inter‑laboratory comparisons; evaluation of uncertainties
associated with analytical methods and sampling phases; statistical and geostatistical
processing of environmental data for the validation of methods and strategies of soil
sampling.

Sabrina Barbizzi joined the TrainMiC® programme in 2008 as a member of Italian team
of authorised TrainMiC® trainers.

Maria Belli
Maria Belli started her career in 1976 working on neutron activation analysis applied
to environmental science. She is a physicist and until 2000 worked on environmental
radioactivity and radio‑ecology. Since 2000, she has been the head of the Environmental
Metrology Unit of the National Environmental Protection Institute of Italy (ISPRA). The
unit she heads produces environmental reference materials and organises inter‑laboratory
comparisons of environmental pollutants. Since 2009, the Environmental Metrology
Unit has been accredited according to ISO/IEC 17025:2005 and ISO Guide 34:2000
(General requirements for the competence of reference material producers).

Maria Belli is the author of about 150 scientific papers and joined the TrainMiC®
programme in 2007 as a member of the Italian team of authorised TrainMiC® trainers.

Antonio Menditto
(refer guest editorial)

Marina Patriarca
(refer guest editorial)

Giancarlo Pistone
Giancarlo Pistone gained his degree in veterinary medicine at the University of Turin
(Italy). He is the head of the Quality Assurance Department, Istituto Zooprofilattico
Sperimentale del Piemonte, Liguria e Valle d’Aosta, and is also the Director of the

15
Practical examples of traceability, measurement uncertainty and validation in chemistry

Cuneo Section of the Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle
d’Aosta.

Giancarlo Pistone has more than 20 years experience in laboratory management and the
training of staff members and veterinarians concerning validation and quality assurance
in analytical chemistry. He is also a lead assessor for the Italian accreditation body in
charge of accrediting laboratories for the official control of food products. He is the
author of some 40 scientific publications and has been involved in the organisation of
more than 50 courses in the field of laboratory accreditation, quality assurance, method
validation and metrological verification of measuring equipment.

Since 2007, Giancarlo Pistone has been an authorised TrainMiC® trainer and member of
the Italian TrainMiC® team.

Michela Sega
Michela Sega gained her masters degree in chemistry and PhD in analytical chemistry at
the University of Turin (Italy). She has been working at the Italian National Institute of
Metrological Research (INRIM) in Turin (Italy) since 1998.

Michela Sega works in the field of metrology in chemistry, particularly in gas analysis
and organic analysis. Her main activity is devoted to the realisation of primary gas
mixtures with the gravimetric method and to the analysis of organic micro‑pollutants by
means of Gas chromatography‑mass spectrometry (GC‑MS).

Michela Sega closely collaborates with the Italian accreditation body for calibration
laboratories (SIT) for amount of substance. She has active collaborations with Italian
national bodies, such as the National Institute of Health and the Italian Environmental
Protection Agency. She is the Italian contact person for the EURAMET Technical
Committee of Metrology in Chemistry (TC‑MC) and is involved in the activities of the
Consultative Committee for Amount of Substance — Metrology in Chemistry (CCQM).

The author of more than 30 scientific papers, Michela Sega has been involved in the
organisation of more than 20 courses in the field of quality assurance, method validation
and metrological verification of measuring equipment.

Since 2008, Michela Sega has been an authorised TrainMiC® trainer and member of the
Italian TrainMiC® team. Currently, she is chairing the EURAMET Technical Committee
on Metrology in Chemistry (TC‑MC).

16
About the authors

Chapter 2

Gordana Horvat
Gordana Horvat has been involved in analytical chemistry since she started working at
the Institute of Public Health ‘Dr Andrija Štampar’ in Zagreb, Croatia, in 1975. Currently,
she works as a head of laboratory for food additives.

Gordana Horvat has a lot of experience in analytical chemistry gained while working
in the field of water and food analysis using GC and HPLC techniques. Her area of
expertise is the development and implementation of new analytical methods in the field
of food testing using liquid chromatography.

Currently, Gordana Horvat is engaged in the implementation of new analytical methods


for the testing of food additives as a part of the harmonisation of Croatian national
legislation to European Union legislation.

Gordana Horvat lectures on food additives at the Faculty of Applied Health Studies.

Snježana Marinčić
Snježana Marinčić works at the Institute of Public Health ‘Dr Andrija Štampar’ in
Zagreb, Croatia, and holds the position of quality manager. As a quality manager, she is
involved in QA/QC of measurements in the field of testing samples from environmental,
food and commonly used objects.

Snježana Marinčić’s long experience in analytical chemistry includes testing in the field of
water examination, mainly wet chemistry, and the application of liquid chromatographic
methods in the field of environmental and food analysis.

Snježana Marinčić lectures and is a trainer on issues related to laboratory accreditation


and metrology in chemistry such as measurement uncertainty evaluation and QA/QC
measures. She actively collaborates with the Croatian Accreditation Agency, where she
is a member of the working group in inter‑laboratory comparisons, and the Croatian
Metrology Society, where she is a member of Management Board.

Snježana Marinčić is a member of the TrainMiC® Editorial Board and is the national
TrainMiC® team leader for Croatia.

17
Practical examples of traceability, measurement uncertainty and validation in chemistry

Chapter 3

Jelena Bebić
Jelena Bebić works at the Directorate of Measures and Precious Metals, Serbia, in
the field of physico‑chemical measurements and reference materials. She gained her
diploma in 2000 at the Faculty of Technology and Metallurgy, University of Belgrade
(Serbia): her area of study was the antimicrobial activity of essential oils related to their
composition. At present, she is finishing her PhD, studying natural antioxidants in plants.

Jelena Bebić’s main interests are measurement methods and techniques in biotechnology
(GC‑MS, UV‑VIS, GC‑FID, GC‑ECD) and measurement standards in the field of
physico‑chemical quantities (density of liquids, refractometry). An important part of her
work is knowledge transfer in QA/QC for analytical laboratories and the dissemination
of knowledge in metrology in chemistry with a focus on measurement uncertainty.

In 2007, Jelena Bebić joined the TrainMiC® as a member of Serbian team of authorised
TrainMiC® trainers.

Nada L. Lazić
Nada L. Lazić started her professional career at the research and development institute
of the rubber shoe and tyre company Borovo Gumitrade D.O.O., Croatia, where she
worked until 1987. Her main research topics were related to polymer engineering, the
reinforcement of rubbers and methods of rubber testing. From 1987 to 1991, she worked
within the same company as a head of the control quality department laboratory.

In 1991, Nada Lazić joined the Institute of General and Physical Chemistry, Belgrade,
Serbia, and is currently working in polymer science and the quality issues related to
methods of water and industrial waste testing, as a research fellow and quality manager.

In 2004, Nada Lazić gained her MSc at the Faculty of Technology and Metallurgy,
University of Belgrade, Serbia, with work on the influence of silica filler structure and
surface characteristics on the properties of rubber properties.

Nada Lazić has published over 20 scientific papers and presented at numerous international
and national conferences and since 2008 she has been an authorised TrainMiC® trainer
and a member of the Serbian TrainMiC® team.

18
About the authors

Chapter 4

Tidža Muhić‑Šarac
Tidža Muhić‑Šarac started work as a researcher at the motor factory Famos, Sarajevo
(Bosnia and Herzegovina). She finished her PhD study in 1999 at the Faculty of Science,
Department of Chemistry, University of Sarajevo (Bosnia and Herzegovina) with her
thesis ‘Extractability of amounts of metals (Fe, Mn, Cu and Zn) from soils in Bosnia and
Herzegovina’. She has continued her analytical work in the quality control of metals,
alloys, water, soils and food.

Currently, Tidža Muhić‑Šarac is employed at the Faculty of Science, Department of


Chemistry, University of Sarajevo (Bosnia and Herzegovina), as an assistant professor.
A major part of her work is teaching analytical chemistry, environmental chemistry,
quality in analytical chemistry and metrology in chemistry. Her research interests include
experimental and theoretical studies of species metals from water and soil.

Tidža Muhić‑Šarac is the TrainMiC® team leader for Bosnia and Herzegovina and
since 2000 she has also been associated to BATA, the Institute of Accreditation BiH, as
technical assessor and technical expert for measurement uncertainty issues.

Munir Mehović
Munir Mehović finished his masters degree in chemical science in 2009 at the Faculty of
Science, Department of Chemistry, the University of Sarajevo (Bosnia and Herzegovina).
His masters thesis was related to inter‑laboratory collaboration and proficiency testing.
He works at the University Džemal Bijedić, Mostar, in the Department of Chemistry at
the Faculty of Education as a senior assistant in analytical chemistry. His main interest
is quality management and quality assurance in testing analytical laboratories. He has
published several papers in the field of environmental chemistry engineering and is
about to start working on his doctoral dissertation.

Munir Mehović joined the TrainMiC® programme in 2006.

Mustafa Memić
Mustafa Memić began his professional career as a chemist at the Institute of the Centre
of Development and Research of New Materials — Energoinvest — in Sarajevo (Bosnia
and Herzegovina) in 1989. Since 1990, he has worked in the Department of Analytical
Chemistry, Faculty of Science at the University of Sarajevo. He finished his PhD in 2007,
with his thesis ‘Study of biodegradation levels of polycyclic aromatic hydrocarbons
(PAHS) and chlorinated phenols by ligninilytic fungi using gas chromatography with
mass spectrometry detection (GC/MS)’. His main research interests are in the area of
trace metals and analysis with FAAS, ET‑AAS, HG‑AAS and UV/VIS spectrometry.

19
Practical examples of traceability, measurement uncertainty and validation in chemistry

As assistant professor, he lectures on analytical chemistry, electro‑analytical chemistry,


spectroscopic methods of analysis, analytical methods in forensic chemistry, and sensors
and analysis. He has supervised more than 10 MSc and more than 20 BSc students. He
has published results of his research work in over 20 scientific papers and presented
them at numerous international conferences. He was actively involved in the project
SIMCA — a scientific cooperation between research institutions for the study of airborne
particles in important cities of the Adriatic area.

Mustafa Memić is a member of the working group of BAS Technical Committees of


the Institute for Standardisation of Bosnia and Herzegovina and is a member of the
TrainMiC® team for Bosnia and Herzegovina.

Chapter 5

Brigita Tepuš
Brigita Tepuš gained her PhD ‘Hybrid removal of atrazine using catalytic ozonisation and
nitrate using ion exchange from drinking water’ at the University of Maribor (Slovenia)
at the Faculty of Chemistry and Chemical Engineering and started her career at the
Komunalno podjetje Ptuj d.d. in the area of domestic water treatment and analytical
chemistry in 2001.

Brigita Tepuš became the head of the laboratory for analytical chemistry in 2003 and now
works in the field of validation and measurement uncertainty in analytical chemistry.

Marjana Simonič
Marjana Simonič gained her PhD at the University of Maribor (Slovenia) and started her
career at the same Faculty of Chemistry and Chemical Engineering, in the Laboratory
for water treatment in 1992. Her main research interests are in the area of water
treatment, membrane separation and water analyses using UV/VIS spectrometry, atomic
spectrometry, HPLC, and other advanced instrumental techniques. She is the author and
co‑author of more than 30 scientific papers.

Marjana Simonič has been the head of the laboratory for water treatment since 2003. Her
research group has accomplished a number of research and applied projects in the field
of water treatment, wastewater treatment and analytical chemistry.

As assistant professor, Marjana Simonič lectures at the University of Maribor, Faculty


of Chemistry and Chemical Engineering in water treatment and chemistry and water
analysis.

20
About the authors

abbreviations and acronyms

Network of organisations in Europe having the objective of establishing


Eurachem a system for the international traceability of chemical measurements
and the promotion of good quality practices.
EURAMET European Association of National Metrology Institutes
ILC Inter‑laboratory comparisons
PT Proficiency testing
QC Quality control
RM Reference materials

21
Chapter 1
simultaneous measurement of the concentration
of retinol and α-tocopherol in human serum
by hplC with UV and fluorimetric detection
Antonella Semeraro, Ilaria Altieri, Elena Amico di Meane, Sabrina Barbizzi,
Maria Belli, Antonio Menditto, Marina Patriarca, Giancarlo Pistone, Michela Sega
• TrainMiC® example summary form (‘blue page’)
• A short introduction to the analytical procedure (‘slides’)
• All the input needed to do the three exercises (‘yellow pages’)
• The solved exercises (‘green pages’)

23
Practical examples of traceability, measurement uncertainty and validation in chemistry

TrainMiC® example summary form

General information about the example


Simultaneous measurement of concentration of retinol and
Measurand α‑tocopherol in human serum by HPLC with UV and fluorimetric
detection

Example No Ex‑22

Antonella Semeraro, Ilaria Altieri, Elena Amico di Meane, Sabrina Barbizzi,


Authors of the example Maria Belli, Antonio Menditto, Marina Patriarca, Giancarlo Pistone, Michela
Sega

Analytical procedure Developed in‑house

Customer’s requirements Working range, linearity, LoQ (from scientific reference)

24
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

Attached files
File
File No, type and name Content of the file attached Remarks
Yes No
Ex‑22‑1‑I‑vitamines‑
1 — I

About the analytical procedure: short Given by the


serum‑HPLC‑2009‑
introduction
ü lecturer
Ver1.ppt
Description of the analytical
PART I
procedure
ü
Each
The customer’s requirements
participant
PART II concerning the quality of the ü receives their
measurement result
own copy to
2 — Yellow

Ex‑22‑2‑Y‑vitamines‑ Validation of the measurement keep


serum‑HPLC‑2009‑ PART III procedure — relevant equations ü
Ver1.doc and measurement data
You may also
Measurement uncertainty of the want to include
PART IV result — relevant equations and ü the relevant
measurement data certificates
here.
Establishing traceability in
PART I
analytical chemistry
ü

Single laboratory validation of


PART II ü
3 — Green

Ex‑22‑3‑G‑ measurement procedures


vitamines‑serum‑
HPLC2009‑Ver1.doc
Building an uncertainty budget ü
Addendum 1: By spreadsheet
PART III approach
ü
Addendum 2: By dedicated
software
ü

History of the example


Version Uploaded on Short description of the change
1 17 September 2009

25
Practical examples of traceability, measurement uncertainty and validation in chemistry

a short introduction to the analytical procedure

26
Simultaneousmeasurement
Simultaneous measurement
oftheconcentration
of the concentration
ofretinolandα‑tocopherol
of retinolinand
human
α‑tocopherol
serumbyHPLCwith
in human
UVandfluorimetric
serum…detection

27
Practical examples of traceability, measurement uncertainty and validation in chemistry

28
Simultaneousmeasurement
Simultaneous measurement
oftheconcentration
of the concentration
ofretinolandα‑tocopherol
of retinolinand
human
α‑tocopherol
serumbyHPLCwith
in human
UVandfluorimetric
serum…detection

29
Practical examples of traceability, measurement uncertainty and validation in chemistry

all the input needed to do the three exercises (yellow pages)

analytical procedure

simultaneous measurement of the concentration of retinol and


α-tocopherol in human serum by hplC with UV and fluorimetric
detection

parT I ...................................................................................................................................31
description of the analytical procedure

parT II .................................................................................................................................34
The customer’s requirements concerning the quality of the measurement result

parT III ................................................................................................................................35


Validation of the measurement procedure — relevant equations and
measurement data

parT IV ................................................................................................................................38
Measurement uncertainty of the result — relevant equations and measurement
data

30
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

PART I. Description of the analytical procedure

Aim
Oxidative damage caused by free radicals plays an important role in the development
of several degenerative pathologies. The vitamins retinol and α‑tocopherol are major
components of the antioxidant system in humans, protecting cell membranes against
peroxidation. Serum concentrations of retinol and α‑tocopherol are the best available
biomarkers of their respective levels in the body.

The aim of the analytical procedure is to quantify vitamins in serum to allow the
investigation of the antioxidant status in relation to the development of degenerative
pathologies.

Measurement principle
Serum samples are extracted using a liquid‑liquid technique. Since retinol and
α‑tocopherol are affected by the oxidising nature of air, ultraviolet light, high temperature
and oxidant agents, t‑butyl‑hydroxytoluene (BHT) is added as a protective factor during
the extraction step.

Retinol and α‑tocopherol are separated on LC‑18 pre‑column and column by isocratic
elution with methanol as the mobile phase at a flow rate of 1 mL min– 1.

The effluent is monitored by measuring its absorbance at 292 nm, to quantify α‑tocopherol,
and its fluorescence, at 340 nm in excitation and 520 nm in emission, to quantify retinol.
To compensate for possible losses of analyte, two internal standards are used: retinyl
acetate for retinol and α‑tocopheryl acetate for α‑tocopherol.

Equipment
— HPLC equipped with UV and fluorimetric detectors
— UV‑IS spectrophotometer
— Analytical balance
— Automatic pipettes
— Centrifuge

Reference materials
Retinol alcohol (≥ 99.0 %), α‑tocopherol (≥ 98.0 %), retinyl acetate (~ 100 %), α‑tocopheryl
acetate (≥ 97.0 %) were purchased from Fluka (Buchs/Schweiz, Switzerland). The last
two compounds are used as internal standards.

Stock solutions, at approximate concentrations of 100 mg L– 1 (retinol), 600 mg L– 1


(α‑tocopherol and retinyl acetate) and 1 000 mg L– 1 (α‑tocopheryl acetate) are prepared

31
Practical examples of traceability, measurement uncertainty and validation in chemistry

in acetonitrile from weighed amounts of vitamins. Tocopherol is a slightly viscous


pale yellow oil and for this reason it is difficult to weigh accurately; to overcome this
problem, concentrations of retinol and α‑tocopherol in the stock solutions are estimated
spectrophotometrically after dilution in ethanol on the basis of their molar absorption
coefficient values. In addition, internal standard solutions (retinyl acetate (5.6 mg L– 1)
and α‑tocopheryl acetate (210 mg L– 1)), to be added to the control and human serum
samples, are prepared by dilution of the respective stock solution in acetonitrile. Stock
solutions are stored in plastic containers covered with aluminium foil and kept at – 20 °C
for up to three months.

Three calibration materials are prepared to assess linearity by diluting the stock solutions
in acetonitrile to the final concentrations of 0.2, 0.6 and 1.0 mg L– 1 for retinol, 5.0,
15.0 and 25.0 mg L– 1 for α‑tocopherol, 0.80 and 30.0 mg L– 1 for retinyl acetate and
α‑tocopheryl acetate respectively. All working standard solutions are protected from
light and kept at + 4 °C for not more than a week.

Reference materials for quality control


Matrix‑matched control materials (CM) were prepared from a pool of
human sera obtained from healthy subjects. Two portions (25 g each) of the
pool, one unspiked and the other spiked with known amounts of the stock
solutions of retinol and α‑tocopherol to the final added concentration of
0.34 mg L–1 and 12.6 mg L–1, respectively, were aliquoted into amber tubes (1.5 mL) and stored
at – 80 °C.

Certified reference materials (CRM)


The two‑level CRM ‘Fat‑soluble vitamins, carotenoids and cholesterol in human serum’
(Standard reference material SRM 968c) was purchased from the NIST (Gaithersburg,
MD, USA).

Reagents
All reagents are at least PA grade:
— Phosphate saline buffer (PBS)
— BHT
— Butanol
— Ethyl acetate
— K2HPO4
— Methanol HPLC grade

32
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

Sample pretreatment
Dilute 200 µL of serum with 300 µL of PBS into an amber tube. Add 50 µL of each
internal standard solution followed after vortexing for 15 s by 250 µL of the butanol
and ethyl acetate solution containing 5 g L– 1 of BHT and further vortexing for 60 s.
Finally add 150 µL of the K2HPO4 saturated solution, vortex for 30 s, then centrifuge at
10 000 rpm for 10 min. The organic upper layer has to be transferred to a 1.5 mL amber
tube and centrifuged again at 8 000 rpm for 5 min, then placed into an amber auto‑sampler
vial and injected (20 µL) into the HPLC apparatus. The auto‑sampler is cooled to
8 °C.

Calibration
In routine work, only one calibration standard (retinol 0.6 mg L– 1; α‑tocopherol
15 mg L– 1) is run at the beginning of the day. The daily measurement of the calibration
standard is used to calculate sample concentrations from the following formula:

Areasample Std
AreaSTDi
C = CSTD × sample
× × DF
AreaSTDi AreaStd

where:
CSTD = Working standard concentration
(0.6 mg L– 1 for retinol and 15 mg L– 1 for α‑tocopherol)
Areasample = Peak area of sample
AreaStd = Peak area of standard
sample
AreaSTDi = Peak area of internal standard for sample
Std
AreaSTDi = Peak area of internal standard for standard
DF (dilution factor) = 1.75

33
Practical examples of traceability, measurement uncertainty and validation in chemistry

PART II. The customer’s requirements concerning quality


of the measurement result

The requirements for linearity, working range and limit of quantification (LoQ) are
defined considering the ranges of values reported for healthy subjects in the scientific
literature as follows:
— working range and linearity:

0.19–0.92 mg L– 1 for retinol


3.1–22.4 mg L– 1 for α‑tocopherol,
— LoQ:
0.19 mg L– 1 for retinol
3.1 mg L– 1 for α‑tocopherol.

Desirable quality specifications for intermediate precision (I %), bias (B %) and total
allowable error (TE %) can be defined based on the components of biological variability,
namely, within and between subjects variation 1. A database of such information is
available online (http://www.westgard.com).

Performance targets defined in this way were:


I % < 6.8 %, B % < 5.8 %, TE % = 17.1 % for retinol
I % < 6.9 %, B % < 5.1 %, TE % = 16.5 % for α‑tocopherol.

The requirement for TE % is intended as a requirement for expended measurement


uncertainty.

1
Fraser, C. G., Scandanavian Journal of Clinical Laboratory Investigation, 59 (1999), 487.

34
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

parT III. Validation of the measurement procedure — relevant


equations and measurement data

Linearity
Calibration curves were studied in the following concentration ranges: 0-1.0 mg L– 1
for retinol and 0-20.0 mg L– 1 for α‑tocopherol. Twelve standard curves were analysed
and the determination coefficients were always greater than 0.998 for retinol and 0.996
for α‑tocopherol. In addition, linearity was verified by visual inspection and residual
analysis.

Limit of detection and quantification


Since retinol and α‑tocopherol are always present in serum, a matrix blank was
simulated by a fourfold diluted serum from normal subjects and analysed 11 times under
repeatability conditions.

The results of the 11 measurements under repeatability conditions are shown in Table 1.

Table 1: Results under repeatability conditions


Retinol α‑tocopherol
Measurement
mg L– 1 mg L– 1
1 0.154 1.651
2 0.140 1.691
3 0.141 1.731
4 0.140 1.609
5 0.146 1.545
6 0.152 1.645
7 0.147 1.581
8 0.153 1.715
9 0.147 1.725
10 0.145 1.427
11 0.144 1.652

Precision

Repeatability and intermediate precision

Two different CMs near the extremes of the physiological range were analysed 10 times
in the same analytical session. The same samples were reanalysed in different analytical
sessions, over a period of three months to evaluate intermediate precision. The means, the
standard deviations and the relative standard deviation (RSD %) are shown in Table 2.

35
Practical examples of traceability, measurement uncertainty and validation in chemistry

Table 2: Repeatability and intermediate precision data

Retinol (mg L– 1) α‑tocopherol (mg L– 1)


CM 1 CM 2 CM 1 CM 2
Repeatability Mean 0.53 0.88 4.0 14.9

SD 0.01 0.03 0.1 0.3

RSD % 2.7 % 3.3 % 2.9 % 2.2 %

n 10 10 10 10

Intermediate Mean 0.53 0.85 4.2 15.7

precision SD 0.03 0.03 0.1 0.4

RSD % 4.6 % 3.6 % 3.2 % 2.4 %

n 24 26 25 26

Dependency of repeatability and intermediate precision on concentration was tested by


means of test F, with critic F value of F9.9 = 3.18, F23.25 = 2.02 and F24.25 = 1.96.

Trueness
Trueness was studied by analysing, under repeatability conditions,
the NIST SRM 968c (n = 5) consisting of two vials at different levels
of concentration. In order to verify the absence of systematic errors,
a significance test was applied (with t = 2.78 (n - 1 = 5 - 1; p = 0.05)) using the ratios
shown in the following formula:

1 − Rm
≤t
u '( Rm)

where:
Rm is the ratio between the mean measured value and the certified value and u(Rm) is the
uncertainty associated with the bias estimate, calculated as follows:

36
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

where:
u(Cr) = Standard uncertainty calculated from the expanded uncertainty associated
to the certified value divided by the stated coverage factor k (k = 2);

  u(C )  2  RSD  2 
u '( Rm) =  r
 +   
  Cr  n  

Cr = Certified value
RSD = Relative standard deviation obtained for replicate measurements of the
CRM
n = Number of repeated measurements of the CRM

Compound Retinol (mg L– 1) α‑tocopherol (mg L– 1)


CRM 1 CRM 2 CRM 1 CRM 2
Cr ± U (Cr) 0.841 ± 0.027 0.484 ± 0.012 7.47 ± 0.47 16.79 ± 0.76
Mean 0.83 0.49 7.5 16.9
SD 0.01 0.02 0.2 0.3
RSD % 1.2 4.0 2.1 1.9
N 5 5 5 5
Rm 0.987 1.012 1.004 1.006
u’(Rm) 0.017 0.022 0.033 0.024
|1‑Rm| /u(Rm) 0.764 0.545 0.121 0.250

U(Cr) = expanded uncertainty (coverage factor = 2 for a confidence level of 95 %).

37
Practical examples of traceability, measurement uncertainty and validation in chemistry

PART IV. Measurement uncertainty of the result:


relevant equations and measurement data

Retinol
Input quantity Value Units Remarks
CSTD 0.60 mg L – 1
Calibration standard concentration
AreaStd 859 007.8 AU Peak area of standard
Areasample 468 383.53 AU Peak area of sample

Area Std Peak area of internal standard in the calibration


STDi 522 950.67 AU
standard
sample
AreaSTDi 535 940.5 AU Peak area of internal standard in the sample

DF 1.75 — Dilution factor

α‑tocopherol

Input quantity Value Units Remarks


CSTD 14.83 mg L – 1
Calibration standard concentration
AreaStd 102 256.8 AU Peak area of standard
Areasample 23 652.8 AU Peak area of sample
Std
AreaSTDi Peak area of internal standard in the calibration
75 853.2 AU
standard
sample
AreaSTDi 72 136.4 AU Peak area of internal standard in the sample

DF 1.75 // Dilution factor

Relevant equations

1. Combined relative standard uncertainty of the result

u(C )
u 'c ( P ) + u 'c ( Rm ) + u ' ( x )
2 2 2
=
C
where:
u 'c (P) = The best estimate of precision
u 'c (Rm) = The uncertainty contribution associated with the bias estimate
u 'c (x) = Any other relevant contribution to uncertainty (e.g. method robustness,
sample variability)

38
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

2. Best estimate of precision contribution, expressed as relative SD


( n1 − 1)( RSD1 )2 + + ( ni − 1)( RSDi )2
u 'c ( P ) =
( n1 − 1) + + ( ni − 1)

This equation is valid when precision is independent on concentration.

3. Uncertainty of the bias estimate


u '1 ( Rm)2 + + u 'i ( Rm)2
u 'c ( Rm) =
i

  u(C )  2  RSD  2 
u 'i ( Rm) =  r
 +   
  Cr  n  

where:
u(Cr) = Standard uncertainty calculated from the expanded uncertainty associated
to the certified value divided by the stated coverage factor k (k = 2)
Cr = Certified value
RSD = Relative standard deviation obtained for replicate measurements of the
CRM
n = Number of repeated measurements of the CRM
i = Number of analysed CRM

4.  Other contributions to uncertainty, u ' ( x ) — relative standard uncertainty of


this contribution

Other contributions such as sample variability or robustness were not taken into account
at this stage.

39
Practical examples of traceability, measurement uncertainty and validation in chemistry

The solved exercises (green pages)

TrainMiC® exercises

Analytical procedure

Simultaneous measurement of the concentration of retinol


and a‑tocopherol in human serum by HPLC with UV and
fluorimetric detection

exerCIse 1:
establishing traceability in analytical chemistry

exerCIse 2:
single laboratory validation of measurement procedures
Part I: General issues
Part II: Parameters to be validated
Part III: Some calculations and conclusions

exerCIse 3:
building an uncertainty budget

40
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

1. Specifying the analyte and measurand

Analyte Retinol and α‑tocopherol

Measurand Concentration of retinol and α‑tocopherol in human serum in mg L– 1

Units mg L– 1

2. Choosing a suitable measurement procedure with associated model equation


The analytes are isolated from the serum samples by extraction with a mixture of
Measurement butanol and ethyl acetate 1:1 (v/v). The extract is analysed by HPLC with UV and
procedure fluorescence detection. Separation is accomplished using LC‑18 pre‑column and
column by isocratic elution with methanol at a flow rate of 1 mL min– 1.

Type of calibration Standard curve Standard addition Internal standard

Model equation:
Areasample Std
AreaSTDi
C = CSTD × sample
× × DF
AreaSTDi AreaStd

where:
CSTD = Calibration standard concentration (0.6 mg L– 1 for retinol, 15 mg L– 1
for α‑tocopherol)
Areasample = Peak area of sample
AreaStd = Peak area of standard
sample
AreaSTDi = Peak area of internal standard for sample
Std
AreaSTDi = Peak area of internal standard for standard

DF = Dilution factor

41
Practical examples of traceability, measurement uncertainty and validation in chemistry

3. List the input quantities according to their influence on the uncertainty of the
result of the measurement (first the most important ones): at this point, your
judgement should be based on your previous experience only.
Area sample
1 Areasample and STD : extraction efficiency, separation efficiency, UV and fluorimetric

quantification, repeatability, vitamins degradation

2 Areastandard and AreaSTDi


Std
: separation efficiency, UV and fluorimetric quantification, repeatability,
vitamins degradation
CSTD: Absorbance (spectroscopic measurement of standard concentrations), Volume (necessary
3
dilutions to obtain calibration standards)
4 DF (Vf /Vi): pipette calibration, repeatability, temperature
5

4. List the reference standards needed and state the information regarding
traceability of the reference value

For the analyte


Retinol alcohol (≥ 99.0 %), retinyl acetate (~ 100 %), Fluka (Buchs/Schweiz, Switzerland)
Name/chemical The standard content is evaluated considering the UV absorbance of the standard
formula/producer solution. Traceability is based on the calibration of the UV spectrophotometer and the
molar absorption coefficient value,
α‑tocopherol (≥ 98.0 %), α‑tocopheryl acetate (≥ 97.0 %) Fluka (Buch/Schweiz,
Switzerland)
Name/chemical
The standard content is evaluated considering the UV absorbance of the standard
formula/producer
solution. Traceability is based on the calibration of the UV spectrophotometer and the
molar absorption coefficient value,
Name/chemical
formula/producer

For the other input quantities


Quantity/equipment/calibration
1 (e.g. mass/balance/calibrated by Volume/automated pipettes/calibrated by an accredited centre
NMI, U  = xx (k = 2))
2 Quantity/equipment/calibration Volume/volumetric flasks/Class A
Absorbance/UV‑VIS spectrophotometer/certified filters and
3 Quantity/equipment/calibration
solutions

UV detection‑relative measurement. Fl intensity/detector/


4 Quantity/equipment/calibration
relative measurement, not directly part of the traceability chain.

42
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

5. Estimating uncertainty associated with the measurement


Are all important parameters included in the model
Yes No 
equation?
Other important parameters:

6. How would you prove traceability of your result?


Analysing certified reference materials: CRM: ‘Fat‑soluble vitamins, carotenoids and cholesterol in
1 human serum’ (Standard reference material SRM 968c), NIST (Gaithersburg, MD, USA).
2 Using traceable calibrated glassware and instrumentation

7. Any other comments, questions …

43
Practical examples of traceability, measurement uncertainty and validation in chemistry

SINGLE LABORATORY VALIDATION OF


MEASUREMENT PROCEDURES

parT I: General IssUes

1. Specify the measurement procedure, analyte, measurand and units


The analytes are isolated from the serum samples by extraction with a
mixture of butanol and ethyl acetate 1:1 (v/v). The extract is analysed by
The measurement procedure HPLC with UV and fluorescence detection. Separation is accomplished using
LC‑18 pre‑column and column by isoctratic elution with methanol at a flow
rate of 1 mL min– 1.
Analyte Retinol and α‑tocopherol

The measurand Concentration of retinol and α‑tocopherol in human serum in mg L– 1

Units mg L– 1

2. Specify the scope


Matrix Serum

Retinol: 0.05–1.0 mg L– 1
Measuring range
α‑tocopherol: 0.95–23.0 mg L– 1

3. Requirements of the measurement procedure


Intended use of the
Assessment of vitamin status in population groups
results
Parameters to be validated Value requested by the customer
LOD
Retinol: < 0.19 mg L– 1
LoQ
α‑tocopherol: < 3.1 mg L– 1
Repeatability
Mark the customer’s
requirements and give Retinol: 3.2-6.8 %
Within‑lab reproducibility
their values α‑tocopherol: 3.8-6.9 %
Retinol: 5.4-7.8 %
Trueness
α‑tocopherol: 5.6-8.9 %
Retinol: 10.7-17.1 %
Measurement uncertainty
α‑tocopherol: 11.9-18.5 %
Other — state

44
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

4. Origin of the measurement procedure


VALIDATION

New in‑house method Full

Modified validated method Partial

Official standard method Confirmation/verification

45
Practical examples of traceability, measurement uncertainty and validation in chemistry

parT II: paraMeTers To be ValIdaTed

5. Selectivity/interference/recovery

Where yes, give further information, for example which CRM, reference method.
CRM/RM: analysis of available CRM or RM
Further information
‘Fat‑soluble vitamins, carotenoids and cholesterol in human serum’ (Standard reference material
SRM 968c) purchased from the NIST
Spike of pure substance

Compare with a reference method

Selectivity, interferences

Test with different matrices

Other — specify

6. Measuring range
Linearity
Upper limit
LOD
LoQ

7. Spread — precision
Repeatability
Reproducibility (within Lab)
Reproducibility (between labs)

46
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

8. Robustness
Variation of parameters

9. Quality control
Control charts

Participation in PT schemes

10. Other parameters to be tested


Working range and testing of homogeneity of variances

R square

Residual standard deviation

Standard deviation of the analytical procedure

Coefficient of variation of the analytical procedure

Measurement uncertainty

47
Practical examples of traceability, measurement uncertainty and validation in chemistry

parT III: soMe CalCUlaTIons and ConClUsIons

11. Calculation of parameters requested by the customer


Parameters
requested to be Calculations
validated
Retinol: 0.015 mg L– 1
α‑tocopherol: 0.29 mg L– 1
LOD
Calculated as: 3 × SD
Retinol: 0.05 mg L– 1
α‑tocopherol: 0.95 mg L– 1
LoQ
Calculated as: 10 × SD

SD
RSD % = × 100
mean

Retinol: 2.7 % at the concentration level of 0.53 mg L– 1; 3.3 % at the


concentration level of 0.88 mg L– 1
Repeatability
α‑tocopherol: 2.9 % at the level of 4.0 mg L– 1; 2.2 % at the level of 14.9 mg L‑1
Retinol: 4.6 % at the concentration level of 0.53 mg L– 1; 3.6 % at the
Within‑lab concentration level of 0.85 mg L– 1
reproducibility
α‑tocopherol: 3.2 % at the level of 4.0 mg L– 1; 2.4 % at the level of 15.7 mg L– 1
Retinol: 98.7 % at the level of 0.83 mg L– 1 (B % = – 1.3 %); 101.2 % at the level of
0.49 mg L– 1 (B % = 1.2 %)
Trueness
α‑tocopherol: 100.4 % at the level of 7.5 mg L– 1 (B % = 0.4 %); 100.6 % at the
level of 16.9 mg L– 1 (B % = 0.6 %)
Retinol: u(C)/C % = 4.6 %; U(C)/C = 9.2 %
Measurement
uncertainty α‑tocopherol: u(C)/C % = 4.1 %; U(C)/C % = 8.2 %

Other —state

48
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

12. Does the analytical procedure fulfil the requirement(s) for the intended use?
Is the
Value requested by the
Value obtained requirement
Parameter customer
during validation fulfilled?
(the same as stated in Question 3)
Yes/No
Retinol: 0.015 mg L– 1
LOD α‑tocopherol:
0.29 mg L– 1
Retinol: 0.05 mg L– 1
Retinol: < 0.19 mg L– 1 Yes
LoQ α‑tocopherol:
α‑tocopherol: < 3.1 mg L– 1 Yes
0.95 mg L– 1
Retinol: 2.7-3.3 %
Repeatability α‑tocopherol:
2.9-2.2 %
Retinol: u’(P) = 6.8  % Retinol: 4.1 % Yes
Within‑lab
reproducibility
α‑tocopherol: u’(P) = 6.9  % α‑tocopherol: 2.8 % Yes
Retinol:
Retinol: B % = 5.8 % Yes
B % = 1.2-1.3 %
Trueness
α‑tocopherol:
α‑tocopherol: B % = 5.1 % Yes
B % = 0.4-0.6 %
Retinol: Yes
Retinol: 10.7-17.1 %
Measurement U(C)/C % = 9.2 %
uncertainty α‑tocopherol:
α‑tocopherol: 11.9-18.5 %
U(C/C) % = 8.2 % Yes

Other

The analytical procedure is fit for the intended use:


Yes No

For measurement uncertainty and traceability, refer to the corresponding pages.

49
Practical examples of traceability, measurement uncertainty and validation in chemistry

BUILDING AN UNCERTAINTY BUDGET

1. Specify the measurand and units


Measurand Concentration of retinol and α‑tocopherol in human serum in mg L– 1
Units mg L– 1

2. Describe the measurement procedure and provide the associated model


equation
Measurement procedure:
The analytes are isolated from the serum samples by extraction with a mixture of butanol
and ethyl acetate 1:1 (v/v). The extracts are analysed by HPLC with UV and fluorescence
detection. Separation is accomplished using LC‑18 pre‑column and column by isocratic
elution with methanol.

Model equation:
Areasample Std
AreaSTDi
C = CSTD × sample
× × DF
AreaSTDi AreaStd

where:
CSTD = Working standard concentration (0.6 mg L– 1
for retinol,
15 mg L– 1 for α‑tocopherol)
Areasample = Peak area of sample
AreaStd = Peak area of standard
sample
AreaSTDi
= Peak area of internal standard for sample

Std
AreaSTDi
= Peak area of internal standard for standard

DF (dilution factor) = 1.75

3. Identify (all possible) sources of uncertainty


Uncertainty of concentration of reference solutions
Uncertainty of measurements of peak area
Method bias
Matrix effect
Other: sample pretreatment
Other:
Other:

50
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

4. Evaluate the value of each input quantity


Retinol:

Input quantity Value Units Remarks


CSTD 0.60 mg L – 1
Calibration standard concentration
AreaStd 859 007.80 AU Peak area of standard
Areasample 468 383.53 AU Peak area of sample
Std Peak area of internal standard for
AreaSTDi 522 950.67 AU
standard
sample
AreaSTDi 535 940.50 AU Peak area of internal standard for sample

DF 1.75 // Dilution factor

α‑tocopherol:

Input quantity Value Units Remarks


CSTD 14.8 mg L – 1
Calibration standard concentration
AreaStd 102 256.80 AU Peak area of standard
Areasample 23 652.80 AU Peak area of sample
Std Peak area of internal standard for
AreaSTDi 75 853.20 AU
standard
sample
AreaSTDi 72 136.40 AU Peak area of internal standard for sample

DF 1.75 // Dilution factor

5. Calculate the value of the measurand, using the model equation


Retinol:
468 383.53 522 950.67
C = 0.60 × × × 1.75 = 0.56 mg L−1
535 940.50 859 007.80

α‑tocopherol:
23 652.8 75 853.2
C = 14.8 × × × 1.75 = 6.3 mg L−1
72 136.4 102 256.8

51
Practical examples of traceability, measurement uncertainty and validation in chemistry

6. Evaluate the combined standard uncertainty (uc) using validation data


Retinol:

1. Precision:
( n1 − 1)( RSD1 )2 + ( n2 − 1)( RSD2 )2 ( 24 − 1)(0.046)2 + ( 26 − 1)(0.036)2
u 'c ( P ) = = = 0.041
( n1 − 1) + ( n2 − 1) ( 24 − 1) + ( 26 − 1)

2. Uncertainty of the bias estimate:

  0.027  2 
  u(C )  2  RSD  2     0 .012 
2 

u '1 ( Rm) =  r
 +   =  2  +    = 0.017
  Cr  n     0.841   5  
  
 

  0.012  2 
  u(C )  2  RSD  2     0 . 04 
2 

u '2 ( Rm) =  r
 +   =  2  +   = 0.022
  Cr  n     0.484   5  
  
 

u1 ( Rm)2 + u2 ( Rm)2 (0.017 )2 + (0.022)2


u 'c ( Rm) = = = 0.0220
2 2

3. Other contributions

None taken into account at this stage.

4. Combined relative standard uncertainty:

u(C )
= u 'c ( P)2 + u 'c ( Rm)2 = (0.041)2 + (0.020)2 = 0.046
C
u(C)/C % = 4.6 %

α‑tocopherol:

1. Precision:
( n1 − 1)( RSD1 )2 + ( n2 − 1)( RSD2 )2 ( 25 − 1)(0.032)2 + ( 26 − 1)(0.024)2
u 'c ( P ) = = = 0.028
( n1 − 1) + ( n2 − 1) ( 25 − 1) + ( 26 − 1)

52
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

2. Uncertainty of the bias estimate:

  0.47  2 
  u(C ) 2
 RSD  
2    0 .21 
2 

u '1 ( Rm) =  r
+ =  2  +   = 0.033
  Cr 
  n     7.47   5  

  
 

  0.76  2 
  u(C )  2
 RSD  
2    0.19 
2 

u '2 ( Rm) =  r
+ =  2  +   = 0.024
  Cr 
  n     16.79   5  

  
 

u1 ( Rm)2 + u2 ( Rm)2 (0.033)2 + (0.024)2


u 'c ( Rm) = = = 0.0229
2 2

3. Other contributions

None taken into account at this stage.

4. Combined relative standard uncertainty:


u(C )
= u 'c ( P)2 + u 'c ( Rm)2 = (0.028)2 + (0.029)2 = 0.041
C
u(C)/C % = 4.1 %

7. Calculate expanded uncertainty (U) and specify the coverage factor k and the
units
Retinol:

U(C)/C = 2 × 0.046 = 0.092

K = 2, confidence level of 95 %

U(C)/C % = 9.2 %

U(C)sample = 0.092 × 0.56 = 0.06 mg L– 1

53
Practical examples of traceability, measurement uncertainty and validation in chemistry

α‑tocopherol:

U(C)/C = 2 × 0.041 = 0.082

k = 2, confidence level of 95 %

U(C)/C % = 8.2 %

U(C)sample = 0.082 × 6.3 = 0.6 mg L– 1

8. Analyse the uncertainty contribution and specify the main three input quantities
contributing the most to Uc
1 Retinol: Precision
α‑tocopherol: the contribution of the uncertainty of the bias estimate and of measurement precision
2
are equivalent
3

9. Prepare your Uncertainty budget report


Retinol:
C = (0.56 ± 0.06) mg L– 1
k = 2, confidence level of 95 %

α‑tocopherol:
C = (6.3 ± 0.5) mg L– 1
k = 2, confidence level of 95 %

54
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…

Further reading

1. Halliwell, B., Gutteridge, J. M. C., Free Radicals in Biology and Medicine, Fourth
edition, Oxford University Press (2006).

2. Joint WHO/FAO Expert Consultation, Diet, Nutrition and Prevention of Chronic


Diseases, WHO, Geneva (2003).

3. Nierenberg, D. W., Lester, D. C., Journal of Chromatography, 345 (1985), p. 275.

4. Semeraro, A. et al., Journal of Chromatography B, Analytical Technologies in the


Biomedical and Life Sciences, 877(11-12) (2009), pp. 1209-1215.

5. BIMP, IEC, IFCC, ISO, IUPAC, IUPAP, OIML, Guide to the Expression of Uncertainty
in Measurement, ISO, Geneva (1995).

6. Eurachem, The Fitness for Purpose of Analytical Methods, Eurachem (1998)


(http://www.eurachem.org).

7. Eurachem/CITAC, Eurachem/CITAC Guide — Quantifying Uncertainty in Analytical


Measurement, Second edition, Eurachem (2000) (http://www.eurachem.org).

8. Fraser, C. G., Scandanavian Journal of Clinical Laboratory Investigation, 59 (1999),


487.

55
Chapter 2
Measurement of the concentration of cyclamate
concentration in soft drinks by a high-performance
liquid chromatographic method
Gordana Horvat, Snježana Marinčić

• TrainMiC® example summary form (‘blue page’)


• A short introduction to the analytical procedure (‘slides’)
• All the input needed to do the three exercises (‘yellow pages’)
• The solved exercises (‘green pages’)

57
Practical examples of traceability, measurement uncertainty and validation in chemistry

TrainMiC® example summary form

General information about the example


Measurement of concentration of cyclamate concentration in soft
Measurand drinks by
a high‑performance liquid chromatographic method
Example No Ex‑17

Authors of the example Gordana Horvat, Snježana Marinčić

HRN EN 12857:2000 — Foodstuffs — Determination of cyclamate


Analytical procedure —High‑performance liquid chromatographic method for orange
juice beverage
Repeatability, reproducibility (HRN EN 12857:2000)
Customer’s requirements Maximum limit for cyclamate in soft drinks (Directive 2003/115/EC
amending Directive 94/35/EC on sweeteners for use in foodstuffs)

58
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

attached files

File
File No, type and
Content of the file attached Remarks
name
Yes No
Ex‑17‑1‑I‑
1 — I

cyclamate‑ Given by the


beverage‑HPLC‑
About the analytical procedure: short introduction ü lecturer
2009‑Ver1.ppt

PART I Description of the analytical procedure ü


Each
The customer’s requirements
participant
PART II concerning the quality of the ü receives their
measurement result
own copy to
2 — Yellow

Ex‑17‑2‑Y‑ Validation of the measurement keep


cyclamate‑ PART III procedure — relevant equations and ü
beverage‑HPLC‑ measurement data
2009‑Ver1.doc
You may
also want
Measurement uncertainty of the
to include
PART IV result — relevant equations and ü the relevant
measurement data
certificates
here.
Establishing traceability in analytical
PART I
chemistry
ü

Ex‑17‑3‑G‑ Single laboratory validation of


3 — Green

PART II
measurement procedures
ü
cyclamate‑
beverage‑HPLC‑
2009‑Ver1.doc
Building an uncertainty budget ü
PART III Addendum 1: By spreadsheet approach ü
Addendum 2: By dedicated software ü

history of the example


Version Uploaded on Short description of the change
1 4 January 2010

59
Practical examples of traceability, measurement uncertainty and validation in chemistry

a short introduction to the analytical procedure

60
Measurementofthe
Measurement ofconcentration
the concentration
ofcyclamate
of concentration
cyclamate concentration
insoftdrinksbyahigh‑performance
in soft drinks by
liquid
a high‑performance…
chromatographicmethod

61
Practical examples of traceability, measurement uncertainty and validation in chemistry

62
Measurementofthe
Measurement ofconcentration
the concentration
ofcyclamate
of concentration
cyclamate concentration
insoftdrinksbyahigh‑performance
in soft drinks by
liquid
a high‑performance…
chromatographicmethod

63
Practical examples of traceability, measurement uncertainty and validation in chemistry

all the input needed to do the three exercises (yellow pages)

analytical procedure

Measurement of the concentration of cyclamate concentration


in soft drinks by a high-performance liquid chromatographic
method

The quality of result should comply with the requirements


of hrn en 12857:2000 Foodstuffs —Measurement of the
concentration of cyclamate — high-performance liquid
chromatographic method

parT I ...................................................................................................................................65
description of the analytical procedure

parT II .................................................................................................................................68
The customer’s requirements concerning the quality of the measurement result

parT III ................................................................................................................................69


Validation of the measurement procedure — relevant equations and
measurement data

parT IV ................................................................................................................................72
Measurement uncertainty of the result — relevant equations and measurement
data

64
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

parT I. description of the analytical procedure

Parts of the text are taken from: HRN EN 12857:2000 — Foodstuffs — Determination
of cyclamate — High‑performance liquid chromatographic method

Scope
This European Standard specifies a high‑performance liquid chromatographic (HPLC)
method for the measurement of sodium cyclamate concentration in various foodstuffs
such as orange nectar, apricot jam, hard candy, fruit yogurt, cream dessert, ice and many
other dietary products in mass concentration from 5 to 600 mg L– 1 cyclamate.

Principle
Sodium cyclamate is extracted from the sample with n‑heptane, converted to
N,N‑dichlorocyclohexylamine and measured by HPLC on the reversed‑phase column
using UV detection at a wavelength of 314 nm.

Reagents
— Methanol, for HPLC
— n‑Heptane, for HPLC
— Sodium carbonate solution, ρ = 50 g L– 1
— Sodium sulphate, anhydrous
— Sodium hypochlorite solution (1.7 % active chlorine) (see HRN EN 12857:2000
for a detailed preparation procedure)
— Sulphuric acid, w(H2SO4) = 50 %
— Cyclamic acid sodium salt — purity (98.5 ± 0.5) %, k = 2
— Sodium cyclamate standard stock solution n — concentration 600 mg L– 1 (prepare
by weighing and dissolving appropriate mass of salt in 1 000 ml deionised water)
— Deionised water

Equipment
High‑performance liquid chromatography system equipped with a pump module,
a degasser, an oven, a UV‑VIS detector, and a chromatography data station.
Chromatographic separation was performed using an RP‑C18 100A (5 μ, 150 mm x
4.5 mm i.d.) stainless steel column. The mobile phase consisted of 80 % methanol and
20 % deionised water. Elution time is 20 min.

The mobile phase flow rate was 1.0 mL min– 1 and the column was thermostated at
40 °C. The sample was injected with a 20 μL sample loop and detection was performed
at 314 nm. Except for above, the usual laboratory equipment was also used: ultrasonic
bath, filtering apparatus, pipettes, volumetric flasks, weighing equipment, separating
funnels, etc.

65
Practical examples of traceability, measurement uncertainty and validation in chemistry

Analytical procedure
Dilute soft drinks (filtering if necessary), with water to give cyclamate contents of
maximum 600 mg L– 1 or take directly for derivatisation. Pipette 20.0 mL of the sample
solution into a separating funnel, add 1.0 mL of sulphuric acid, 10.0 mL of n‑heptane
and 2.5 mL sodium hypochlorite solution, and shake vigorously for 1 min. Discard the
lower, aqueous phase, dry the n‑heptane layer with sodium sulphate and filter through a
fluted filter paper.

Soft drink sample

Sample preparation
Preparation of
- extraction
calibration solutions
- derivatisation

Measurement of
prepared sample by
Calibration of HPLC
HPLC using UV
detection of 314 nm

Measurement result

Figure 2.1: Flow chart of the analytical procedure

66
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

Calibration
Identify cyclamate in the sample test solution by comparing the retention time of the
analyte in the sample solution with that of the standard substance. For the measurement
by the external standard method, integrate the chromatographic peak areas and compare
the results with the corresponding values for the standard substance using a calibration
graph.

Expression of results
Estimate the concentration, in mg L– 1, of the cyclamate in soft drinks beverages using
the chromatographic peak area as follows:
As − B0
c =
cyc
B1

For determination of B1 and B0 the following equations were used:


n

∑ (c i − c )( Ai − A)
1
n
1
n

B1 =
i =1
n B0 = A − B1 × c c= ∑
n i=1 c i A= ∑
n i=1 A i
∑ (c i − c )2
i =1

Input quantities:

Quantity Units Definition


Chromatographic peak area of the sample chromatographic
As 1
peak
Chromatographic peak area corresponding to calibration
Ai 1
solution (i= 1, 2, 3, 4 and 5)
A 1 Average of chromatographic peak area
ccyc mg L– 1 Concentration of cyclamate in sample from calibration data
Concentration of calibration solutions on ith level
ci mg L– 1
(C1,…, Cn)
c mg L– 1 Average concentration of calibration solutions
B1 mAU Slope of the calibration curve
B0 mAU Intercept of the calibration curve

67
Practical examples of traceability, measurement uncertainty and validation in chemistry

parT II. The customer’s requirements concerning quality of the


measurement result

Extract from Directive 2003/115/EC of the European Parliament and of the Council of
22 December 2003 amending Directive 94/35/EC on sweeteners for use in foodstuffs: the
parametric value (maximum limit) for cyclamate in soft drinks: 250 mg L– 1 (Directive
2003/115/EC)

Extract from HRN EN 12857:2000 — Determination of cyclamate by HPLC method —


for orange juice beverage are presented as the analytical requirements:

Cyclamate
Repeatability Reproducibility
No of average
Sample No of labs sr (mg L– 1) sR (mg L– 1)
values concentration
RSDr (%) RSDr (%)
(mg L– 1)
Orange juice sr 5.5 sR 8.6
10 48 178.3
beverage RSDr 3.1 RSDR 4.9

68
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

parT III. Validation of the measurement procedure — relevant


equations and measurement data

Verification of measurement procedure was carried out in order to verify the application
of standard HRN EN 12857:2000 (Determination of cyclamate by HPLC method).

Precision
A working standard at a concentration level of 239 mg L– 1 cyclamate was measured with
three repetitions over a period of six months.

Repeatability of measurement procedure (s1) is calculated by pooling of standard


deviations over K days.

Intermediate precision (s2) (Reproducibility within laboratory, Rw) was calculated from
the mean of those repetitions.

( )
K K
1 1 2
s1 = ∑ s2
K k =1 k
s2 = ∑ Y −Y
K − 1 k =1 k
K
1
Y = ∑Y
K k =1 k

69
Practical examples of traceability, measurement uncertainty and validation in chemistry

Table 1: Measurement data

x1 x2 x3 xmean St.dev. Variance s(pooled)


255.170 255.121 257.131 255.807 1.146591 1.31467 5.732054
235.663 237.055 243.620 238.779 4.249524 18.05846 5.732054
242.911 241.665 242.911 242.496 0.719378 0.517505 5.732054
247.156 236.692 228.331 237.393 7.701243 59.30914 5.732054
248.806 246.713 252.362 249.294 2.8559 8.156164 5.732054
246.870 229.966 233.761 236.866 8.869359 78.66552 5.732054
240.983 246.327 248.856 245.389 4.019501 16.15638 5.732054
229.211 234.415 236.471 233.366 3.742022 14.00273 5.732054
231.979 231.279 229.764 231.007 1.132214 1.281908 5.732054
246.851 246.219 245.887 246.319 0.489718 0.239824 5.732054
229.520 217.993 223.618 223.710 5.764055 33.22433 5.732054
247.700 250.456 251.710 249.955 2.051347 4.208025 5.732054
225.720 227.313 227.445 226.826 0.960095 0.921783 5.732054
229.113 227.229 229.493 228.612 1.212405 1.469925 5.732054
252.967 251.516 247.599 250.694 2.7768 7.710619 5.732054
230.985 231.166 230.413 230.855 0.393055 0.154492 5.732054
mean 239 245.3915
s2 9.7 32.85644
RSD 0.041 s1 5.7
RSD 0.024

NB: In order to calculate pooled st.dev. (repeatability), the xmean and the st.dev. for the
value of each line (x1, x2, x3) was calculated; hence, rounding is done at the very end.

Recovery of analytical procedure R


cobserved − cmatrix
R=
cspiked

70
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

Input quantities:

Quantity Units Definition


Difference between the observed concentration value and
cobserves− matrix mg L– 1
concentration of matrix sample (soft drink)
cspiked mg L– 1 Calculated sum of spiked sample concentration

Table 2: Measurement data

cspiked cobs− mx
R
(mg L– 1) (mg L– 1)
354.5 357.76 1.009
408.8 413.05 1.01
418.2 411.97 0.985
450.8 452.44 1.004
473.2 467.42 0.988
684.2 690.12 1.009
877.1 873.93 0.996
894.3 902.97 1.01
1150.0 1140.14 0.991

Rmean 1.000
St.dev. 0.0096

Recovery of analytical procedure was determined as following: during the procedure


with soft drink, certain amounts of cyclamate were added for each of the nine
concentration levels shown in Table 2. Samples gained by this method were prepared
three times according to the procedure description. Samples gained by this procedure
were chromatographically analysed twice. The differences between mean values of
measured concentrations and the amounts of cyclamate in the matrix (which were used
in procedure) are shown in column cobs‑mx.

71
Practical examples of traceability, measurement uncertainty and validation in chemistry

PART IV. Measurement uncertainty of the result:


relevant equations and measurement data

Relevant equations

1. Preparation of calibration solutions


mCRM × wCRM c1 × Vi p
c1 = ci =
V1000 Vi f

Quantity Units Definition


Mass weight of CRM — sodium cyclamate for preparing
mCRM* mg
stock standard solution
wCRM Purity of CRM
V1000f mL Volume of stock solution
c1 mg L– 1 Concentration of cyclamate stock standard solution
Volume of working solutions used to prepare the calibration
Vi p mL
solution (i = 10 and 2 mL)
Vi f mL Final volume of calibration solutions (I = 25, 50 and 250 ml)
Concentration of calibration solution used to prepare the
ci mg L– 1
calibration curve (i = 2, 3, 4 and 5)

* The conversion factor for converting the amount of sodium cyclamate to the amount
of cyclamate is 0.8906.

2. Model equation

As − B0
ccyc =
B1

n _

∑ (c i − c )( Ai − A)
n n
= i =1 1 1
B 1 n B0 = A − B1 × c C= ∑C A= ∑Α
∑ (c i − c )2 n i=1 i n i=1 i
i =1

72
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

Measurement data
Std‑unc
Input quantity Units Value
(u)
C1 mg L– 1 606.65 1.8
C2 mg L – 1
242.66 0.78
Concentration of calibration solutions C3 mg L– 1 121.3 0.338
C4 mg L– 1 24.27 0.09
C5 mg L – 1
4.85 0.02
A1 — 2660 395 25 680
A2 — 1084 098 7090
Absorbance of the calibration solutions/
A3 — 530 536 11 679
chromatographic peak area
A4 — 102 309 828
A5 — 18 015 171
Concentration average C mg L– 1 199.9 0.40

Chromatographic peak area average A mAU 879 070 5800

Recovery R — 1 0.0032

Intermediate precision (Within lab reproducibility) Rw mg L– 1 48.2 1.97

Absorbance of the sample solution As mAU 212 217 701

Intercept of the calibration curve B0 mAU 314 10 899


Slope of the calibration curve B1 mAU 4395 45.3
Concentration of cyclamate in sample solution ccyc mg L– 1 48.2 3.2

Explanation of measurement uncertainty calculation:


As − B0 1
c = × + Rw
cyc
B1 R

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Input quantities:
Quantity Units Definition
ccyc mg L– 1 Concentration of cyclamate in sample from calibration data
Chromatographic peak area corresponding to soft drink
As mAU
sample
B0 mAU Intercept of the calibration curve
B1 mAU Slope of the calibration curve
R mAU Recovery factor
Intermediate precision (within lab reproducibility)
Rw mg L– 1

u ( ccyc )  u ( A − B0 ) 
2 2 2
 u( B1 )   u( R ) 
 B  +  R  +  A − B  + ( u( Rw ) )
2
=
ccyc 1 0

u( A − B0 ) = u( A)2 + u( B0 )2

B0 = Asr − B1 × Csr

u( B0 ) = u( A)2 + u( B1 × C )2

 u(C )
2 2
u( B1 × C )  u( B1 ) 
=  B  +  C 
B1 × C 1

 u(C )
2 2
 u( B1 ) 
u( B1 × C ) = B1 × C ×  +
 B1   C 

 u ( A − B0 ) 
2 2 2
 u( B1 )   u( R ) 
+ ( u( Rw ) )
2
u( ccyc ) = ccyc   +  +
 B1   R   A − B0 

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

The solved exercises (green pages)

TrainMiC® exercises

analytical procedure

Measurement of the concentration of cyclamate concentration


in soft drinks by a high-performance liquid chromatographic
method

The quality of result should comply with the requirements of


hrn en 12857:2000 Foodstuffs — determination of cyclamate
— high-performance liquid chromatographic method

exerCIse 1:
establishing traceability in analytical chemistry

exerCIse 2:
single laboratory validation of measurement procedures
Part I: General issues
Part II: Parameters to be validated
Part III: Some calculations and conclusions

exerCIse 3:
building an uncertainty budget
Addendum I: By spreadsheet approach
Addendum II: By dedicated software

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Practical examples of traceability, measurement uncertainty and validation in chemistry

ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

1. Specifying the analyte and measurand


Analyte Cyclamate

Measurand Concentration of cyclamate in soft drinks

Units mg L– 1

2. Choosing a suitable measurement procedure with associated model equation


HRN EN 12857:2000 Foodstuffs — Determination of cyclamate —
Measurement procedure
High‑performance liquid chromatographic method
Type of calibration Standard curve Standard addition Internal standard

Model equation:
The concentration of cyclamate, ccyc, is calculated by:
As − B0
ccyc =
B1

where As is a measured area of the sample chromatographic peak, B1 is the slope of the
calibration curve and B0 is the intercept of the calibration curve. For the determination of
B1 and B0 the following equations were used:
n

∑ (C i − C )( Ai − A)
B = i =1
n
B0 = A − Â1 × C
1

∑ ( Ci − C ) 2
i =1

n n
1 1
A= ∑Α
n i=1 i
C= ∑C
n i=1 i

where Ci are the concentration of reference solutions on ith level (C1,…,Cn), and Ai
represents areas of corresponding chromatographic peaks. Take into account all the
dilution steps.

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

3. List the input quantities according to their influence on the uncertainty of the
result of the measurement (the most important ones first): at this point, your
judgement should be based on your previous experience only.
1 Intermediate precision (Within lab. reproducibility)
2 Recovery
3 Calibration — mass, weighing
4 Calibration — volume, volumetric flasks and pipettes
5 Calibration — purity of Cyclamic acid sodium salt

4. List the reference standards needed and state the information regarding
traceability of the reference value

For the analyte


1 Name/chemical formula/producer Cyclamic acid sodium salt, purity (98.5 ± 0.5) %, k = 2
2 Name/chemical formula/producer

For the other input quantities


Quantity/equipment/calibration
Mass/balance calibrated by an accredited laboratory and
1 (e.g. mass/balance/calibrated by NMI,
regularly checked by calibrated weights
U = xx (k = 2))
Volume/Volumetric flasks — Class A quality, Calibrated by
2 Quantity/equipment/calibration
producer and checked by the laboratory
Volume/Volumetric pipettes, Calibrated by producer and
3 Quantity/equipment/calibration
regularly checked by the laboratory
4 Quantity/equipment/calibration

5. Estimating uncertainty associated with the measurement


Are all important parameters included in
Yes No
the model equation?
Other important parameters: Intermediate precision (Within‑lab. reproducibility); Recovery

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Practical examples of traceability, measurement uncertainty and validation in chemistry

6. How would you prove traceability of your result?


1 Participation in a proficiency testing scheme
2
3

7. Any other comments, questions …

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

SINGLE LABORATORY VALIDATION


OF MEASUREMENT PROCEDURES

parT I: General IssUes

1. Specify the measurement procedure, analyte, measurand and units


According to HRN EN 12857:2000, the determination of concentrations of
The measurement procedure sodium cyclamate in soft drinks by a method of HPLC with UV detection at
314 nm
Analyte Cyclamate

The measurand Concentration of cyclamate in soft drinks

Units mg L– 1

2. Specify the scope


Matrix Soft drinks beverages

Measuring range 5‑600 mg L– 1

3. Requirements of the measurement procedure


Intended use of the results
Parameters to be validated Value requested by the customer
LOD
LoQ
at 178.3 mg L– 1, sr = 5.5 mg L– 1
Mark the customer’s Repeatability
or 3.1 %
requirements and give their at 178.3 mg L– 1, sR = 8.6 mg L– 1
values Reproducibility
or 4.6 %
Trueness
Measurement uncertainty
Other — state

4. Origin of the measurement procedure


VALIDATION
New in‑house method Full
Modified validated method Partial
Official standard method Confirmation/verification

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Practical examples of traceability, measurement uncertainty and validation in chemistry

parT II: paraMeTers To be ValIdaTed

5. Selectivity/interference/recovery
Where yes, give further information, for example which CRM, reference method.

CRM/RM: analysis of available CRM or RM

Further information

Spike of pure substance

Compare with a reference method

Selectivity, interferences

Test with different matrices

Other — specify

6. Measuring range
Linearity
Upper limit
LOD
LoQ

7. Spread – precision
Repeatability
Reproducibility (within Lab)
Reproducibility (between labs)

8. Robustness
Variation of parameters

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

9. Quality control
Control charts
Participation in PT schemes

10. Other parameters to be tested


Working range and testing of homogeneity of variances
R square
Residual standard deviation
Standard deviation of the analytical procedure
Coefficient of variation of the analytical procedure
Measurement uncertainty

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Practical examples of traceability, measurement uncertainty and validation in chemistry

parT III: soMe CalCUlaTIons and ConClUsIons

11. Calculation of parameters requested by the customer


Parameters requested to be validated Calculations
LOD
LoQ
Repeatability
K
1 At a concentration of 239 mg L– 1, sr = 5.73 or RSDr = 2.4 %
s1 = ∑ s2
K k =1 k
Within‑lab reproducibility

( )
K
1 2
s2 = ∑ Y −Y
K − 1 k =1 k At a concentration of 239 mg L– 1, sr = 9.69 or RSDR = 4.1 %
K
1
Y = ∑Y
K k =1 k

Trueness
The measurement uncertainty at a level of 48.2 mg L– 1 is
Measurement uncertainty
estimated to be 14.7 %.
Other — state

12. Does the analytical procedure fulfil the requirement(s) for the intended use?
Value requested by
Is the requirement
the customer Value obtained
Parameter fulfilled?
(the same as stated in during validation
Yes/No
Question 3)
LOD

LoQ

Repeatability RSDr = 5.5 % RSDr = 2.4 % Yes


Within‑lab
RSDR = 8.6 % RSDR = 4.1 % Yes
reproducibility
Trueness
Measurement
uncertainty
Other

The analytical procedure is fit for the intended use:


Yes No

For measurement uncertainty and traceability, refer to the corresponding pages.

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

BUILDING AN UNCERTAINTY BUDGET

1. Specify the measurand and units


Measurand Concentration of cyclamate in soft drink
Units mg L– 1

2. Describe the measurement procedure and provide the associated model


equation

Measurement procedure:
Concentrations of sodium cyclamate in soft drinks were measured by a method using
HPLC with UV detection. Tested quantities were measured by application of an external
standard, combined with a calibration curve. For the final result, recovery is not taken
into account.

The concentration of cyclamate, ccyc, is calculated by:

Model equation:

As − B0
ccyc =
B1
n _

∑ (ci − c )( Ai − A) 1
n


n
1
B1 = c=
i =1
n B0 = A − B1 × c n i=1 c i A= ∑Α
n i=1 i
∑ (c i − c )2
i =1

Input quantities:
Quantity Units Definition

As 1 Peak area of the sample chromatographic peak


Peak area corresponding to calibration solution
Ai 1
(i =1, 2, 3, 4 and 5)
A 1 Average of peak area
ccyc mg L– 1 Concentration of cyclamate in sample from calibration data
Concentration of calibration solutions on ith level
ci mg L– 1
(C1,…,Cn)
c mg L– 1 Average concentration of calibration solutions
B1 mAU mg L– 1 Slope of the calibration curve
B0 mAU Intercept of the calibration curve

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Practical examples of traceability, measurement uncertainty and validation in chemistry

3. Identify (all possible) sources of uncertainty


Uncertainty of concentration of reference solutions
Uncertainty of measurements of peak area
Method bias
Matrix effect
Other: uncertainty of preparation, measurement of calibration solutions and constructing the
calibration graph
Other: uncertainty of intermediate precision (Within lab. reproducibility)
Other: uncertainty of recovery

4. Evaluate values of each input quantity


Input quantity Value Units Remarks
C 199.95 mg L– 1

A 879 071 mAU

B1 4395 mAU mg L– 1
B0 314.126 mAU
R 1 —
RW 48.21 mg L– 1

5. Evaluate the standard uncertainty of each input quantity


Standard
Input quantity Units Remarks
uncertainty
C 0.39534 mg L– 1

A 5820 mAU

B1 45.24 mAU mg L– 1


B0 10896 mAU
R 0.003206 —
Rw 1.98 mg L– 1

6. Calculate the value of the measurand, using the model equation


As − B0 , where c = 48.2 mg L– 1
ccyc = cy
B1

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

7. Calculate the combined standard uncertainty (uc) of the result and specify units

Using: Mathematical solution; Spreadsheet approach; Commercial software

Standard
Input quantity Value Units Remarks
uncertainty
As 212 217 701 mAU Average As of three replicates

B1 4395 45.24 mAU L mg‑1


B0 314.126 10 896 mAU
R 1 0.003206 —
Intermediate
precision
(Within lab. 48.2 1.98 mg L– 1
Reproducibility)
Rw

As − B0 1
c = × + Rw
cyc
B1 R

u( c )  u ( A − B0 ) 
2 2 2
 u( B1 )   u( R ) 
 B  +  R  +  A − B  + ( u( Rw ) )
2
=
c 1 0

u( A − B0 ) = u( A)2 + u( B0 )2

u( B0 ) = u( A)2 + u( B1 × C )2

 u(C )
2 2 2 2
u( B1 × C )  u( B1 )   45.24   0, 39534 
=  B  +  C  =  4395  +  199.95  = 1.098658 × 10
−4
B1 × C 1

 u(C )
2 2
 u( B1 ) 
u( B1 × C ) = B1 × C ×   +
 B1   C 

u( B1 × C ) = 4395 × 199.95 × 1.098658 × 10−4 = 9 211.1

u( B0 ) = 58202 + 9211.12 = 10 896

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Practical examples of traceability, measurement uncertainty and validation in chemistry

u( A − B0 ) = 7012 + 108962 = 10 919

 u ( A − B0 ) 
2 2 2
 u( B1 )   u( R ) 
+ ( u( Rw ) )
2
u( c ) = c   +  +
 B1   R   A − B0 

2 2 2 2
 45.24   0.003206   10919   1.98 
u ( c ) = 48.2  +  +  211 903  +  48.2 
 4395   1
u( c ) = 48.2 × 0.06677
u( c ) = 3.22

8. Calculate expanded uncertainty (Uc) and specify the coverage factor k and the
units

(Uc) = k × 3.22
(Uc) = (48.2 ± 7.1) mg L– 1 (k = 2.2)

9. Analyse the uncertainty contribution and specify the main three input quantities
contributing the most to Uc
1 u(B0) — uncertainty of intercept of calibration curve
2 u(Rw) — uncertainty of intermediate precision (within lab. reproducibility)
3 U(B1) — uncertainty of slope of calibration curve

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

10. Prepare your Uncertainty budget report


Std‑unc
Input quantity Units Value
(u)
C1 mg L– 1 606.65 1.78
C2 mg L– 1 242.66 0.78
Concentration of calibration solutions C3 mg L – 1
121.3 0.37
C4 mg L – 1
24.27 0.09
C5 mg L– 1 4.85 0.02
A1 — 2 660 395 25 680
A2 — 1 084 098 7 090
Absorbance of the calibration solutions/
A3 — 530 536 11 679
chromatographic peak area
A4 — 102 309 828
A5 — 18 015 171
Concentration, average C mg L – 1
199.95 0.40

Chromatographic peak area, average A mAU 879 071 5820

Recovery R — 1 0.0032
Intermediate precision (Within lab
Rw mg L– 1 48.2 1.98
reproducibility)

Absorbance of the sample solution As mAU 212 217 701

Intercept of the calibration curve B0 mAU 314 10 896


Slope of the calibration curve B1 mAU mg L cyc– 1
4 395 45.24
Concentration of cyclamate in sample
ccyc mg L– 1 48.2 3.22
solution

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Further reading

1. HRN EN 12857:2000, Foodstuffs — Determination of cyclamate — High‑performance


liquid chromatographic method.

2. Eurachem/CITAC Guide (2000), Quantifying Uncertainty in Analytical Measurement


(http://www.european‑accreditation.org).

3. Eurolab, Technical Report 01/2006, Guide to the Evaluation of Measurement


Uncertainty for Quantitative Test Results (http://www.eurolab.org/pub/i_pub.html).

4. Guthrie, W., Liu, Hung‑kung, Hands‑on workshop on estimating and reporting


measurement uncertainty, NIST, Measurement Science Conference (2007).

5. NIST/SEMATECH, e‑Handbook of Statistical Methods (http://www.nist.gov/stat.


handbook/).

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

addendum I: Measurement uncertainty calculation:


spreadsheet approach (excel)

Calibration solution C1

u(m st) u(ω) u(V 1 L)


0.01800 0.00289 0.00024
Input quantity Value
m st 612.78 612.79800 612.78000 612.78000
ω 0.99 0.99000 0.99289 0.99000
V1L 1.00 1.00000 1.00000 1.00024
C1 606.65 606.67 608.42 606.50
uc 1.77724 0.00032 3.13621 0.02207
df 112702 0.0 % 99.3 % 0.7 %
k 2 0.99000 612.78000 – 606.50364
U 3.48337 0.57 % Urelat., %

Calibration solution C2
u(c‑stock) u(V‑10 p) u(V‑25 t)
1.77724 0.00816 0.02449
Input quantity Value
c‑stock 606.65 608.42724 606.65000 606.65000
V‑10 10.00 10.00000 10.00816 10.00000
V‑25 25.00 25.00000 25.00000 25.02449
C2 242.66 243.37 242.86 242.42
uc 0.7753 0.50537 0.03926 0.05642
df 1464978 84.1 % 6.5 % 9.4 %
k 2 0.40000 24.26600 – 9.69690
U 1.51951 0.63 % Urelat., %

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Calibration solution C3
u(c‑stock) u(V‑10 p) u(V‑50 t)
1.77724 0.00816 0.02449
Input quantity Value
c‑stock 606.65 608.42724 606.65000 606.65000
V‑10 10.00 10.00000 10.00816 10.00000
V‑50 50.00 50.00000 50.00000 50.02449
C3 121.33 121.69 121.43 121.27
uc 0.37375 0.12634 0.00981 0.00353
df 1279972 90.4 % 7.0 % 2.5 %
k 2 0.20000 12.13300 – 2.42541
U 0.73253 0.60 % Urelat., %

Calibration solution C4
u(c‑stock) u(V‑2 p) u(V‑50 t)
1.77724 0.00408 0.02449
Input quantity Value
c‑stock 606.65 608.42724 606.65000 606.65000
V‑2 2.00 2.00000 2.00408 2.00000
V‑50 50.00 50.00000 50.00000 50.02449
C4 24.27 24.34 24.32 24.25
uc 0.08746 0.00505 0.00245 0.00014
df 757936 66.1 % 32.1 % 1.8 %
k 2 0.04000 12.13300 – 0.48508
U 0.17141 0.71 % Urelat., %

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Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

Calibration solution C5
u(c‑stock) u(V‑2 p) u(V‑250 t)
1.77724 0.00408 0.06124
Input quantity Value
c‑stock 606.65 608.42724 606.65000 606.65000
V‑2 2.00 2.00000 2.00408 2.00000
V‑250 250.00 250.00000 250.00000 250.06124
C5 4.85 4.87 4.86 4.85
uc 0.01737 0.00020 0.00010 0.00000
df 737259 67.0 % 32.5 % 0.5 %
k 2 0.00800 2.42660 – 0.01941
U 0.03404 0.70 % Urelat., %

C average
u(C 1) u(C 2) u(C 3) u(C 4) u(C 5)
1.77724 0.77530 0.37375 0.08746 0.01737
Input quantity Value
C1 606.65 608.43 606.65 606.65 606.65 606.65
C2 242.66 242.66 243.44 242.66 242.66 242.66
C3 121.30 121.30 121.30 121.67 121.30 121.30
C4 24.27 24.27 24.27 24.27 24.36 24.27
C5 4.85 4.85 4.85 4.85 4.85 4.87
Result 199.95 200.30 200.10 200.02 199.96 199.95
uc 0.39534 0.12634 0.02404 0.00559 0.00031 0.00001
df 171956 80.8 % 15.4 % 3.6 % 0.2 % 0.0 %
k 2 0.20000 0.20000 0.20000 0.20000 0.20000
U 0.77485 0.39 % Urelat., %

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Practical examples of traceability, measurement uncertainty and validation in chemistry

A average
u(A 1) u(A 2) u(A 3) u(A 4) u(A 5)
171 828 11679 7090 25680
Input quantity Value
A1 18015 18186 18015 18015 18015 18015
A2 102309 102309 103137 102309 102309 102309
A3 530536 530536 530536 542215 530536 530536
A4 1084098 1084098 1084098 1084098 1091188 1084098
A5 2660395 2660395 2660395 2660395 2660395 2686075
Result 879071 879105 879236 881406 880489 884207
uc 5820 1170 27423 5455962 2010724 26378496
df 3.15 0.0 % 0.1 % 16.1 % 5.9 % 77.9 %
k 3 0.20000 0.20000 0.20000 0.20000 0.20000
U 18522 2.11 % Urelat., %

B0
u(A‑average) u(C‑average) u(B 1)
5820 0.39534 45.24
Input quantity Value
A‑average 879071 884891 879071 879071
C‑average 199.95 199.95 200.34 199.95
B1 4395 4395 4395 4440.21
Result 314.126 6134.13 – 423.38 – 8731.43
uc 10896 33872400 3018931 81822102
df 4.52 28.5 % 2.5 % 68.9 %
k 3 1.00000 – 4394.96901 – 199.94600
U 30251 9630.19 % Urelat., %

92
B1

u u
u(C 1) u(A 1) u(C 2) u(A 2) u(C 3) u(A 3) u(C 4) u(A 4) u(C 5) u(A 5)
(C‑ average) (A‑ average)
1.77724 25680 0.78 7090 0.37375 11679 0.09 828 0.01737 171 0.39534 5820
Input
Value
quantity
C1 606.65 608.43 606.65 606.65 606.65 606.65 606.65 606.65 606.65 606.65 606.65 606.65 606.65
A1 2660395 2660395 2686075 2660395 2660395 2660395 2660395 2660395 2660395 2660395 2660395 2660395 2660395
C2 242.66 242.66 242.66 243.44 242.66 242.66 242.66 242.66 242.66 242.66 242.66 242.66 242.66
A2 1084098 1084098 1084098 1084098 1091188 1084098 1084098 1084098 1084098 1084098 1084098 1084098 1084098
C3 121.30 121.30 121.30 121.30 121.30 121.67 121.30 121.30 121.30 121.30 121.30 121.30 121.30
A3 530536 530536 530536 530536 530536 530536 542215 530536 530536 530536 530536 530536 530536
C4 24.27 24.27 24.27 24.27 24.27 24.27 24.27 24.36 24.27 24.27 24.27 24.27 24.27
A4 102309 102309 102309 102309 102309 102309 102309 102309 103137 102309 102309 102309 102309
C5 4.85 4.85 4.85 4.85 4.85 4.85 4.85 4.85 4.85 4.87 4.85 4.85 4.85
A5 18015 18015 18015 18015 18015 18015 18015 18015 18015 18015 18186 18015 18015
C‑ average 199.95 199.95 199.95 199.95 199.95 199.95 199.95 199.95 199.95 199.95 199.95 200.35 199.95
A‑average 879071 879071 879071 879071 879071 879071 879071 879071 879071 879071 879071 879071 884891
Result 4395 4382 4438 4394 4396 4395 4391 4395 4394 4395 4395 4395 4395
uc 45.24 172.44665 1857.29046 0.30898 1.56132 0.27695 14.36647 0.07666 0.36029 0.00375 0.01895 0.00021 0.00000
df 2.43 8.4 % 90.7 % 0.0 % 0.1 % 0.0 % 0.7 % 0.0 % 0.0 % 0.0 % 0.0 % 0.0 % 0.0 %
k 4 – 7.38893 0.00168 – 0.71696 0.00018 1.40807 – 0.00032 3.16564 – 0.00072 3.52315 – 0.00081 – 0.03657 0.00000
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…

U 194.65 4.43 % Urelative, %

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Practical examples of traceability, measurement uncertainty and validation in chemistry

ccyc

u(A) u(B0) u(B1) u(R) u(Rw)


701 10896 45.24 0.003 1.980
Input quantity Value

As 212217 212918 212217 212217 212217 212217

B0 314.126 314.126 11210.126 314.126 314.126 314.126


B1 4395 4395 4395 4440.24 4395 4395
R 1.000 1.00000 1.00000 1.00000 1.00320 1.00000
Rw 0.000 0.00 0.00 0.00 0.00 1.97
Result 48.2 48.37 45.74 47.72 48.06 50.18
uc 3.21825 0.02544 6.14634 0.24132 0.02365 3.88090
df 11.42 0.2 % 59.6 % 2.3 % 0.2 % 37.6 %
k 2.20 0.00023 – 0.00023 – 0.01086 – 48.06074 1.00000
U 7.06981 14.69 % Urelat., %

u(A)
u(B0)
u(B1)
u(R)
u(Rw )

NB: Linear approximation of the calibration curve is made in the broad concentration
interval.
Therefore, u(B0) and u(B1) are the most significant sources of error. Measurement
uncertainties of expected results are still acceptable for their intended use.

94
Chapter 3
Measurement of the concentration of arsenic
in groundwater by flame atomic absorption
spectrometry (hydride technique)
Nada L. Lazić, Jelena Bebić
• TrainMiC® example summary form (blue page)
• A short introduction to the analytical procedure (slides)
• All the input needed to do the three exercises (yellow pages)
• The solved exercises (green pages)

95
Practical examples of traceability, measurement uncertainty and validation in chemistry

TrainMiC® example summary form

General information about the example


Measurement of concentration of arsenic in groundwater by flame atomic
Measurand
absorption spectrometry (hydride technique)

Example No Ex‑25

Authors of the example Nada L. Lazić, Jelena Bebić

ISO 11969:1996 — Water quality — Determination of arsenic — Atomic


Analytical procedure
absorption spectrometric method (hydride technique)
Recovery maximum limit 10 %
Customer’s requirements Limit of quantification minimum 5 µg L– 1
Relative expanded measurement uncertainty maximum 10 %, k  = 2

96
Measurement of the concentration of arsenic…

Attached files
File
File No, type and name Content of the file attached Remarks
Yes No
Ex‑25‑1‑I‑As‑water‑ About the analytical procedure: short Given by the
1 — I

AAS‑2010‑Ver1.ppt introduction
ü lecturer
Description of the analytical
PART I
procedure
ü
The customer’s requirements
PART II concerning the quality of the ü Each participant
measurement result receives their own
2 — Yellow

Validation of the measurement copy to keep


EX‑25‑2‑Y‑As‑water‑
AAS‑2010‑Ver1.doc procedure — relevant
PART III
equations and measurement
ü
data
You may also
Measurement uncertainty of
want to include
PART IV the result — relevant equations ü the relevant
and measurement data
certificates here.
Establishing traceability in
PART I
analytical chemistry
ü

Single laboratory validation of


PART II ü
3 — Green

measurement procedures
EX‑25‑3‑G‑As‑water‑
AAS‑2010‑Ver1.doc Building an uncertainty budget ü
Addendum 1: By spreadsheet
PART III approach
ü
Addendum 2: By dedicated
software
ü

History of the example


Version Uploaded on Short description of the change
1 4 January 2011

97
Practical examples of traceability, measurement uncertainty and validation in chemistry

a short introduction to the analytical procedure

Determination of arsenic in ground


water by flame atomic absorption
spectrometry (hydride technique)

Scope of the presentation


• The analytical procedure and the customer’s requirements
• About ‘the chemistry’ and the measurement method
• Model equation

The analytical procedure and the


customer’s requirements

• Analytical procedure:
ISO 11969:1996, Determination of arsenic, Atomic absorption
method (hydride technique)

• Customer`s requirements:
The customer's requirements concerning quality of the measurement
result are following:
Recovery maximum limit 10 %
Limit of quantification minimum 5 g L-1
Relative expanded measurement uncertainty maximum 10 %, k = 2

98
Measurementofthe
Measurement of the
concentration
concentration
ofarsenic
of in
arsenic…
groundwaterbyflameatomicabsorptionspectrometry(hydridetechnique)

Measurement method
• As is determined by flame atomic absorption spectrometry
(hydride technique), by GBC Avanta AAS Ver 2.0
• Sample is prepared by adding HCl and KJ, in specified amounts
according to the Standard ISO 11969:1996.
• Calibration is done with the blank solution and 6 equidistant
calibration solutions (working solutions) for an appropriate
concentration range.
• For plotting of a calibration curve or for the calculation of the
calibration function the resulting values of the analyte
concentrations of the calibration solutions are used.
• Test sample is analysed in the same way as the calibration
solutions with the FAAS.
• Concentration of As is calculated using the calibration curve and
measurement results.

Flowchart of the
analytical procedure

Collection and Preparation of calibration


preparation of sample standards C1 to C6

Absorbance measurement Absorbance measurement

Calculation of calibration curve

Calculation of sample
concentration using the
calibration curve

99
Practical examples of traceability, measurement uncertainty and validation in chemistry

Preparation of
standards and samples
• Standards are prepared from stock solution
CAs = (1000 ± 2) mg L-1 by dilution to
concentrations of 10 g L-1, 20 g L-1, 40 g L-1,
60 g L-1, 80 g L-1 and 100 g L-1
• Sample is prepared by adding HCl and KJ, in
specified amounts according to the Standard ISO
11969:1996.
• Procedural blank is subjected to exactly the same
sample preparation procedure as the analysed
sample.

100
Measurementofthe
Measurement of the
concentration
concentration
ofarsenic
of in
arsenic…
groundwaterbyflameatomicabsorptionspectrometry(hydridetechnique)

Model equation and equation for


measurement uncertainty calculation

C = [(Asample - b0)/b1] (1/R)

U = k u (CAs )

101
Practical examples of traceability, measurement uncertainty and validation in chemistry

All the input needed to do the three exercises (yellow pages)

analytical procedure

Measurement of the concentration of arsenic in groundwater by


flame atomic absorption spectrometry (hydride technique)

PART I ................................................................................................................... 103


description of the analytical procedure

PART II .................................................................................................................. 108


The customer’s requirements concerning the quality of the measurement result

PART III ................................................................................................................. 109


Validation of the measurement procedure — relevant equations and
measurement data

PART IV ................................................................................................................. 110


Measurement uncertainty of the result — relevant equations and measurement
data

102
Measurement of the concentration of arsenic…

parT I. description of the analytical procedure

Task description
One of the important parameters for the measurement of concentration of groundwater
quality is the arsenic content. The maximum allowed concentration of arsenic in these
waters, Class I and II, is given in Directive 98/83/EC of 3 November 1998 on the quality
of water intended for human consumption. The parametric value (maximum limit) for
arsenic in drinking water is 10 µg L– 1.

The internationally recognised method for the measurement of concentratione of arsenic


in drinking, ground and surface waters is by flame atomic absorption spectrometry
(hydride technique) as provided in ISO 11969:1996 (Water quality — Determination
of arsenic — Atomic absorption spectrometric method (hydride technique)). This
standard defines methods for the measurement of concentration of arsenic, including
the organically bound arsenic in drinking water, groundwater and surface water in the
concentration range 1–10 µg L– 1.

Scope
This example defines a standard method for measurement of concentration of arsenic in
groundwater by Flame atomic Absorption Spectrometry (FAAS) (hydride technique),
based on ISO 11969:1996.

The procedure is optimised to fit for purpose for the extended concentration range. The
range of application is in mass concentration 4–100 µg L– 1.

The experimental protocol is shown in Figure 3.1.

Figure 3.1: The experimental protocol

Collection and preparation of Preparation of calibration


sample standards C1 to C6

Absorbance measurement
Absorbance measurement

Calculation of calibration
curve

Calculation of sample
concentration using
the calibration curve

103
Practical examples of traceability, measurement uncertainty and validation in chemistry

The method is based on the measurements of atomic absorption of arsenic formed by


thermal decomposition of arsenic (III) hydride.

Under the conditions in this method, only arsenic (III) is transformed to hydride. To
avoid errors in the measurement, the oxidation state should be transformed to arsenic
(III) prior to measurement.
Arsenic (III) is transformed to gaseous arsenic (III) hydride (AsH3) by reaction with
sodium tetrahydroborate, in an acidic environment, obtained with hydrochloric acid.

Absorbance is measured at 193.7 nm wavelength.

Interferences
Most organic compounds can interfere in the measurement of concentration of arsenic
by FAAS. They should be removed by digestion, prior to the sample preparation, in
accordance with ISO 11969:1996.

Other potential interfering ions are shown in Table1.

Table 1: Influence of other ions on the measurement of concentration of As by FAAS


Concentration in mg L– 1 for measurement of
Interfering ion
of 1 µg L– 1 As
Cu2+ > 2.0
Sb5+ > 0.2
Se 4+
> 0.05
NO3− > 100

Reagents
1. E. Merck class p.a. — HCl, ρ = 1.15 g mL– 1.
2. E. Merck class p.a, KJ solution.
3. As stock solution, Backer, CAs= (1 000 ± 2) mg L—1.
4. As standard solution, 10 mg L– 1 (pipette 1 mL of stock solution in 100 mL
volumetric flask, and make up to a volume with water).
5. As calibration solution, 1 000 µg L– 1 (pipette 10 mL of standard solution in
100 mL volumetric flask, and make up to a volume with water).
6. As working solutions from 10 to 100 µg L– 1 (pipette into a series of volumetric
flasks of 100 mL, 1 mL, 2 mL, 4 mL, 6 mL, 8 mL, 10 mL respectively of
1 000 µg L– 1 calibration solution, and make up to a volume with water).
7. CRM: QC Check Sample (WP‑Water Pollution), QWPTM‑2X20, VHG Labs,
CAs = (49.375 ± 0.171) µg L– 1.

104
Measurement of the concentration of arsenic…

Apparatus
‑ Flame atomic absorption spectrometer equipped for hydride technique with
hollow cathode lamp for arsenic
‑ Pipette variable 0–20 mL gravimetrically checked (d = 0.01 mL)
‑ Volumetric flask 100 mL (laboratory glassware Class A; certified d = 0.1 mL for
20 °C)
‑ Polypropylene vials 12 g

Sampling and pre-treatment


Sampling should be carried out in accordance with ISO 5667‑1:1980 (Water
quality — Sampling — Part 1: Guidance on the design of sampling programmes),
ISO 5667‑2:1991(Water quality — Sampling — Part 2: Guidance on sampling
techniques) and ISO 5667‑3:1994 (Water quality — Sampling — Part 3: Guidance on
the preservation and handling of samples).

Containers of glass, PE, PP and PTFE are suitable for sample collection. Clean all
containers with HNO3 (φHNO3 = 10 %) and rinse with water.

Preservation of samples is carried out in accordance with ISO 5667‑3:1994.

Procedure
Instrument set‑up
As is measured by Flame atomic absorption spectrometry (hydride technique) — GBC
Avanta AAS Version 2.0.

Instrumental parameters are described in Table 2.

Table 2: Instrumental parameters for FAAS measurement of concentration of As


FAAS parameters Values
Lamp current 8.00 Ma
Wavelength 193.70 nm
Slit width 1.00 nm
Slit height Normal
Instrument mode Abs. BC on

Prior to the instrumental measurement, calibration function for the measurement must be
established according to ISO 8466‑1:1990 (Water Quality — Calibration and evaluation
of analytical methods and estimation of performance characteristics — Part 1: Statistical
evaluation of the linear calibration function).

105
Practical examples of traceability, measurement uncertainty and validation in chemistry

The sample is prepared by adding HCl and KJ, in specified amounts according to
ISO 11969:1996. The procedural blank is subjected to exactly the same sample
preparation procedure as the analysed sample.

Calibration
Perform the calibration with the blank solution and six equidistant calibration solutions
(working solutions) for an appropriate concentration range, at least six replicates for
each solution. The linearity of the calibration curve is often limited.

The absorbance of the blank is not subtracted but all the measurements are made against
the blank. To plot a calibration curve or for the calculation of the calibration function, use
the resulting values of the analyte concentrations of the calibration solutions (working
solutions).

Analyse the test sample in the same way as the calibration solutions with the FAAS.
Perform at least six replicates.

Instrumental measurement parameters are described in Table 3.

Table 3: Instrumental measurement parameters for the FAAS measurement of


concentration of As

Instrumental measurement parameters Values


Measurement mode Integration
Sample introduction Manual
Read time 3.00 s
Replicates 3
Calibration mode Conc. Least Squares
Measure sample blank after calibration Yes
Flame type Air‑acetylene
Fuel flow 2.000 L min– 1
Oxidant flow 10.00 L min– 1
Burner angle 0o
Workhead height 15.00 mm

106
Measurement of the concentration of arsenic…

Calculation

Mass concentration of arsenic in µg L– 1 in the solution is calculated from absorbance,


as follows:
A − b0
C=
b1

where A, b1 and b0 are the measured absorbance of the sample, the slope of the linear
least squares calibration curve in L µg– 1 and the calculated blank respectively.

The slope of the linear least squares calibration curve b1 and calculated blank b0 are
calculated from equations:

∑( C ) ( )
n

i − C × Ai − A
b1 = i =1

∑( C )
n
2
i −C
i =1

b0 = A − b1 × C
n
1
A= ∑A
n i=1 i
n
1
C= ∑C
n i=1 i

where Ci and Ai are concentrations of calibration (working) solutions on ith level


(C1,...,Ci,...,Cn) in µg L– 1 and absorbances of ith calibration solution (A1,..., Ai,..., An).

Take into account all the dilution steps.

107
Practical examples of traceability, measurement uncertainty and validation in chemistry

PART II. The customer’s requirements concerning quality of


the measurement result

Arsenic is a natural element that occurs naturally in rocks and soil and is used for a variety
of purposes in industry and agriculture. The World Health Organisation (WHO), the
Department of Health and Human Services (DHHS), and the Environmental Protection
Agency (EPA) have determined that inorganic arsenic can cause cancer in humans.

It is possible to be exposed to arsenic through the following.


• Food: Arsenic in the air can be washed into the ground when it rains,
contaminating crops and fields. Although arsenic can build up in the tissues of
fish and shellfish, this form of arsenic will not hurt people.

• Water: Water may have arsenic in it if there are high levels of arsenic in the rocks
through which the water flows, or if there is a leaking hazardous waste site close
by. Some chemicals containing arsenic can dissolve in water.

• Air: Sawdust and smoke from burning arsenic‑preserved wood may contaminate
the air you breathe.
Once released, arsenic remains in the environment for a long time. It is widely believed
that naturally occurring arsenic dissolves out of certain rock formations when groundwater
levels drop significantly. Surface arsenic‑related pollutants enter the groundwater system
by gradually moving with the flow of groundwater from rain, melting snow, and so on.

In the sample provided by the customer for analysis, it is presumed that the concentration
of arsenic is high, above the maximum allowed limit of 10 µg L– 1. The sample is
collected from the groundwater in the area where there are natural deposits of arsenic.
This groundwater will be subjected to water treatment, in order to remove excess of
arsenic.

The customer’s requirements concerning quality of the measurement result are:


‑ recovery maximum limit 10 %,
‑ limit of quantification minimum 5 µg L– 1,
relative expanded measurement uncertainty maximum 10 %, k = 2.

108
Measurement of the concentration of arsenic…

PART III. Validation of the measurement procedure — relevant


equations and measurement data

In the present case study, the methodology for the validation of the measurement
procedure, ISO 11969:1996 (Water quality — Determination of arsenic — Atomic
absorption spectrometric method (hydride technique)) is presented. For the calculations,
the emphasis is on the parameters that are required by the customer. However, for the
purpose of this exercise, recovery (R) and limit of quantification (LoQ) will be calculated
only.

Measurement data:
Cobserved 48.880 µg L– 1

CCRM 49.375 µg L– 1

Abl
– 0.0014
– 0.0009
– 0.0023
– 0.0026
– 0.0023
0.0008
0.0007
– 0.0013
– 0.0048
0.0060
b0 0.01537

b1 0.00473 L µg– 1

Equations:
n

∑x ∑( x )
2
i −x A − b0
x= i =1
s=
i
ALOQ = Abl + 10 × sbl LOQ = LOQ

n n−1 b1

cobserved
R=
cCRM

109
Practical examples of traceability, measurement uncertainty and validation in chemistry

PART IV. Measurement uncertainty of the result: relevant


equations and measurement data (2)

In the present study, the methodology for the evaluation of the uncertainty measurement
of the result of arsenic concentration measurement in groundwater is presented. Arsenic
concentration was measured using FAAS. The necessary relevant information was
obtained from the method validation data, quality control data and equipment calibration
certificates. The method of measurement is described together with the measurement
equation, selected traceable reference standards and the associated measurement
uncertainty. The major sources of uncertainty of the result of measurement were identified
and the combined uncertainty was calculated. The identification of the main uncertainty
sources represents the basis for the target operation to reduce the measurement uncertainty
of this measurement. Sampling and sample preparation steps have not been taken into
consideration for the purpose of estimating the uncertainty budget.1

Experimental protocol
Sampling

Sample preparation for FAAS measurement

Optimise all parameters of FAAS

Steps of preparation of calibration solutions and FAAS calibration

Aspiration of calibration standards and samples

FAAS measurement of arsenic concentration

Result

1
You may also want to include the relevant certificates here.

110
Measurement of the concentration of arsenic…

Measurement data and intermediate results


Quantity Units Value Std. unc. RSu (%)
Standards C1 μg L– 1 10.00 0.08 0.80
C2 μg L– 1 20.00 0.13 0.65
C3 μg L – 1
40.00 0.24 0.60
C4 μg L – 1
60.00 0.36 0.60
C5 μg L– 1 80.00 0.48 0.60
C6 μg L– 1 100.00 0.59 0.59
Absorbances A1 0.0529 0.0022 4.16
A2 0.1109 0.0038 3.43
A3 0.2145 0.0103 4.80
A4 0.3042 0.0115 3.78
A5 0.3926 0.0115 2.93
A6 0.4824 0.0135 2.80
Sample 0.3028
0.3021
0.3012
0.2834
0.2664
0.2685
A 0.2874 0.0070 2.44
Recovery R 0.99 0.01 1.00

Concentration average Caver μg L– 1 51.67 0.15 0.29


Absorbance average Aaver 0.2596 0.0037 1.43
Slope of the linear LSCC b1 L μg ‑1
0.00473 0.000127 2.68

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Equations:
The input quantities in the model equation:

A − b0 C Concentration of arsenic (μg L– 1)


C= A Measured absorbance of the sample
b1
b0 Calculated blank
b1 Slope of the linear least square
∑( C ) ( )
n

i − C × Ai − A calibration curve
b1 = i =1
Ci Concentration of calibration
∑( C )
n

−C
2
solutions (μg L– 1)
i
i =1 Ai Aabsorbances of calibration
solutions
1
n
CAs Final result for concentration
A= ∑A
n i=1 i
b0 = A − b1 × C
of arsenic (μg L– 1)
R Recovery
n Cobserved Measured concentration of the
1
C= ∑C
n i=1 i
analyte in CRM (μg L– 1)
CCRM Concentration of CRM (μg L– 1)
A − b0 1 cobserved
CAs = × R=
b1 R cCRM

Equations used for measurements uncertainty calculations

u ( b0 ) = (u( A)) + (u( b × C ))


2 2
1

u( b × C )  u(C )
2
 u( b )
2
1
= +   1

b1 × C b1  C 

u( c )  u ( b1 )   u( R )  u ( A − b0 ) 
2 2 2

=  b  +  R  +  A − b 
c 1 0

u ( A − b0 ) = (u( A)) + (u( b )) 2 2


0

112
Measurement of the concentration of arsenic…

The solved exercises (green pages)

TrainMiC® exercises

Analytical procedure

Measurement of the concentration of arsenic in groundwater


by flame atomic absorption spectrometry (hydride technique)

exerCIse 1:
establishing traceability in analytical chemistry

exerCIse 2:
single laboratory validation of measurement procedures
Part I: General issues
Part II: Parameters to be validated
Part III: Some calculations and conclusions

exerCIse 3:
building an uncertainty budget
Addendum I: By spreadsheet approach
Addendum II: By dedicated software

113
Practical examples of traceability, measurement uncertainty and validation in chemistry

ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

1. Specifying the analyte and measurand


Analyte Arsenic

Measurand Concentration of arsenic in groundwater

Units µg L– 1

2. Choosing a suitable measurement procedure with associated model equation


Measurement ISO 11969:1996 — Water quality — Determination of arsenic — Atomic absorption
procedure spectrometric method (hydride technique)
Type of calibration Standard curve Standard addition Internal standard

Model equation:

The mass concentration of arsenic in µg L– 1 in the solution is calculated from the


absorbance as follows:
A − b0
C=
b1
where A, b1 and b0 are the measured absorbance of the sample, the slope of the linear
least squares calibration curve in L µg– 1 and the calculated blank respectively.

The slope of the linear last squares calibration curve b1 and calculated blank b0 are
calculated from equations:

∑( C ) ( )
n

i − C × Ai − A
b1 = i =1

∑( C )
n
2
i −C
i =1

b0 = A − b1 × C
n
1
A= ∑A
n i=1 i
n
1
C= ∑C
n i=1 i

Where Ci and Ai are concentrations of calibration (working) solutions on ith level


(C1,...,Ci,...,Cn) in µg L– 1 and absorbances of ith calibration solution (A1,..., Ai,.., An).

114
Measurement of the concentration of arsenic…

3. List the input quantities according to their influence on the uncertainty of the
result of the measurement (the first most important ones): at this point, your
judgement should be based on your previous experience only.
1 Uncertainty of recovery factor
2 Uncertainty of concentration of calibration solutions
3 Uncertainty of absorbance measurement
4 Uncertainty of volumes
5

4. List the reference standards needed and state the information regarding
traceability of the reference value

For the analyte


1 Name/chemical formula/producer As stock solution, Backer, CAs = (1000 ± 2) mg L– 1
2 Name/chemical formula/producer

For the other input quantities


Quantity/equipment/calibration Calibrated volumetric flasks — Class A and pipettes,
1 (e.g. mass/balance/calibrated by NMI,
with established traceability
U = xx (k = 2))
2 Quantity/equipment/calibration Absorbance — calibrated FAAS
3 Quantity/equipment/calibration
4 Quantity/equipment/calibration

5. Estimating uncertainty associated with the measurement


Are all important parameters included in the model
Yes No
equation?
Other important parameters: Interferences, with‑in lab reproducibility

6. How would you prove traceability of your result?


1 Analysis of matrix CRM
2 Participation in inter‑laboratory comparisons
3

115
Practical examples of traceability, measurement uncertainty and validation in chemistry

7. Any other comments, questions …

116
Measurement of the concentration of arsenic…

SINGLE LABORATORY VALIDATION OF


MEASUREMENT PROCEDURES

parT I: General IssUes

1. Specify the measurement procedure, analyte, measurand and units


The measurement ISO 11969:1996 — Water quality — Determination of arsenic — Atomic absorption
procedure spectrometric method (hydride technique)
Analyte Arsenic
The measurand Concentration of arsenic in groundwater
Units µg L– 1

2. Specify the scope


Matrix Groundwater
Measuring range 4–100 µg L– 1 arsenic

3. Requirements of the measurement procedure


Intended use of the results Assessing the quality of groundwater prior to water treatment process
Parameters to be validated Value requested by the customer
LOD
LoQ 5 µg L– 1
Repeatability
Mark the customer’s
requirements and give Within‑lab
their values reproducibility
Trueness Recovery max 10 %
Measurement
% U(C) = 10, k = 2
uncertainty
Other — state

4. Origin of the measurement procedure


VALIDATION

New in‑house method Full

Modified validated method Partial

Official standard method Confirmation/verification

117
Practical examples of traceability, measurement uncertainty and validation in chemistry

parT II: paraMeTers To be ValIdaTed

5. Selectivity/interference/recovery

Where yes, give further information, for example which CRM, reference method.
CRM/RM: analysis of available CRM or RM
Further information
CRM: QC Check Sample (WP‑Water Pollution), QWPTM‑2X20, VHG Labs, CAs = (49.375 ± 0.171) µg L– 1
Spike of pure substance

Compare with a reference method

Selectivity, interferences

Test with different matrices

Other — specify

6. Measuring range
Linearity
Upper limit
LOD
LoQ

7. Spread — precision
Repeatability

Reproducibility (within Lab)

Reproducibility (between labs)

118
Measurement of the concentration of arsenic…

8. Robustness
Variation of parameters

9. Quality control
Control charts
Participation in PT schemes

10. Other parameters to be tested


Working range and testing of homogeneity of variances
R square
Residual standard deviation
Standard deviation of the analytical procedure
Coefficient of variation of the analytical procedure
Measurement uncertainty

119
Practical examples of traceability, measurement uncertainty and validation in chemistry

parT III: soMe CalCUlaTIons and ConClUsIons

11. Calculation of parameters requested by the customer


Parameters requested
Calculations
to be validated
LOD

LoQ = 5 µg L– 1

Calculations
Ablank (aver) = – 0.00081
LoQ
s(A) 0.002895
A(LoQ) 0.028136

LoQ 2.7 µg L– 1

Repeatability

Within‑lab
reproducibility
Recovery < 10 %
Trueness
R = Cobserved/CCRM = 0.99
R = 1 %
Measurement
uncertainty

Other — state

120
Measurement of the concentration of arsenic…

12. Does the analytical procedure fulfil the requirement(s) for the intended use?
Value requested by the Is the requirement
Value obtained
Parameter customer fulfilled?
during validation
(the same as stated in Question 3) Yes/No
LOD
LoQ LoQ = 5 µg L– 1 2.7 µg L– 1 Yes
Repeatability
Within‑lab
reproducibility
Trueness R < 10 % 1 % Yes
Measurement
uncertainty
Other

The analytical procedure is fit for the intended use:


Yes No

For measurement uncertainty and traceability, refer to the corresponding pages.

121
Practical examples of traceability, measurement uncertainty and validation in chemistry

BUILDING AN UNCERTAINTY BUDGET

1. Specify the measurand and units


Measurand Concentration of arsenic in groundwater
Units µg L– 1

2. Describe the measurement procedure and provide the associated model


equation

Measurement procedure:
ISO 11969:1996 — Water quality — Determination of arsenic — Atomic absorption
spectrometric method (hydride technique)

Model equation:

A − b0 1
CAs = ×
b1 R

The input quantities in the model equation:

A Measured absorbance of the sample


b0 Calculated blank
b1 Slope of the linear least square calibration curve
CAs Final result for concentration of arsenic (μg L– 1)
R Recovery

3. Identify (all possible) sources of uncertainty


Uncertainty of concentration of reference solutions
Uncertainty of measurements of peak area
Method bias
Matrix effect
Other: uncertainty of measurements of absorbances of the sample
Other: preparation, measurement of calibration solutions and constructing the calibration graph
Other:

122
Measurement of the concentration of arsenic…

4. Evaluate values of each input quantity


Input quantity Value Units Remarks
A 0.2874
b0 0.01537
b1 0.00473 L µg– 1
R 0.99

5. Evaluate the standard uncertainty of each input quantity


Standard
Input quantity Units Remarks
uncertainty
A 0.0070
b0 0.00755
b1 0.000127 L µg‑1
R 0.01

6. Calculate the value of the measurand using the model equation


A − b0 1
CAs = × = 58.09 µg L– 1
b1 R

7. Calculate the combined standard uncertainty (uc) of the result and specify units
using:

Mathematical solution Spreadsheet approach Commercial software

Standard
Input quantity Value Units Remarks
uncertainty
A 0.2874 0.0070
b0 0.01537 0.00755
b1 0.00473 0.000127 L µg‑1
R 0.99 0.01

u(CAs) = 2.735 µg L– 1

123
Practical examples of traceability, measurement uncertainty and validation in chemistry

8. Calculate expanded uncertainty (Uc) and specify the coverage factor k and the
units

Uc = 2 × u(CAs) = 5.5 µg L– 1

% U(C) = (5.5/58.09) × 100 = 9.5 %

9. Analyse the uncertainty contribution and specify the main three input quantities
contributing the most to Uc
1 Calibration curve
2 Measurement of the absorbance of the sample
3 Recovery of the method

10. Prepare your Uncertainty budget report

124
Measurement of the concentration of arsenic…

Further reading

1. ISO 11969:1996 Water quality — Determination of arsenic — Atomic absorption


spectrometric method (hydride technique).

2. ISO 8466‑1:1990 Water Quality — Calibration and evaluation of analytical methods


and estimation of performance characteristics — Part 1: Statistical evaluation of the
linear calibration function.

3. ISO 5667‑1:1980 — Water quality — Sampling — Part 1: Guidance on the design of


sampling programmes.

4. ISO 5667‑2:1991 — Water quality — Sampling — Part 2: Guidance on sampling


techniques.

5. ISO 5667‑3:1994 — Water quality — Sampling — Part 3: Guidance on the preservation


and handling of samples.

6. Kratgen, J., ‘Calculating standard deviation and confidence intervals with a universally
applicable spreadsheet technique’, Analyst, 119 (1994), pp. 2161-2165.

7. Guide to the Expression of Uncertainty in Measurements (GUM), First edition (1995),


Geneva, Switzerland.

8. Eurachem/CITAC Guide CG4, Quantifying Uncertainty in Analytical Measurement,


Second edition (2000).

125
Practical examples of traceability, measurement uncertainty and validation in chemistry

addendum I: Measurement uncertainty calculation:


spreadsheet approach (excel)

126
Measurement of the concentration of arsenic…

127
Practical examples of traceability, measurement uncertainty and validation in chemistry

128
Measurement of the concentration of arsenic…

129
Practical examples of traceability, measurement uncertainty and validation in chemistry

130
Chapter 4
Measurement of the mass fraction of sodium
chloride in milk products by Volhard’s method
Tidža Muhić‑Šarac, Munir Mehović, Mustafa Memić

• TrainMiC® example summary form (blue page)


• A short introduction to the analytical procedure (slides)
• All the input needed to do the three exercises (yellow pages)
• The solved exercises (green pages)

131
Practical examples of traceability, measurement uncertainty and validation in chemistry

TrainMiC® example summary form

General information about the example

Measurand Mass fraction of NaCl in milk products

Example No Ex‑16

Authors of the example Tidža Muhić‑Šarac, Munir Mehović, Mustafa Memić

Analytical procedure Volhard titration

Repeatability, 0.10 % m/m NaCl


Customer’s requirements
Expanded measurement uncertainty, 0.20 % NaCl (k  = 2)

132
Measurement of the mass fraction of sodium chloride…

attached files
File
File No,
Content of the file attached Remarks
type and name
Yes No
Ex‑16‑1‑I‑NaCl‑
1 — I

milk products‑ Given by the


Volhard‑2007‑
About the analytical procedure: short introduction ü lecturer
Ver1.ppt

PART I Description of the analytical procedure ü


Each
The customer’s requirements participant
PART II concerning the quality of the ü receives their
Ex‑16‑2‑Y‑ measurement result own copy to
2 — Yellow

NaCl‑milk Validation of the measurement keep


products‑ PART III procedure — relevant equations and ü
Volhard‑2007‑ measurement data
Ver1.doc
You may also
Measurement uncertainty of the want to include
PART IV result — relevant equations and ü the relevant
measurement data certificates
here.
Establishing traceability in analytical
PART I
chemistry
ü
Ex‑16‑3‑G‑ Single laboratory validation of
3 — Green

NaCl‑milk PART II
measurement procedures
ü
products‑
Volhard‑2007‑ Building an uncertainty budget ü
Ver1.doc
PART III Addendum 1: By spreadsheet approach ü
Addendum 2: By dedicated software ü

history of the example


Version Uploaded on Short description of the change
1 18 May 2008

133
Practical examples of traceability, measurement uncertainty and validation in chemistry

a short introduction to the analytical procedure

Determination of sodium chloride


in milk products by
Volhard’s method

Scope of the presentation


• The analytical procedure and the customer’s requirements
• About ‘the chemistry’ and the measurement method
• Model equation

The analytical procedure and the


customer’s requirements

• Method defined in Official Yournal of SFRJ,


No. 32/83 “Methods for establishing quality of
milk and milk products”

• Concentration of NaCl in milk products defined


in Official Yournal of SFRJ, No. 51/82
“Quality of milk and milk products”

• Amounts of NaCl in milk products must be


adjusted according to the specifications

134
Measurement of the mass fraction of sodium chloride…
chloride in milk products by Volhard’s method

About the analytical principle

• The method is based on the volume measurement of


KSCN standard solution used for back titration of the
aliquots of AgNO3 standard solution added in excess to
the test sample solution containing NaCl besides saturated
FeNH4(SO4)2 · 12 H2O as the indicator in the nitric acid
environment.

Titration: NaCl + AgNO 3 AgCl(s) + NaNO3

Back titration: AgNO3 + KSCN AgSCN(s) + KNO3

About the analytical procedure


• Transfer the weighed samples into Erlenmeyer flasks.

• Add 20 mL of distilled water.

• Add 10.00 mL (25.00 mL) of 0.1 mol L-1 AgNO3 standard solution
and 10 mL of HNO3 solution, respectively.

• Warm the sample to the boiling point and add 10 mL of 10 %


KMnO4 solution. In case that the sample solution becomes
decolourised, add more KMnO4 solution until the colour brown is
obtained.

• Add oxalic acid into the mixture up to decolourisation, then add 100
mL of distilled water, 5 mL of Fe(NH4)(SO4)2 indicator solution and
stir all components well.

• Titrate immediately with 0.1 mol L-1 of KSCN standard solution until
the colour red-brown is obtained and remains stable for at least 30
seconds.

135
Practical examples of traceability, measurement uncertainty and validation in chemistry

Flow chart of the


analytical procedure

More equations (1)


a) Primary standard solution 0.1 M NaCl prepared
(5.85g L-1)
mNaCl PNaCl
cNaCl = (2)
M NaCl V1

b) Standard solution 0.1 M AgNO3

c NaCl V2
cAgNO3 = (3)
V3

c) Standard solution 0.1 M KSCN


cAgNO3 V4 (4)
cKSCN =
V5

136
Measurement of the mass fraction of sodium chloride…
chloride in milk products by Volhard’s method

More equations (2)

From the equations (2) and (3) it follows that:

mNaCl PNaCl V2
cAgNO3 =
M NaCl V1 V3

From the equations (2), (3) and (4) it follows that:

mNaCl PNaCl V2 V4
cKSCN =
M NaCl V1 V3 V5

More equations (3)

Chloride content expressed as mass fraction wNaCl (in %)

(CAgNO3 VAgNO3 CKSCN VKSCN ) M NaCl 100


WNaCl =
msample

cAgNO3 V6 = n1 cKSCN V7 = n2

(n1 n2 ) M NaCl 100


WNaCl =
msample

137
Practical examples of traceability, measurement uncertainty and validation in chemistry

Measurement uncertainty evaluation


Comprehensive equation (containing all sources of uncertainty)

mNaCl PNaCl V2 V6 mNaCl PNaCl V2 V4 V7 M NaCl 100


WNaCl =
M NaCl V1 V3 M NaCl V1 V3 V5 msample

u (WNaCl )
2 2
u ( n) u (msample)
+ (rep )
2
= +
WNaCl n msample

138
Measurement of the mass fraction of sodium chloride…

all the input needed to do the three exercises (yellow pages)

analytical procedure

Measurement of the mass fraction of sodium chloride in milk


products by Volhard’s method

PART I ............................................................................................................................... 140


Description of the analytical procedure

PART II .............................................................................................................................. 145


The customer’s requirements concerning the quality of the measurement result

PART III ............................................................................................................................. 146


Validation of the measurement procedure — relevant equations and
measurement data

PART IV ............................................................................................................................ 149


Measurement uncertainty of the result — relevant equations and
measurement data

139
Practical examples of traceability, measurement uncertainty and validation in chemistry

parT I. description of the analytical procedure

Task description

Table salt as an additive to milk products


According to the regulations on the quality of milk products, common, stone and sea salt
are placed to the market under the term ‘table salt’: table salt must contain at least 95 %
NaCl as a dry residue and not more than 5 % of water.

Table salt is one of the oldest additives used for the preparation of foodstuffs. Salt is used
both as a flavour improving means and as a preservative.

In milk products, depending on the product type, the salt content can vary from 0.05 % in
butter up to 8 % in some kinds of cheese as, for example Travnicki cheese. As far as the
organoleptic intensity of salt‑containing products is concerned, it has been determined
that product saltiness is not correlated to the chemically measured salt mass fraction in
the products. Saltiness emerges as a result of a series of factors: colloidal condition of
proteins, quantity of water and grease, pH value, etc.

Table salt is an important means of preserving foodstuffs due to its capability for
absorbing water from the foodstuffs, for its bacteriostatic action and for its capability of
inactivating enzymes to a certain extent.

Estimation of measurement uncertainty


Testing laboratories should have, and apply, procedures for measurement uncertainty
estimation. In certain cases, the nature of a testing method can hinder the rigorous
measurement uncertainty estimation that is justified from the statistical and measuring
point of view.

In those cases, the laboratory should provide the procedures for the identification of all
the components of uncertainty, then an accurate estimation should be made and objective
reporting of the results obtained about uncertainty provided. Reasonable estimation
should be based on knowledge of the performance of the method and on the measurement
scope and should make use of, for example, previous experience and validation data.

Measurement uncertainty is a parameter attributed to the results of analysis and it


describes the result’s dispersion around the estimated average. It is specific for every
single method in the same laboratory and it is constant for one single parameter measured
(very often for a certain area of concentration) as long as the resources related to the
estimation remain constant.

140
Measurement of the mass fraction of sodium chloride…

Principle of the measurement method


This method describes how to determine sodium chloride which can be added to butter,
clotted cream, milk cream and all kinds of cheese.

The method is based on the volume measurement of KSCN standard solution used for
back titration of the aliquots of AgNO3 standard solution added in excess to the test
sample solution containing NaCl besides saturated FeNH4(SO4)2·12H2O as the indicator
in the nitric acid environment. The chemical reactions in the solution are:
Titration: NaCl + AgNO3 → AgCl(s) + NaNO3

Back titration: AgNO3 + KSCN → AgSCN(s) + KNO3

Analytical procedure
1. Transfer the weighed samples into Erlenmeyer flasks.
2. Add 20 mL distilled water.
3. Add 10.00 mL (25.00 mL) 0.1 mol L– 1 AgNO3 standard solution and 10 ml
HNO3 solution respectively.
4. Warm the sample to the boiling point and add 10 mL 10 % KMnO4 solution.
In the case that the sample solution becomes decolourised, add more KMnO4
solution until the brown colour is obtained.
5. Add oxalic acid to the mixture up to decolourisation, then add 100 mL distilled
water, 5 mL Fe(NH4)(SO4)2 indicator solution and stir all components well.
6. Titrate immediately with 0.1 mol L– 1 KSCN standard solution until the red‑
brown colour is obtained and remains stable for at least 30 s.

Calculation of the result


(CAgNO3 × VAgNO3 − CKSCN × VKSCN ) × M Cl × 100
WNaCl = (1)
msample

Analysis of the method procedure stages


All steps of the method in the measurement of the concentration of sodium chloride in
milk products including the relevant standard measuring uncertainty are presented in
Scheme 1.

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Preparation of standard solutions


KSCN solution is standardised by standard AgNO3 solution previously standardised
against standard sodium chloride solution. Solid sodium chloride is used as a primary
standard.

Standardisation of the solutions is made through the following stages:


I. Solid sodium chloride is dried at 120 °C for 2 hours. Approximately 5.85 g is
weighed (accuracy 0.1 mg) to achieve a 0.1 mol L– 1 concentration as close as
possible.

II. The weighed NaCl is transferred into a 1 000 mL volumetric flask filled with
distilled water up to the calibration mark. NaCl concentration is estimated using
the following equation:

mNaCl × PNaCl
cNaCl = (2)
M NaCl × V1

III. A 10.0 mL aliquot of NaCl solution is taken and put into Erlenmeyer flasks, then
titrated using the previously prepared AgNO3 solution contained in the 50.0 mL
burette. The end‑point of titration is detected using K2CrO4 indicator solution.
Concentration of AgNO3 is estimated using the following equation:

cNaCl × V2
cAgNO3 = (3)
V3

IV. A 10.0 mL aliquot of AgNO3 standard solution is put into Erlenmeyer flasks and
titrated using the previously prepared KSCN solution contained in the 50.0 mL
burette. The end‑point of titration is detected using Fe(NH4)(SO4)2 indicator
solution. KSCN concentration is estimated using the following equation:

cAgNO3 × V4
cKSCN = (4)
V5

From equations (2) and (3) it follows that:

mNaCl × PNaCl × V2
cAgNO3 = (5)
M NaCl × V1 × V3

142
Measurement of the mass fraction of sodium chloride…

From equations (2), (3) and (4) it follows that:

mNaCl × PNaCl × V2 × V4
cKSCN = (6)
M NaCl × V1 × V3 × V5

Assuming that:

mNaCl × PNaCl × V2 × V6
n1 = (7) and
M NaCl × V1 × V3

mNaCl × PNaCl × V2 × V4 × V7
n2 = (8)
M NaCl × V1 × V3 × V5

3.2. Finally, it results in (mass fraction WNaCl in %):

( n1 − n2 ) × M NaCl × 100
WNaCl = (9)
msample

where:
V1 = Volume of primary standard NaCl solution (mL)
= Volume of NaCl solution aliquot used for standardising AgNO3
V2
solution (mL)
V3 = Volume of AgNO3 solution used for NaCl aliquot titration (mL)
= Volume of AgNO3 solution aliquot used for standardising
V4
KSCN solution (mL)
= Volume of KSCN solution used for AgNO3 solution aliquot
V5
titration (mL)
V6 = Volume of standard AgNO3 solution aliquot (mL)
= Volume of standard KSCN solution used for the AgNO3 aliquot
V7
back titration
m = Mass of NaCl (g)
P = Purity of NaCl (99.95 % m/m)

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Sheme 1: Volhard,s method determination of NaCl in milk products, with standard


measurement uncertainty types

START

144
Measurement of the mass fraction of sodium chloride…

parT II. The customer’s requirements concerning quality of the


measurement result

1. Repeatability; S = 0.10 % m/m NaCl


2. Expanded measurement uncertainty; U = 0.20 % m/m NaCl, k = 2

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Practical examples of traceability, measurement uncertainty and validation in chemistry

PART III. Validation of the measurement procedure — relevant


equations and measurement data

Validation is achieved by comparing the results achieved by another method (Mohr’s


method for the measurement of the mass fraction of chloride ions)

(a) Relevant equation for Volhard’s method:


(CAgNO3 × VAgNO3 − CKSCN × VKSCN ) × 10−3 × M NaCl × 100
WNaCl =
msample

Repeatability of method (Volhard’s method)


Mass c(AgNO3) V(AgNO3) c(KSCN) V(KSCN) M(NaCl) W(NaCl)
No
(g) (mol L– 1) (mL) (mol L– 1) (mL) (g mol– 1) (% m/m)
1 3.9360 0.0999 10.00 0.0998 6.20 58.443 0.56
2 3.7713 0.0999 10.00 0.0998 6.25 58.443 0.58
3 3.8413 0.0999 10.00 0.0998 6.30 58.443 0.56
4 4.3738 0.0999 10.00 0.0998 6.00 58.443 0.53
5 3.8024 0.0999 10.00 0.0998 6.70 58.443 0.51
6 3.5438 0.0999 10.00 0.0998 6.70 58.443 0.54
7 4.2133 0.0999 10.00 0.0998 6.00 58.443 0.56
8 4.2466 0.0999 10.00 0.0998 6.00 58.443 0.55
9 4.1013 0.0999 10.00 0.0998 6.20 58.443 0.54
10 3.8678 0.0999 10.00 0.0998 6.55 58.443 0.52
Average: 0.546
St.dev.: 0.02162

(b) Relevant equation for Mohr’s method:

( cAgNO3 × VAgNO3 ) × 10−3 × M NaCl × 100


WNaCl =
msample

146
Measurement of the mass fraction of sodium chloride…

Repeatability of method (Mohr’s method)


Mass V(AgNO3) c(AgNO3) M(NaCl) w(NaCl)
No
(g) (mL) (mol L– 1) (g mol– 1) (% m/m)
1 0.4735 5.02 0.0100 58.443 0.62
2 0.4974 4.93 0.0100 58.443 0.58
3 0.4594 4.64 0.0100 58.443 0.59
4 0.5121 5.00 0.0100 58.443 0.57
5 0.4563 4.68 0.0100 58.443 0.60
6 0.5763 5.62 0.0100 58.443 0.57
7 0.5597 5.75 0.0100 58.443 0.60
8 0.4627 4.60 0.0100 58.443 0.58
9 0.4811 4.62 0.0100 58.443 0.56
10 0.6506 6.56 0.0100 58.443 0.59
Average: 0.586
St.dev.: 0.0529

Comparison of results:

Methods Average St.dev. n


Volhard 0.546 0.0216 10
Mohr 0.586 0.0529 10

F-test (comparability of variances)

Discussion of equivalence of variances for two methods.

H0: variances do NOT differ significantly


H1: variances are significantly different

Degrees of freedom for a 95 % level of confidence; v1 = v2 = 9 ; Ftab = 3.18; Fexp = 2.44

( 0.0529 )
2
s12
F ( v1 , v2 ) = F= = 2.44
s22
( 0.0216 )
2

Fexp = 2.44 < Ftab = 3.18

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Null hypothesis about variances cannot be rejected at a level of confidence of 95 % and


was confirmed by F‑test for Volhard’s and Mohr’s methods.

t-test (equivalence of mean values)


Comparison of average values for two methods.

H0: mean values do NOT differ significantly


H1: mean values are significantly different

Degrees of freedom for a 95 % level of confidence; v1 = v2 = 9 ; v1 + v2 = 9 + 9 = 18 ;

ttab = 2.101; texp = 2.100

x1 − x2 n1 + n2
t= × = 2.100
s n1 × n2

n1 n2

∑( x − x1 ) + ∑ ( xi − x2 )
2 2

s= i =1
i
i =1
=
(n 1 − 1 ) × s12 + ( n2 − 1 ) × s22
= 0.0425
n1 + n2 − 2 n1 + n2 − 2

texp = 2.100 < ttab = 2.101

Null hypothesis about average values cannot be rejected at a level of confidence of 95 %


and was confirmed by t‑test for Volhard’s and Mohr’s methods.

148
Measurement of the mass fraction of sodium chloride…

ParT IV. Measurement uncertainty of the result: relevant


equations and measurement data 

1. Combined standard uncertainty calculation


Combined standard measurement uncertainties are calculated depending on their type,
A or B, i.e. according to the distribution function of the measuring data (triangle,
rectangular or normal distribution).

(a) Relevant equation to calculate combined uncertainty

( n1 − n2 ) × M NaCl × 100
WNaCl =
msample

Comprehensive equation (containing all sources of uncertainty)

 m × PNaCl × V2 × V6 mNaCl × PNaCl × V2 × V4 × V7  M NaCl × 100


WNaCl =  NaCl − ×
 M NaCl × V1 × V3 M NaCl × V1 × V3 × V5  msample

(b) Measurement uncertainty for AgNO3 amount of substance (n1)


Uncertainty sources for AgNO3 amount of substance amount (n1), according to the
equation
mNaCl × PNaCl × V2 × V6
n1 =
M NaCl × V1 × V3

as well as the values of combined standard and relative standard uncertainties are shown
in Table 1.

Table 1: Uncertainty elements for n1


Standard
Input quantity Value Units Remarks
uncertainty
m(NaCl) 5980.4 mg 0.085 Mass of NaCl as primary standard
P(NaCl) 0.9995  % 0.00029 Purity of NaCl
V1 1000 mL 0.6 Volume of volumetric flask
V2 10.0 mL 0.04 Volume of NaCl aliquot
V3 10.24 mL 0.05 Volume of AgNO3 for NaCl titration
V6 10.0 mL 0.04 Volume of AgNO3 for analysis

n1 = 0.001 mol

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Practical examples of traceability, measurement uncertainty and validation in chemistry

u ( n1 )  u ( mNaCl )   u ( PNaCl )   u ( V1 )   u ( V2 )   u ( V3 )   u ( V6 ) 
2 2 2 2 2 2

=   +   +   +   +   +  
n1  mNaCl   PNaCl   V1   V2   V3   V6 

(c) Measurement uncertainty for KSCN amount of substance (n2)


Uncertainty sources for KSCN amount of substance (n2), according to the equation

mNaCl × PNaCl × V2 × V4 × V7
n2 =
M NaCl × V1 × V3 × V5

as well as the values of combined standard and relative standard uncertainties are shown
in Table 2.

Table 2: Uncertainty elements for n2


Standard
Input quantity Value Units Remarks
uncertainty
m(NaCl) 5980.4 mg 0.085 Mass of NaCl as primary standard
P(NaCl) 0.9995  % 0.00029 Purity of NaCl
V1 1000 mL 0.6 Volume of volumetric flask
V2 10.0 mL 0.04 Volume of NaCl aliquot
V3 10.24 mL 0.05 Volume of AgNO3 for NaCl titration
V4 10.0 mL 0.04 Volume of AgNO3 titration
V5 10.01 mL 0.05 Volume of KSCN for AgNO3 titration
V7 6.29 mL 0.05 Volume of KSCN for AgNO3 titration

n2 = 0.0006 mol

u ( n2 )  u ( mNaCl )   u ( PNaCl )   u ( V1 )   u ( V2 )   u ( V3 )   u ( V4 )   u ( V5 )   u ( V7 ) 
2 2 2 2 2 2 2 2

=   +   +   +   +   +   +   +  
n2  mNaCl   PNaCl   V1   V2   V3   V4   V5   V7 

(d) Measurement uncertainty of a difference of amounts of substance n1 and n2


If n1 – n2 = Δn, then u(Δn) is calculated as uncertainty of the subtraction results

u ( ∆n ) = u ( n1 ) + u ( n2 )
2 2

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Measurement of the mass fraction of sodium chloride…

(e) Calculation of total combined uncertainty for method


Measurement of sources of uncertainty for the described method for the measurement of
the mass fraction of NaCl in milk products, according to the equation

( n1 − n2 ) × M NaCl × 100
WNaCl =
msample

as well as the values of combined standard and relative uncertainties are shown in
Table 3.

Table 3: Uncertainty elements and values of combined standard uncertainty in the


measurement of the mass fraction of NaCl according to Volhard’s method
Combined
Input quantity Remarks Value Units
Uncertainty u(x)
n1 AgNO3 amount of substance 0.001 mol
n2 KSCN amount of substance 0.0006 mol
∆n Amount of substance 0.0004 mol
WNaCl Mass fraction NaCl 0.546  % m/m
Expanded
uncertainty
(P = 95 %; k = 2)

From Eq. (9) it results that the complete combined uncertainty of measurement according
to Volhard’s method can be expressed as:
2
 u( ∆n)   u( msample ) 
2
u(WNaCl )
=   +   + ( rep )2
WNaCl  ∆n   msample 

Calculation of expanded measurement uncertainty of Volhard’s method:


The expanded measurement uncertainty represents the product of complete measurement
uncertainty (for one method) and the k factor. From the probability of finding the
measurement results, the k factor can have value 2 for the probability of finding the
measurement results of 95 % or value 3 for probability of finding the measurement
results of 99 %. The factor of value 2 is used most frequently for the calculation of
expanded measurement uncertainties.
U = k × u(x)

151
Practical examples of traceability, measurement uncertainty and validation in chemistry

The solved exercises (green pages)

TrainMiC® exercises

analytical procedure

Measurement of the concentration of sodium chloride in milk


products by Volhard’s method

exerCIse 1:
establishing traceability in analytical chemistry

exerCIse 2:
single laboratory validation of measurement procedures
Part I: General issues
Part II: Parameters to be validated
Part III: Some calculations and conclusions

exerCIse 3:
building an uncertainty budget
Addendum I: By spreadsheet approach
Addendum II: By dedicated software

152
Measurement of the mass fraction of sodium chloride…

ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

1. Specifying the analyte and measurand

Analyte Chloride expressed as sodium chloride (NaCl)

Measurand Chloride mass fraction expressed as total sodium chloride in milk cream

Units Mass fraction wNaCl in %

2. Choosing a suitable measurement procedure with associated model equation


Measurement
Volumetric method (titration and back titration)
procedure

Type of calibration Standard curve Standard addition Internal standard

Model equation:
1.Standard solutions

1.1. Stock solution‑prepared from pure (99.95 % m/m) NaCl (0.1 M; 5.85 g L– 1)

cNaCl × V2
cAgNO3 = (2)
V3

1.2. Standard solution of AgNO3 (0.1 M; 17.0 g L– 1)

cNaCl × V2
cAgNO3 = (3)
V3

1.3. Standard solution of KSCN (0.1 M; 9.7 g L– 1)

cAgNO3 × V4
cKSCN = (4)
V5

1.4. From equations (2) and (3) it follows that:

mNaCl × PNaCl × V2
cAgNO3 = (5)
M NaCl × V1 × V3

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Practical examples of traceability, measurement uncertainty and validation in chemistry

1.5. From equations (2), (3) and (4) it follows that:

mNaCl × PNaCl × V2 × V4
cKSCN = (6)
M NaCl × V1 × V3 × V5

2. Combined model equation for the calculation of mass fraction WNaCl (in %):

(CAgNO3 × VAgNO3 − CKSCN × VKSCN ) × M NaCl × 100


WNaCl =
msample

 m × PNaCl × V2 × V6 mNaCl × PNaCl × V2 × V4 × V7  M NaCl × 100


WNaCl =  NaCl − ×
 M NaCl × V1 × V3 M NaCl × V1 × V3 × V5  msample

where:
V1 = Volume of primary standard NaCl solution (mL)
= Volume of NaCl solution aliquot used for standardising AgNO3
V2
solution (mL)
V3 = Volume of AgNO3 solution used for NaCl aliquot titration (mL)
= Volume of AgNO3 solution aliquot used for standardising
V4
KSCN solution (mL)
= Volume of KSCN solution used for AgNO3 solution aliquot
V5
titration (mL)
V6 = Volume of standard AgNO3 solution aliquot (mL)
= Volume of standard KSCN solution used for the AgNO3 aliquot
V7
back titration
m = Mass of NaCl (g)
P = Purity of NaCl (99.95 % m/m)

3. List the input quantities according to their influence on the uncertainty of the
result of the measurement (first the most important ones): at this point, your
judgement should be based on your previous experience only.
1 Repeatability
2 Volume of the analysed solution (pipettes, volumetric flasks and burette) (mL)
3 Concentration of standard solution‑purity of NaCl (99.95 %)
4 Mass of homogenised sample (g)
5

154
Measurement of the mass fraction of sodium chloride…

4. List the reference standards needed and state the information regarding
traceability of the reference value

For the analyte


1 Name/chemical formula/producer Purity of NaCl, Merck, min 99.95 %
2 Name/chemical formula/producer

For the other input quantities


Quantity/equipment/calibration
Balance calibrated by CRM etalons (certified mass
1 (e.g. mass/balance/calibrated by NMI, U = xx
pieces) in laboratory
(k = 2))
Volumetric flask — Class A or B quality (certificate) and
2 Quantity/equipment/calibration
calibration with water in laboratory
3 Quantity/equipment/calibration
4 Quantity/equipment/calibration

5. Estimating uncertainty associated with the measurement


Are all important parameters included in the
Yes No
model equation?
Matrix effect, interferences by Br‑, I‑, CN‑
Other important parameters:
(but there are not present in a milk products)

6. How would you prove traceability of your result?


1 By the use of purity reagents (p.a. quality)
2 By the use of calibrated balance
3 By the use of volumetric flasks — Class A or B quality calibrated in laboratory

7. Any other comments, questions …

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Practical examples of traceability, measurement uncertainty and validation in chemistry

SINGLE LABORATORY VALIDATION OF


MEASUREMENT PROCEDURES

parT I: General IssUes

1. Specify the measurement procedure, analyte, measurand and units


The measurement
Volumetric method (back titration)
procedure
Analyte Chloride expressed as sodium chloride (NaCl)
The measurand Chloride content expressed as total sodium chloride in milk cream
Units Mass fraction WNaCl in %

2. Specify the scope


Matrix Proteins, fats and lactose
Measuring range 0.1‑10 % m/m

3. Requirements of the measurement procedure


Intended use of the results
Parameters to be validated Value requested by the customer
LOD
LoQ
Mark the customer’s Repeatability S = 0.1 % m/m NaCl
requirements and give
their values In‑lab reproducibility
Trueness
Measurement uncertainty U = 0.2 % m/m NaCl (k = 2)
Other — state

4. Origin of the measurement procedure


VALIDATION
New in‑house method Full
Modified validated method Partial
Official standard method Confirmation/verification

156
Measurement of the mass fraction of sodium chloride…

parT II: paraMeTers To be ValIdaTed

5. Selectivity/interference/recovery

Where yes, give further information, for example which CRM, reference method.

CRM/RM: analysis of available CRM or RM


Further information
Spike of pure substance
Purity of NaCl (value of certificate 99.95 %)
Compare with a reference method

Selectivity, interferences

Test with different matrices

Other — specify
Compare with another method (Mohr’s method for the measurement of the concentration of
chloride)
(a) t‑test (test of equivalent average values for two methods)
(b) F‑test (test of equivalent variances for two methods)

6. Measuring range
Linearity
Upper limit
LOD
LoQ

7. Spread — precision
Repeatability
Reproducibility (within Lab)
Reproducibility (between labs)

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Practical examples of traceability, measurement uncertainty and validation in chemistry

8. Robustness
Variation of parameters

9. Quality control
Control charts
Participation in PT schemes

10. Other parameters to be tested


Working range and testing of homogeneity of variances
R square
Residual standard deviation
Standard deviation of the analytical procedure
Coefficient of variation of the analytical procedure
Measurement uncertainty

158
Measurement of the mass fraction of sodium chloride…

parT III: soMe CalCUlaTIons and ConClUsIons

11. Calculation of parameters requested by the customer


Parameters requested to be
Calculations
validated
LOD
LoQ
n

Repeatability ∑(x i − x )2
S= n =1
; S = 0.0216 % m/m
n −1
Within‑lab reproducibility
t‑test

Trueness x1 − x2 n1 + n2
t= × ; tab > texp ; 2.101 > 2.100
s n1 × n2

U = k × u (x); k = 2, 95 % level of confidence


Measurement uncertainty
U = 2 × 0.017 = 0.034 % m/m
F‑test

s12
Other — state F ( v1 , v2 ) = ; Ftab > Fexp ; 3.18 > 2.44
s22

12. Does the analytical procedure fulfil the requirement(s) for the intended use?
Value requested by the Is the requirement
Value obtained
Parameter customer fulfilled?
during validation
(the same as stated in Question 3) Yes/No
LOD
LoQ
Repeatability 0.1 % m/m NaCl ± 0.0216 % m/m NaCl Yes
Within‑lab
reproducibility
Trueness
Measurement U = repeatability × 2
± 0.034 % m/m NaCl Yes
uncertainty U = 0.2 % m/m NaCl
Other

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Practical examples of traceability, measurement uncertainty and validation in chemistry

The analytical procedure is fit for the intended use:


Yes No

For measurement uncertainty and traceability, refer to the corresponding pages.

160
Measurement of the mass fraction of sodium chloride…

BUILDING AN UNCERTAINTY BUDGET

1. Specify the measurand and units


Measurand Total amounts sodium chloride in milk cream
Units Mass fraction wNaCl in %

2. Describe the measurement procedure and provide the associated model


equation

Measurement procedure:
1. Transfer the weighed samples into Erlenmeyer flasks.
2. Add 20 mL distilled water.
3. Add 10.00 mL (25.00 mL) 0.1 mol L– 1 AgNO3 standard solution and 10 mL
HNO3 solution respectively.
4. Warm the sample to the boiling point and add 10 mL 10 % KMnO4 solution.
In the case that the sample solution becomes decolourised, add more KMnO4
solution until the brown colour is obtained.
5. Add oxalic acid into the mixture up to decolourisation, then add 100 mL distilled
water, 5 mL Fe(NH4)(SO4)2 indicator solution and stir all components well.
6. Titrate immediately with 0.1 mol L– 1 of KSCN standard solution until the red‑
brown colour is obtained and remains stable for at least 30 s.

Model equation:
1. Standard solutions

1.1. Stock solution‑prepared from pure (99.95 % m/m) NaCl, (0.1 M; 5.85 g L– 1)

mNaCl × PNaCl
cNaCl = (2)
M NaCl × V1

1.2. Standard solution of AgNO3 (0.1 M; 17.0 g L– 1)

cNaCl × V2
cAgNO3 = (3)
V3

1.3. Standard solution of KSCN (0.1 M; 9.7 g L– 1)


cAgNO3 × V4
cKSCN = (4)
V5

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Practical examples of traceability, measurement uncertainty and validation in chemistry

1.4. From equations (2) and (3) it follows that:

mNaCl × PNaCl × V2
cAgNO3 = (5)
M NaCl × V1 × V3

1.5. From equations (2), (3) and (4) it follows that:

mNaCl × PNaCl × V2 × V4
cKSCN = (6)
M NaCl × V1 × V3 × V5

2. Combined model equation for the calculation of mass fraction wNaCl (in %):

(CAgNO3 × VAgNO3 − CKSCN × VKSCN ) × M NaCl × 100


WNaCl =
msample

( n1 − n2 ) × M NaCl × 100
WNaCl =
msample

3. Model equation for calculation combined uncertainty (with all sources uncertainty)

 m × PNaCl × V2 × V6 mNaCl × PNaCl × V2 × V4 × V7  M NaCl × 100


WNaCl =  NaCl − ×
 M NaCl × V1 × V3 M NaCl × V1 × V3 × V5  msample

3. Identify (all possible) sources of uncertainty


Uncertainty of concentration of reference solutions
Uncertainty of measurements of peak area
Method bias
Matrix effect
Other: uncertainty of measurements of sample mass
Other: uncertainty of volume of the analysed solutions
Other:

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Measurement of the mass fraction of sodium chloride…

4. Evaluate values of each input quantity


Input quantity Value Units Remarks
m(NaCl) 5980.4 mg Mass of NaCl as primary standard
P(NaCl) 0.9995  % Purity of NaCl
V1 1000 mL Volume of volumetric flask
V2 10.0 mL Volume of NaCl aliquot
V3 10.24 mL Volume of AgNO3 for NaCl titration
V4 10.0 mL Volume of AgNO3 aliquot
V5 10.01 mL Volume of KSCN for AgNO3 titration
m(sample) 3969.8 mg Sample mass
V6 10.0 mL Volume of AgNO3 for analysis
V7 6.29 mL Volume of KSCN for AgNO3 titration
rep 1 Repeatability of analysis

5. Evaluate the standard uncertainty of each input quantity


Standard
Input quantity Units Remarks
uncertainty
m(NaCl) 0.085 mg Mass of NaCl as primary standard
P(NaCl) 0.0003  % Purity of NaCl
V1 0.66 mL Volume of volumetric flask
V2 0.04 mL Volume of NaCl aliquot
V3 0.05 mL Volume of AgNO3 for NaCl titration
V4 0.04 mL Volume of AgNO3 aliquot
V5 0.05 mL Volume of KSCN for AgNO3 titration
m(sample) 0.06 mg Sample mass
V6 0.04 mL Volume of AgNO3 for analysis
V7 0.05 mL Volume of KSCN for AgNO3 titration
rep 0.0216 Repeatability of analysis

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Practical examples of traceability, measurement uncertainty and validation in chemistry

6. Calculate the value of the measurand, using the model equation


 m × PNaCl × V2 × V6 mNaCl × PNaCl × V2 × V4 × V7  M NaCl × 100
WNaCl =  NaCl − ×
 M NaCl × V1 × V3 M NaCl × V1 × V3 × V5  msample

WNaCl = 0.546 % m/m = 0.55 % m/m

7. Calculate the combined standard uncertainty (uc) of the result and specify units
using:

Mathematical solution Spreadsheet approach Commercial software

Standard
Input quantity Value Units Remarks
uncertainty
Total amount of sodium chloride in
WNaCl 0.546 0.017  % m/m
milk cream

8. Calculate expanded uncertainty (Uc) and specify the coverage factor k and the
units

Uc = u × k ( k = 2) U c = 0.017 × 2
U c = 0.034 % m/m

9. Analyse the uncertainty contribution and specify the main three input quantities
contributing the most to Uc
1 Repeatability — 50.7 % to the expanded uncertainty
2 Volume of KSCN for AgNO3 titration (burette) — 19.64 % to the expanded uncertainty
3 Volume of AgNO3 for analysis (pipette) — 12.59 % to the expanded uncertainty

10. Prepare your Uncertainty budget report

WNaCl = (0.55 ± 0.034) % m/m

164
Measurement of the mass fraction of sodium chloride…

Further reading

1. ISO/BIPM/IEC/IFCC/IUPAC/IUPAP/OIML, International vocabulary of basic and


general terms in metrology, Third edition, VIM (2008) (International Vocabulary of
Metrology — Basic and General Concepts and Associated Terms, ISO (2008)).

2. Eurachem/CITAC GUIDE, Quantifying Uncertainty in Analytical Measurement,


Second edition, Eurachem/CITAC (2000) (http://www.eurachem.info).

3. Eurolab Technical Report No 1/2006, Guide to the Evaluation of Measurement


Uncertainty for Quantitative Test Results, Eurolab (2006) (http://www.eurolab.org).

4. Eurolab Technical Report No 1/2007, Measurement Uncertainty Revisited: Alternative


Approaches to Uncertainty Evaluation, Eurolab (2006) (http://www.eurolab.org).

5. NORDTEST Technical Report 537, Handbook for calculation of measurement


uncertainty in environmental laboratories, NORDTEST (2003) (http://www.nordtest.
org).

6. EA Guideline EA‑4/02, Expression of the uncertainty of measurement in calibration,


EA (1999) (http://www.european‑accreditation.org).

7. Parkany, M., Quality Assurance for Analytical Laboratories, Royal Society of


Chemistry, London, United Kingdom (1993).

8. Eurachem/CITAC Guide, Traceability in chemical measurement, Eurachem/CITAC


(2003) (http://www.eurachem.info).

9. Eurachem/CITAC GUIDE, Guide to Quality in Analytical Chemistry, Eurachem/


CITAC (2002) (http://www.eurachem.org) or CITAC (http://www.citac.cc/).

10. EN ISO/IEC 17025:2005 General requirements for the competence of testing and
calibration laboratories.

11. ‘Quality of milk and milk products’, Official Journal SFRJ, 51/82 (1982).

165
166
RSD
Units Parameter value unc V1 V2 V3 V4 V5 V6 V7 P(NaCl) m(NaCl) m(sample) Rep
(%)
mL V1 1000 0.65 0.07 1000.65 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
mL V2 10 0.04 0.40 10 10.04 10 10 10 10 10 10 10 10 10
mL V3 10.24 0.05 0.49 10.24 10.24 10.29 10.24 10.24 10.24 10.24 10.24 10.24 10.24 10.24
mL V4 10 0.04 0.40 10 10 10 10.04 10 10 10 10 10 10 10
mL V5 10.01 0.05 0.50 10.01 10.01 10.01 10.01 10.06 10.01 10.01 10.01 10.01 10.01 10.01
mL V6 10 0.04 0.40 10 10 10 10 10 10.04 10 10 10 10 10
mL V7 6.29 0.05 0.79 6.29 6.29 6.29 6.29 6.29 6.29 6.34 6.29 6.29 6.29 6.29
P(NaCl) 0.9995 0.0003 0.03 0.9995 0.9995 0.9995 0.9995 0.9995 0.9995 0.9995 0.99979 0.9995 0.9995 0.9995
mg m(NaCl) 5980.4 0.085 0.00 5980.4 5980.4 5980.4 5980.4 5980.4 5980.4 5980.4 5980.4 5980.485 5980.4 5980.4
mg m(sample) 3969.8 0.06 0.00 3969.8 3969.8 3969.8 3969.8 3969.8 3969.8 3969.8 3969.8 3969.8 3969.86 3969.8
rep 1 0.0216 2.16 1 1 1 1 1 1 1 1 1 1 1.0216
M(NaCl) silent
spreadsheet approach (excel)

W(NaCl) 0.546 0.017 3.0 0.546099 0.548639 0.543798 0.54276 0.55105 0.552335 0.53911 0.54661 0.546461 0.54645 0.55826 Func
(k = 1) 0.000355 – 0.00218 0.002655 0.0037 – 0.0046 – 0.00588 0.00734 – 0.0002 – 7.8E‑06 8.3E‑06 – 0.0118 Diff
1.26E‑07 4.78E‑06 7.05E‑06 1.4E‑05 2.1E‑05 3.46E‑05 5.4E‑05 2.5E‑08 6.03E‑11 6.8E‑11 0.00014 Diff^2
0.00027 Sum
V1 V2 V3 V4 V5 V6 V7 P(NaCl) m(NaCl) m(sample) rep
0.0 % 1.7 % 2.6 % 5.0 % 7.7 % 12.6 % 19.6 % 0.0 % 0.0 % 0.0 % 50.74 % Index
100 % Sum

 m × PNaCl × V2 × V6 mNaCl × PNaCl × V2 × V4 × V7  M NaCl × 100


WNaCl =  NaCl − ×
 M NaCl × V1 × V3 M NaCl × V1 × V3 × V5  msample
addendum I: Measurement uncertainty calculation:
Practical examples of traceability, measurement uncertainty and validation in chemistry
Chapter 5
Measurement of the concentration of total organic
carbon (ToC) in waste water
Brigita Tepuš, Marjana Simonič

• TrainMiC® example summary form (‘blue page’)


• A short introduction to the analytical procedure (‘slides’)
• All the input needed to do the three exercises (‘yellow pages’)
• The solved exercises (‘green pages’)

167
Practical examples of traceability, measurement uncertainty and validation in chemistry

TrainMiC® example summary form

General information about the example

Measurand Concentration of total organic carbon (TOC) in waste water

Example No Ex‑09

Authors of the example Brigita Tepuš, Marjana Simonič

SIST ISO 8245:1999 — Water quality — Guidelines for the determination


Analytical procedure
of total (TOC) and dissolved organic carbon (DOC)

Customer’s requirements LoQ, measurement uncertainty

168
Measurement of the concentration of total organic carbon (TOC) in waste water

Attached files

File
File No, type and name Content of the file attached Remarks
Yes No
Ex‑09‑1‑I‑TOC‑
1 — I

About the analytical procedure: short Given by the


wastewater‑NDIR‑
introduction
ü lecturer
2007‑Ver1.ppt
Description of the analytical
PART I
procedure
ü
Each
The customer’s requirements
participant
PART II concerning the quality of the ü receives their
measurement result
own copy to
2 — Yellow

Ex‑09‑2‑Y‑TOC‑ Validation of the measurement keep


wastewater‑NDIR‑ PART III procedure — relevant equations ü
2007‑Ver1.doc and measurement data
You may also
Measurement uncertainty of the want to include
PART IV result — relevant equations and ü the relevant
measurement data certificates
here.
Establishing traceability in
PART I
analytical chemistry
ü

Single laboratory validation of


PART II ü
3 — Green

Ex‑09‑3‑G‑TOC‑ measurement procedures


wastewater‑NDIR‑
2007‑Ver1.doc
Building an uncertainty budget ü
Addendum 1: By spreadsheet
PART III approach
ü
Addendum 2: By dedicated
software
ü

History of the example


Version Uploaded on Short description of the change
1 18 May 2008

169
Practical examples of traceability, measurement uncertainty and validation in chemistry

a short introduction to the analytical procedure

Determination of Total Organic


Carbon (TOC) in wastewater by
combustion

Scope of the presentation


• The analytical procedure and the customer’s requirements
• Chemical reaction and measurement procedure
• Model equation
• Measurement uncertainty

The analytical procedure and the


customer’s requirements

The standard measurement procedure 'Water quality-


Guidelines for the determination of total organic carbon
(TOC) and dissolved organic carbon (DOC) ISO 8245:2000
was followed, which gives guidance for the determination of
TOC in wastewater.
Customer requirements are related to the quality of the
results, accuracy and uncertainty, in accordance with
standard SIST ISO 8245, 1996. Measurement uncertainty
must be below 15 % (general accepted).

170
Measurement of the concentration of total organic carbon (TOC) in waste water

Measurement procedure

1. The organic carbon present in wastewater is


oxidized by combustion at 720 C to CO2.

2. The content of CO2 is determined - non-dispersive


infrared (NDIR) was used (various analytical
techniques can be used for this purpose).

Analytical procedure for the


determination of TOC
Weighting of potassium
hydrogen phthalate (PHP)

Stock reference solution


(CTOC)

Working reference solutions


Sample preparation
(CTOC1)
(homogeneized, acidified)

Working solutions (CTOC2)


Sample dilution

Addition of acid

Sparging of the sample

Measuring concentration of CTOC-sample mg L-1

171
Practical examples of traceability, measurement uncertainty and validation in chemistry

Model equation

Measurand: TOC in wastewater


Unit: mg L-1
A B0
CTOC-sample =
B1

CTOC-sample - concentration of TOC in sample, mg L-1


A - peak area of sample
B1 - slope of calibration curve
B0 - intercept of calibration curve

Model equation and equation for


measurement uncertainty calculation

A B0
CTOC sample = Fd Fr Fh FR Fs
B1

Fd - dilution factor of the sample


Fr - repeatability factor
Fh - stability factor
FR - recovery factor
Fs - factor of sampling procedure

172
Measurement of the concentration of total organic carbon (TOC) in waste water

Cause-effect diagram for the TOC


determination

CTOC-curve Peak areacurve CTOC-sample


gas flow
dilution 1
reagent purity volume
temperature volume mass
CO 2 concentration Peak area
calib. repeatability
dilution 2
pressure dilution
temperature volume volume temperature
temperature
linear least square temperature volume
regression
CTOC

repeatability storage recovery sampling


(precision study) (trueness study)

173
Practical examples of traceability, measurement uncertainty and validation in chemistry

All the input needed to do the three exercises

analytical procedure

Measurement of the concentration of total organic carbon


(TOC) in waste water

The standard measurement procedure in sIsT Iso 8245:1999 —


Water quality — Guidelines for the determination of total (ToC)
and dissolved organic carbon (doC)

parT I ................................................................................................................................ 175


description of the analytical procedure

parT II .............................................................................................................................. 179


The customer’s requirements concerning the quality of the measurement result

parT III ............................................................................................................................. 180


Validation of the measurement procedure — relevant equations and
measurement data

parT IV ............................................................................................................................. 181


Measurement uncertainty of the result — relevant equations and
measurement data

174
Measurement of the concentration of total organic carbon (TOC) in waste water

PART I. Description of the analytical procedure

SIST ISO 8245:1999 — Water quality — Guidelines for the determination of total (TOC)
and dissolved organic carbon (DOC)

Scope
This International Standard describes the measurement procedure for the measurement
of concentration of total carbon (TC), total inorganic carbon (TIC) and total organic
carbon (TOC) in drinking water, groundwater, surface water, sea water and waste water.

This measurements procedure applies to water samples containing organic carbon


content from 0.3 to 1 000 mg L– 1. The lower range is only applicable in special cases, for
example in the case of drinking water; however, for measurements from 0.3 to 5 mg L– 1,
highly sensitive instruments are required. The concentrations above 1 000 mg L– 1 may
be measured after appropriate dilution of the sample solution.

The concentration of purgeable organic substances, such as benzene, toluene, cyclohexane


and chloroform, can also be measured using this method.

Principle
Carbon present in water in organic form (organic C) can be oxidised to carbon dioxide
by combustion, by the addition of an appropriate oxidant, by UV radiation or any other
high‑energy radiation.

The final measurement of the concentration of CO2 is carried out by analytical techniques,
for example infrared spectrometry, titration, thermal conductivity, conductometry,
coulometry, CO2‑sensitive sensors or flame ionisation detection.

Interference
In addition to organic carbon, the water sample may contain carbon dioxide or ions
of carbonic acid. Prior to the measurement of TOC, it is essential that all inorganic
carbon is removed by purging the acidified sample with a gas which is free from
CO2 and organic compounds. Purgeable organic substances such as benzene, toluene,
cyclohexane, and chloroform, may partly escape upon stripping. In the presence of listed
organic compounds, the TOC concentration is measured separately or by subtracting
(TC - TIC = TOC).

Inorganic carbon is removed by acidification and purging or is measured separately.

Reagents
Reagents should be used according to the manufacturer’s instructions, and should, if
necessary, be pretreated. Potassium hydrogen phthalate PHP (Merck, Germany) was
dried to constant mass at 105 °C in this case.

175
Practical examples of traceability, measurement uncertainty and validation in chemistry

Standard stock solution


Pure potassium hydrogen‑phthalate should be used for the preparation of standard stock
solution. A stock reference solution (CTOC) with a concentration of 1 000 mg L– 1 was
prepared by weighing the PHP and dissolving it in a volumetric flask with deionised
water.

Working reference solutions


Prepare working reference solutions by the appropriate dilution of the stock solution.
Pipette 100 mL of the potassium hydrogen phthalate stock solution into a 1 000 mL
volumetric flask, dilute to the mark with water and mix ( CTOC ). 1

Working solutions
Prepare working solutions by appropriate dilution of the working reference solution.
Pipette an appropriate volume of the potassium hydrogen phthalate working reference
solution into a 100 mL (one‑mark) volumetric flask, dilute to the mark with water and
mix (CTOC ).
2

Blank calibration solution


Carry out the complete procedure using a sample of pure water. The maximum acceptable
TOC of dilution water is 0.1 mg L–1 carbon (UV treatment). The TOC of the water
used for dilutions and the preparation of calibration standards should be negligible in
comparison to the TOC in the sample.

Procedure
An acidified sample (pH < 2, c(HCl) = 0.2 mol L– 1, pH is measured by pH meter MA
5741, ISKRA) is analysed by combustion.

The principle is to oxidise the organic carbon (C) in wastewater to carbon dioxide by
combustion at 720 °C. There are two methods for TOC concentration measurement: a
differential and a direct method. In addition to organic carbon, the wastewater sample
may contain carbon dioxide and/or ions of carbonic acid. Prior to the TOC concentration
measurement, it is essential that total inorganic carbon (TIC) is removed by purging
the acidified sample with a gas which is free from CO2 and organic compounds. In
the presence of these substances, the TOC concentration is measured separately or the
differential method

(TOC = TC – TIC). (1)

The remaining TC is measured to determine total organic carbon and the result is
generally referred to as TOC. However, in the TOC (VCPN, Schimadzu) [2], this value is

176
Measurement of the concentration of total organic carbon (TOC) in waste water

referred to a non‑purgeable organic carbon that is present in a sample in a non‑volatile


form (NPOC) to distinguish it from the TOC value obtained by calculating the difference
between TC and TIC (Eq. (2)). In this case, it can be assumed that purgeable organic
substances are not present in the samples, thus

TOC(VCPN) = NPOC = TOC (2)

and the direct method can be applied.

The measurement of the concentration of CO2 can be carried out by a number of different
methods. In this case, a non‑dispersive infrared detection (NDIR) was applied [1], where
the carrier gas delivers the sample combustion products to the cell of a non‑dispersive
infrared gas analyser, where the carbon dioxide is detected. The detector is filled with
sample gas component (CO2) to a specified concentration (8 % CO2 in N2) and divided
into two connected chambers. A micro‑flow sensor is fitted in the connection tube of the
chambers. The incident radiation is absorbed selectively only in the specific absorption
bands of the CO2 gas in the detector. The absorbed energy is instantaneously transformed
to thermal energy through molecular collision [2]. Due to the selectivity of the detector,
absorbed energy variation depends on the CO2 concentration in the sample cell and causes
temperature and pressure difference between the two detector chambers. This results in a
gas flow between the two chambers which is detected by the micro‑flow sensor in‑between
the chambers. Since the beam is interrupted at a specified frequency by a rotating sector
measurement, signal variation is also periodic and generates AC voltage in the mV range,
which is then processed on the NDIR board (peak area). The quantity of rays absorbed is
proportional to the density of the gas according to the Lambert-Beer Law.

Sampling and sample pretreatment


Water samples should be withdrawn in clean glass bottles which should be filled
completely. In case of storage at 4 °C in a refrigerator, the samples should be acidified to
pH 2 if bacteriological activity is to be expected. For storage and for expulsion of carbon
dioxide 0.2 M hydrochloric acid (HCl) was used. Samples should be analysed within
one week. The samples should be homogenised, in this case by ultrasonic treatment
with sufficient efficiency (Figure 5.1). (NB: Samples should be representative; avoid the
contamination of the sample with organic substances.)

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Weighting of PHP

Stock reference
solution (CTOC1)

Sample preparation Working reference


(homogeneized, solutions (CTOC1)
acidified)

Working solutions
Sample dilution (CTOC2)

Addition of acid

Sparging of the
sample

Measuring concentration of
CTOC-sample, mg L-1

Figure 5.1: Flow chart of the analytical procedure


Calculation of the measurement results
Calculate the mass concentration of TOC from the calibration curve.

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Measurement of the concentration of total organic carbon (TOC) in waste water

parT II. The customer’s requirements concerning quality of the


measurement result

EXTRACT FROM THE EU WATER DIRECTIVE

Regulation (EC) No 166/2006 of the European Parliament and of the Council of


18 January 2006 concerning the establishment of a European Pollutant Release
and Transfer Register and amending Council Directives 91/689/EEC and 96/61/EC
(50 000 kg TOC/year).

Customer requirements are related to the quality of the results, accuracy and uncertainty,
in accordance with SIST ISO 8245:1999, measurement uncertainty must be below 15 %
(generally accepted level).

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Practical examples of traceability, measurement uncertainty and validation in chemistry

PART III. Validation of the measurement procedure — relevant


equations and measurement data

LoQ measurement for TOC concentration measurement

The method described in the above mentioned standard applies to those water samples
containing carbon content from 0.3 to 500 mg L– 1. In these experiments, the limit of
detection (LOD) for carbon was determined at 0.2 mg L– 1. The LOD was determined by
means of standard additions (10 replicates were made).

LoQ = mean concentration in blank samples + 10 × standard deviation (3)

The limit for quantification (LoQ) regarding carbonate was determined at 0.3 mg L– 1.

RSD = (100S)/xp, (4)

where S is the standard deviation and xp is the mean value of concentration measured.

LoQ was calculated from 10 replicates made for a series of five different concentrations.
RSD was determined to be 10 %. A range between 0.3 and 500 mg L– 1 was applied for
TOC concentration measurement. Higher concentration of TOC can be measured by
appropriate dilution.

Selectivity was determined by the standard addition method. Standard solutions of


TOC were added to the water sample (which contained neither TOC). The recovery was
between 93 and 105 % for the determination of TOC.

Table 1: Data required for LoQ determination


Standard Standard Standard Standard
Sample 1
Replicate No  addition 1 addition 2 addition 3 addition 4
(mg L– 1)
(mg L– 1) (mg L– 1) (mg L– 1) (mg L– 1)
1 1.09 0.55 1.95 0.27 1.38
2 1.09 0.50 1.96 0.24 1.41
3 0.98 0.47 1.91 0.24 1.43
4 1.00 0.45 1.90 0.22 1.41
5 1.05 0.49 1.87 0.25 1.34
6 1.11 0.53 1.94 0.20 1.37
7 1.03 0.46 1.90 0.26 1.37
8 0.97 0.48 1.93 0.26 1.36
9 1.10 0.57 1.97 0.19 1.37
10 1.00 0.48 1.89 0.23 1.52

Calculate the average value of concentrations for each standard addition (1 to 4) and
correspondent RSDs. Then plot RSD (%) against average concentration values. By linear
regression a line is obtained. Determine LoQ from the line at a RSD value of 10 %.

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Measurement of the concentration of total organic carbon (TOC) in waste water

PART IV. Measurement uncertainty of the result: relevant


equations and measurement data (1)

Potassium hydrogen phthalate PHP (Merck, Germany) was dried to constant mass at
105 °C. A stock reference solution ( CTOC ) with a concentration of 1 000 mg L– 1 was
prepared by weighing the PHP and dissolving it in a volumetric flask with deionised
water. Concentration of the stock reference solution ( CTOC ) was calculated from Eq. (5):1

mPHP × PPHP × M C × 8
CTOC = , (5)
V1000 × M PHP

where:
mPHP = Mass of potassium hydrogen phthalate (g)
PPHP ‑ = Purity of potassium hydrogen phthalate
M PHP = Molecular weight of potassium hydrogen phthalate (g mol‑1)
MC = Molecular weight of carbon (g mol‑1)
V1000 = Volume of one mark volumetric flask (L)
8 = Conversion factor

From the stock reference solution, working reference solutions ( CTOC ) and working
solutions ( CTOC ) were prepared by further dilutions in two dilution steps.
1

First step: three different working reference solutions ( CTOCi ) in three different ranges
1

were prepared; calculated using Eq. (6):


CTOC CTOC × Vp (6)
CTOC1 = =
FD V100

By further dilution (second dilution step), the working solutions ( CTOC ), used for the
2

construction of each curve were prepared; calculated using Eq. (7):


CTOC1 CTOC1 × Vp
CTOC1 = = , (7)
FD V100

where:
VD = Dilution factor of working reference solutions and working solutions
V100 = Volume of one mark volumetric flask (L
VP = Volume of pipettes (L)

The same preparation procedure followed for the working solution was used for the
samples. Depending on the concentration range of TOC in wastewater samples, the
calibration curves were established as described in Table 2.

(1) You may also want to include the relevant certificates here.

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Table 2: Calibration working reference solution concentrations

Expected concentration
Working reference solution
range of TOC in
concentrations (mg L– 1)
wastewater sample (mg L– 1)
0.2‑5 (Range 1) 0.2; 0.5; 1.0; 2.5; 5.0
2‑100 (Range 2) 2; 12.5; 25; 50; 100
100‑500 (Range 3) 100; 125; 166.7; 250; 500

A new calibration curve was prepared always when new batches of reagents were used.

Prior to the measurement of the TOC in water sample, the sample was homogenised
using a homogeniser (Heidolf DIAX 900). Afterwards, the sample was diluted, poured
into a 40 mL vial and acidified with 2 M HCl.

The analytical signals of each working reference solution as well as the sample
analytical signals were measured in five replicates. The analytical signal is the link to the
concentration of the analyte in the solutions.

Identification of the uncertainty sources and their quantification


Concentration of TOC in sample ( CTOC−sample ) was calculated using the following equation:
A − B0
CTOC−sample = × Fd × Fr × Fh × FR × Fs , (8)
B1

where:
CTOC−sample = concentration of TOC in sample (mg L– 1)
A = Peak area of sample (V)
B1 = Slope of calibration curve (V L mg‑1)
B0 = Intercept of calibration curve (V)
Fd = Dilution factor of the sample (1)
Fr = Repeatability factor (1)
Fh = Stability factor (1)
FR = Recovery factor (1)
Fs = Factor of sampling procedure (1)

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Measurement of the concentration of total organic carbon (TOC) in waste water

Sources of uncertainty are thus:


u( A) = Uncertainty of measurement of peak area
u( B0 ) = Uncertainty of intercept of calibration curve
u( B1 ) = Uncertainty of slope of calibration curve
u( Fd ) = Dilution of sample
u( Ft ) = Repeatability
u( FR ) = Recovery
u( Fh ) = Store conditions
u( Fn ) = Sampling procedure

The sources of uncertainty in TOC determination is schematically presented in Figure 2.

Measurement uncertainty of peak area


The standard measurement uncertainty of the peak areas of solutions (Ai) and of the
sample (A) were estimated as the standard deviations (SD) of the mean value: absorbance
was measured in five replicates. The contribution to combined and expanded uncertainty
is seen in Figure 2.

Uncertainty of linear regression


The slope of the linear least squares calibration curve B1 and calculated blank B0 were
calculated using Eqs (9)-(12):
n

∑ (C TOC2 − CTOC2 ) × ( Ai − A)
B1 = i =1 , (9)
n

∑ (C TOC2 − CTOC2 )2
i =1

B0 = A − B1 × CTOC−sample , (10)
n
1
A= ∑ Ai ,
n i=1
(11)

where Ai is raw data (peak area) of working solutions,


n
1
CTOC2 = ∑ CTOC2 ,
n i=1
(12)

where CTOC is the concentration of working solutions (mg L– 1)


2

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Uncertainty of the concentration of working solutions


The uncertainty associated with weighing of the reagent (u( mPHP )) was obtained from the
Calibration certificate of the balance used (ucal.cer. ) , while the uncertainty associated with
the repeatability of the reagent weighing ( Fr ) was obtained from successive weighing
operations based on the data from 10 repetitions (Eq. (13)). Fr was included in the
precision of the method. Because two successive weightings are used, the uncertainties
have to be counted twice:
2 sdr
u( mPHP )2 = ucal.cer
2
. +( )2 , (13)
3

where sdr is the digital resolution and ucal.cer is the balance Calibration certificate.

The uncertainty associated with the volume of the volumetric flask depends on the
uncertainty of the volumetric flask volume itself (u(scer)), the uncertainty associated with
the use of volumetric equipment (u(V)) at a temperature different from that of calibration
and the repeatability of volume delivery of the reference stock solution (Eq. (14)). The
limits of the volumetric flask volume accuracy were indicated by the manufacturer
to be of type B uncertainty with no data on distribution. Triangular distribution was
used and values were divided by 6 . The uncertainty due to the temperature effect
was calculated from an estimate of the temperature range and the volume expansion
coefficient. A temperature variation of ± 5 °C was assumed as a reasonable estimate
with a 95 % confidence. A volumetric expansion coefficient of water was 2.1 × 10– 4 °C.
The repeatability of the volume delivery by stock reference solution preparation was
determined experimentally by a series of fill and weight experiments on a volumetric
flask. All uncertainty contributions were then combined to obtain the uncertainty of the
volume of the volumetric flask (u(V1000)). All contributions of repeatability, including
the repeatability of the volume deliveries were combined in an individual source of
uncertainty (Fr).
spt 5 × 2.1 × 10−4 × V
u(V1000 )2 = ( )2 + ( )2 = u( scer )2 + u(V )2 , (14)
6 3

where:
spt = Producer tolerance
V = Volume (mL)
u(scer) = Uncertainty of the volumetric flask volume

The uncertainty associated with the reagent purity (u(PPHP)) was calculated from the
producer specifications (Eq. (15)). As there was no additional information about the
uncertainty value available, a rectangular distribution was assumed. The purity was
given by the manufacturer’s certificate. Since there were no data on distribution, the

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Measurement of the concentration of total organic carbon (TOC) in waste water

value was divided by 3 (assuming rectangular distribution).


sps
u( PPHP )2 = ( )2 , (15)
3

where sps is the producer specification.

Potassium hydrogen phthalate (PHP) has the empirical formula C8H5O4K. The
uncertainty of the compound molar mass (u( M C )) can be evaluated by combining
the uncertainty in the atomic weights of its constituent elements (u( M PHP )) .
A table of atomic weights including uncertainty estimates is published by IUPAC in
the Journal of Pure and Applied Chemistry [3]. The molar mass standard uncertainty
was found by dividing the IUPAC quoted uncertainty by 3 assuming that it forms the
bounds of a rectangular distribution. So the uncertainties of the PHP molar mass and
carbon atomic weight are calculated by Eqs (16) and (17) respectively:
2
 8 × st (C ) 
u( M C ) = 
2
 (16)
 3 

2 2 2 2
 8 × st (C )   5 × st ( H )   4 × st (O )   st ( K ) 
u( M PHP )2 =   +  +  +  (17)
 3   3   3   3 

where st is the tolerance (atom).

The uncertainty of each step was calculated from a volumetric equipment uncertainty
and the uncertainty associated with the use of volumetric equipment at temperatures
different from those of calibration, uncertainty of the stock reference solution in first
dilution step and working reference solution in the second dilution step.

The concentration uncertainty of the stock reference solution is associated with the
uncertainty of the potassium hydrogen phthalate mass, the purity of the reagent, volume
of the stock solution volumetric flask, potassium hydrogen phthalate molar mass and
carbon atomic weight (Table 2, spreadsheet Ex‑09‑2‑Y‑TOC). The stock reference
solution concentration uncertainty u(CTOC ) was calculated from Eq. (18) (spreadsheet
Ex‑09‑2‑Y‑TOC):

u( mPHP )2 u( PPHP )2 u( M C )2 u(V1000 )2 u( M PHP )2


u(CTOC ) = CTOC × 2
+ 2
+ + + (18)
mPHP PPHP M C2 2
V1000 2
M PHP

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Practical examples of traceability, measurement uncertainty and validation in chemistry

Table 2 and Eq. (18) (spreadsheet Ex‑09‑2‑Y‑TOC)

After diluting the stock reference solution (first dilution), the working reference solutions
CTOC in three different ranges were calculated using Eq. (6). CTOC ‑ values were measured
1 1

at 4.97 mg L– 1 (range 1), at 99.4 mg L– 1 (range 2) and at 497 mg L– 1 (range 3, Table 3),
respectively. The concentration uncertainty of the working reference solution in all three
instrument ranges is associated with the uncertainty of the stock solution concentration
of potassium hydrogen phthalate, the volumes of pipettes Vp (Vp: 0.5, 10 or 50 ml), and
the volume of the 100 ml working solutions volumetric flask. Uncertainties u(Vp)2 and
u(V100)2 were calculated from Eq. (19) by considering corresponding volumes:

2
u(CTOC )2 u(Vp ) u(V100 )2
u(CTOC ) = CTOC × 2
+ + (19)
CTOC Vp2 V10200

Table 3 and Eq. (19) (spreadsheet Ex‑09‑2‑Y‑TOC)

The concentration uncertainty of the working reference solutions were calculated using
Eq. (17), and were measured to be 0.000017 g L– 1 (range 1), 0.00031 g L– 11 (range 2),
and 0.00154 g L– 1 (range 3), respectively.

By the second step of working reference solution dilutions, the working solution
concentrations ( CTOC ) were calculated in three different ranges using Eq. (7). In this way,
2

three different curves (for three different ranges) were developed. The concentrations of
working solutions are presented in Table 1. The concentration uncertainty of the working
calibration solutions regarding all three ranges is associated with the uncertainty of the
working reference solution (5, 100 or 500 mg L– 1), the volumes of pipettes (0.2, 4, 10,
12.5, 20, 25, 50 or 50 mL), and volume of the 100 mL working solution volumetric flask.

The working solution uncertainty was calculated by using the corresponding values of
concentrations and volumes in Eq. (20):

u(CTOC1 )2 u(Vp )2 u(V1000 )2


u(CTOC2 ) = CTOC2 × 2
+ + (20)
CTOC
1 1 Vp2 2
V100

Table 4 and Eq. (20) (spreadsheet Ex‑09‑2‑Y‑TOC)

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Measurement of the concentration of total organic carbon (TOC) in waste water

Dilution of the sample


The standard uncertainty of the sample dilution factor ( Fd ) was estimated from the
uncertainty of the volumetric equipment and the uncertainty associated with the
temperatures different from those of calibration (similarly as described above), and
then the calculations of combined and expanded uncertainty were made, as presented in
Table 5 (spreadsheet Ex‑09‑2‑Y‑TOC).

Uncertainty due to repeatability


The precision was investigated at concentrations covering the full concentration
range specified in the scope of the procedure [4]. Samples of different concentration
levels (0.18, 6.1, 78.3, 120, 235 and 1 973 mg L– 1 TOC) were prepared. Ten replicates
were made at each concentration level and relative standard deviations (RSDs) were
calculated (0.031, 0.046, 0.011, 0.022, 0.044 and 0.026, respectively). It was found that
there is no significant difference between the RSDs at different concentration levels:
this indicates that the precision is proportional to the analyte concentration. The relative
standard deviations were pooled to give a single estimation which can be applied to the
concentration range covered by the precision study (Eq. (21)).

u( Fr ) = RSDpool =
(n − 1 ) × RSD12 + ( n2 − 1 ) × RSD22 + 
= 3.21 % (21)
1

(n 1 − 1 ) + ( n2 − 1 ) + 

RSD1 is relative standard deviation calculated for the sample at concentration level 1 and
n1 is the number of replicates for this sample.

Uncertainty due to recovery


Trueness is estimated in terms of overall recovery, i.e. the ratio of the observed value
to the expected (true) value. The latter can be evaluated in number of ways, in this case
from the results of between laboratory samples, Eqs (22)-(24):

RMS bias =
∑ (bias ) i
2

= 1.7 % (22)
n

SR 6.5
u(Cref ) = = = 0.97 % (23)
n 45

FR = u( bias ) = 2
+ u(Cref )2 = 1.7 2 + 0.97 2 = 1.92 % ,
RMS bias (24)

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Practical examples of traceability, measurement uncertainty and validation in chemistry

where:

RMS bias = Relative mean spread


biasi = The sum of square of all biases between reference and analysed samples
in the inter‑laboratory tests
n = Average number of participated laboratory
SR = The inter‑laboratory’s standard deviation

Uncertainty associated with storage conditions


The standard uncertainty associated with the effect of storage conditions on the results
was evaluated experimentally. The samples were preserved by acidification to Ph < 2
with HCl, stored in the dark at temperatures between 2 and 5 °C, and analysed within
eight days. Triangular distribution was assumed and the stated uncertainty was divided
by 6 (Eq. (25)):
2
 s 
u( Fd )2 =  t  , (25)
 6

where st is the tolerance.

Uncertainty due to sampling


Standard deviation (sd) [5] [6] (replicate analyses of the same sample prepared in a
laboratory), sub‑sampling transport standard deviation (analyses of replicate samples
taken in the field from the bulk sample) and total sampling standard deviation (analysis
of bulk samples obtained by separate application of the sampling procedure) were
determined. The comparison between the different sd estimates described above can
be used to identify the most important sources of measurement uncertainty. For each
measurement 10 replicates (n) were made and calculations were made using Eq. (21).

Calculation of combined and expanded uncertainty


Generally, the result of a measurement is determined from other quantities and
the relationship between result y and the values of the input parameters can be
expressed by a model of Eq. (26), where x represents model input parameters
(CTOC , CTOC Ai , Fd , Ft , Fh , FR , FS ) . The uncertainty of the result u( y ) depends on the
1 2

uncertainty of the input parameters ( xi ) and is described by Eq. (27) (variables are
independent). ∂y / ∂xi is a sensitivity coefficient, the partial differential of y with respect
to xi and u( xi ) denotes the uncertainty in y arising from the uncertainty in xi . These
sensitivity coefficients describe how the value of y varies with changes in the parameters
xi :

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Measurement of the concentration of total organic carbon (TOC) in waste water

y = f ( x1 ,, xn ) (26)
2
n
 ∂y 
u( yTOC )2 = ∑   × u ( xi )
2
(27)
i =1  ∂x i 

Standard uncertainty of input parameters (three different ranges) is presented in Table 5.

The uncertainty of CTOC‑sample was measured by combining the standard uncertainty of


CTOC , CTOC Ai , Fd , Ft , Fh , FR and FS Uncertainties were combined by using the rule for
1 2

propagation of errors. To calculate the expanded uncertainty of the result of measurement


at the 95 % confidence level, the result of combined uncertainty (U) was multiplied by a
coverage factor k =2 [7].

The measurement results were determined at 2.29 mg L– 1 in the first range, at 50.6 mg L– 1
in the second and at 246 mg L– 1 in the third respectively. The evaluated combined
uncertainties were calculated at 0.12 mg L– 1 in the first range, at 2.6 mg L– 1 in the second
and at 12.5 mg L– 1 in the third respectively. To obtain an expanded uncertainty at the
95 % confidence level, the combined uncertainty was multiplied by the coverage factor
k of 2. Therefore, the expanded uncertainties of the measurement results were calculated
at (2.29 ± 0.24) mg L– 1 (10.4 %) in the first range, at (50.6 ± 5.2) mg L– 1 (10.2 %) in
the second range and at (246 ± 24.9) mg L– 1 (10.1 %) in the third range respectively. A
10 % relative expanded uncertainty of TOC measurement from 0.2 to 500 mg L– 1 TOC
was calculated. The largest contributions are due to repeatability, recovery and sampling.

CTOC-curve Peak areacurve CTOC-sample


Gas flow
Dilution 1
Reagent purity Volume
Temperature volume mass
CO2 concentration Peak area
Calibration repeatability
Dilution 2
Pressure Dilution
Temperature volume Volume temperature
Temperature
Linear least square Temperature volume
regression

CTOC

Repeatability Storage Recovery Sampling


(precision study) (trueness study)

Figure 2: Cause–effect diagram for the TOC concentration measurement

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Practical examples of traceability, measurement uncertainty and validation in chemistry

The solved exercises (green pages)

TrainMiC® ExErCisEs

Analytical procedure

Measurement of the concentration of total organic carbon


(TOC) in waste water

exerCIse 1:
establishing traceability in analytical chemistry

exerCIse 2:
single laboratory validation of measurement procedures
Part I: General issues
Part II: Parameters to be validated
Part III: Some calculations and conclusions

ExERCISE 3:
building an uncertainty budget
Addendum I: By spreadsheet approach
Addendum II: By dedicated software

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Measurement of the concentration of total organic carbon (TOC) in waste water

ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

1. Specifying the analyte and measurand


Analyte TOC (total organic carbon)

Measurand Total organic carbon in wastewater

Units mg L– 1

2. Choosing a suitable measurement procedure with associated model equation


Measurement SIST ISO 8245:1999 — Water quality — Guidelines for the determination of total
procedure (TOC) and dissolved organic carbon (DOC)

Type of calibration Standard curve Standard addition Internal standard

Model equation:
A − B0 ,
CTOC−sample = × Fd × Fr × Fh × FR × Fs
B1

where:
CTOC−sample = Concentration of TOC in sample (mg L– 1)
A = Peak area of sample
B1 = Slope of calibration curve
B0 = Intercept of calibration curve
Fd = Dilution factor of the sample
Fr = Repeatability factor
Fh = Stability factor
FR = Recovery factor
Fs = Factor of sampling procedure

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Practical examples of traceability, measurement uncertainty and validation in chemistry

3. List the input quantities according to their influence on the uncertainty of the
result of the measurement (first the most important ones): at this point, your
judgement should be based on your previous experience only.
1 Dilution factor of the sample
2 Repeatability factor
3 Stability factor
4 Recovery factor
5 Factor of sampling procedure
6 The peak areas of solutions
7 Intercept and slope of calibration curve

4. List the reference standards needed and state the information regarding
traceability of the reference value

For the analyte


potassium hydrogen phthalate (C8H5KO4) (stock solution), pure
1 Name/chemical formula/producer substance, RM, Merck
purity ≥ 99.5 %

For the other input quantities

Quantity/equipment/calibration
1 Class A for pipettes and glassware
(e.g. mass/balance/calibrated by NMI, U = xx (k = 2))

2 Quantity/equipment/calibration Calibration of balance

5. Estimating uncertainty associated with the measurement


Are all important parameters included in the model
Yes No
equation?
Other important parameters:

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Measurement of the concentration of total organic carbon (TOC) in waste water

6. How would you prove traceability of your result?


1 Aquacheck and Kemijski Inštitut Ljubljana

2
3

7. Any other comments, questions …

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Practical examples of traceability, measurement uncertainty and validation in chemistry

SINGLE LABORATORY VALIDATION


OF MEASUREMENT PROCEDURES

parT I: General IssUes

1. Specify the measurement procedure, analyte, measurand and units


Measurement procedure is the measurement of the concentration of TOC based on
ISO 8245:1999. The analyte is TOC (total organic carbon). The measurand is TOC and
units are: TOC in wastewater (mg L– 1).

TOC is the sum of organically bound carbon present in water (drinking, wastewater,
The measurement etc.) bonded to dissolved or suspended matter. Cyanate, elemental carbon and
procedure thiocyanate will also be measured.

The procedure is combustion of organic carbon in water to carbon dioxide by


Pt‑catalyst. The final measurement of the concentration of CO2 is carried out by a
number of different procedures. In this case, by infrared detector, which produces
raw data (area counts mV s‑1).
Analyte TOC
The measurand Total organic carbon in wastewater
Unit mg L– 1

2. Specify the Scope


Matrix Matrix is wastewater
The measuring range is 0.3 to 500 mg L 1 (separated into three ranges: 0.3‑5, 2‑100,
Measuring range
and 100‑500 mg L– 1, respectively).

3. Requirements of the measurement procedure


Intended use of the results To analyse wastewater according to Slovenian legislation
Parameters to be validated Value requested by the customer
LOD
LoQ 0.3 mg L– 1
Mark the customer’s Repeatability
requirements and give their
values Within‑lab reproducibility
Trueness
Measurement uncertainty 10 % (0.3‑500 mg L– 1), K  = 2, 95 %.
Other — state

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Measurement of the concentration of total organic carbon (TOC) in waste water

4. Origin of the measurement procedure


VALIDATION
New in‑house method Full
Modified validated method Partial
Official standard method Confirmation/verification

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Practical examples of traceability, measurement uncertainty and validation in chemistry

parT II: paraMeTers To be ValIdaTed

5. Selectivity/interference/recovery

Where yes, give further information, for example which CRM, reference method.
CRM/RM: analysis of available CRM or RM
Further information

Spike of pure substance

Compare with a reference method

Selectivity, interferences

Test with different matrices

Other — specify

6. Measuring range
Linearity
Upper limit
LOD
LoQ

7. Spread — precision
Repeatability
Reproducibility (in‑lab)
Reproducibility (between labs)

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Measurement of the concentration of total organic carbon (TOC) in waste water

8. Robustness
Variation of parameters

9. Quality control
Control charts
Participation in PT schemes

10. Other parameters to be tested


Working range and testing of homogeneity of variances
R square
Residual standard deviation
Standard deviation of the analytical procedure
Coefficient of variation of the analytical procedure
Measurement uncertainty

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Practical examples of traceability, measurement uncertainty and validation in chemistry

parT III: soMe CalCUlaTIons and ConClUsIons

11. Calculation of parameters requested by the customer


Parameters requested to be validated Calculations
LOD
LoQ 0.3 mg L– 1
Repeatability
Within‑lab reproducibility
Trueness
Measurement uncertainty 10 %
Other — state

LoQ calculation:

Table 1: Data for LoQ determination.

Standard Standard Standard Standard


Sample 1
Replicate No  addition 1 addition 2 addition 3 addition 4
(mg L– 1)
(mg L– 1) (mg L– 1) (mg L– 1) (mg L– 1)
1 1.09 0.55 1.95 0.27 1.38
2 1.09 0.50 1.96 0.24 1.41
3 0.98 0.47 1.91 0.24 1.43
4 1.00 0.45 1.90 0.22 1.41
5 1.05 0.49 1.87 0.25 1.34
6 1.11 0.53 1.94 0.20 1.37
7 1.03 0.46 1.90 0.26 1.37
8 0.97 0.48 1.93 0.26 1.36
9 1.10 0.57 1.97 0.19 1.37
10 1.00 0.48 1.89 0.23 1.52
average 1.04 0.50 1.92 0.24 1.40
s 0.05 0.04 0.03 0.03 0.05
RSD (%) 5.07 8.15 1.65 10.65 3.59

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Measurement of the concentration of total organic carbon (TOC) in waste water

Limit of quantification is 0.3 mg L– 1 (Figure 1).

12

10

8
RSD (%)

4
0.3 mg L-1
2

0
0.0 0.5 1.0 1.5 2.0 2.5

Concentration (mg L-1)

Figure 5.2: Determination of limited of quantification

12. Does the analytical procedure fulfil the requirement(s) for the intended use?
Value requested by
Is the requirement
the customer Value obtained
Parameter fulfilled?
(the same as stated in during validation
Yes/No
Question 3)
LOD
LoQ 0.3 mg L– 1 0.3 mg L– 1 Yes
Repeatability
Within‑lab
reproducibility
Trueness
Measurement
10 % 10 % Yes
uncertainty
Other

The analytical procedure is fit for the intended use:


Yes No

For measurement uncertainty and traceability, refer to the corresponding pages.

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BUILDING AN UNCERTAINTY BUDGET

1. Specify the measurand and units


Measurement procedure is measurement of the concentration of TOC
Measurand based on ISO 8245:1999. The analyte is TOC (total organic carbon).
The measurand and units are: TOC in wastewater (mg L– 1).
Units mg L– 1

2. Describe the measurement procedure and provide the associated model


equation

Measurement procedure:
Matrix is wastewater and the measuring range is 0.3-500 mg L– 1.

Model equation:

A − B0
CTOC−sample = × Fd × Fr × Fh × FR × Fs , (8, yellow spreadsheet)
B1

where:
CTOC−sample = Concentration of TOC in sample (mg L– 1)
A = Peak area of sample (V)
B1 = Slope of calibration curve (V L mg‑1)
B0 = Intercept of calibration curve (V)
Fd = Dilution factor of the sample (—)
Fr = Repeatability factor (—)
Fh = Stability factor (—)
FR = Recovery factor (—)
Fs = Factor of sampling procedure (—)

200
Measurement of the concentration of total organic carbon (TOC) in waste water

3. Identify (all possible) sources of uncertainty

u( A) — Uncertainty of measurement of peak area

u( B0 ) — Uncertainty of intercept of calibration curve

u( B1 ) — Uncertainty of slope of calibration curve

u( Fd ) — Dilution of sample

u( Ft ) — Repeatability

u( FR ) — Recovery

u( Fh ) — Store conditions

u( Fn ) — Sampling procedure

4. Evaluate values of each input quantity


Table 2: Uncertainty components of the stock reference solution.
Input
Value Units Remarks
quantity
mPHP 2.125 g mass of potassium hydrogen phthalate
PPHP 0.995 No unit purity of potassium hydrogen phthalate
V1000 1 L volume of one mark volumetric flask
MPHP 204.2212 g mol – 1
molecular weight of potassium hydrogen phthalate
MC 12.0107 g mol – 1
molecular weight of carbon
CTOC 0.994 g L– 1 stock reference solution concentration

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Table 3: Uncertainty components of the working reference solution (ranges 1, 2 and 3).
Input
Value Units Remarks
quantity
Range 1
0.994 g L– 1
CTOC Stock reference solution concentration
V0.5 0.0005 Ll Volume of pipette
V100 0.1 L Volume of (one mark) volumetric flask
CTOC1 4.97 g L – 1
Working reference solution concentrations in range 1
Range 2
CTOC 0.994 g L– 1 Stock reference solution concentration
V10 0.01 Ll Volume of pipette
V100 0.1 L Volume of (one mark) volumetric flask
CTOC1 99.4 g L– 1 Working reference solution concentrations in range 2
Range 3
CTOC 0.994 g L– 1 Stock reference solution concentration
V50 0.05 L Volume of pipette
V100 0.1 L Volume of (one mark) volumetric flask
CTOC1 497 g L– 1 Working reference solution concentrations in range 3

Table 4: Uncertainty components of the working solutions


Input
Value Units Remarks
quantity
Range 1
V4 0.004 L Volume of pipette
V10 0.01 L Volume of pipette
V20 0.02 L Volume of pipette
V50 0.05 L Volume of pipette
V100 0.1 L Volume of pipette
CTOC1 0.00497 g L– 1 Working reference solution concentrations in range 1
V100(flask) 0.1 L Volume of one mark volumetric flask (all 3 ranges)
C 0.2 0.00019 g L – 1
Working solution concentration in range 1
C 0.5 0.00049 g L– 1 Working solution concentration in range 1
C1 0.00099 g L– 1 Working solution concentration in range 1

202
Measurement of the concentration of total organic carbon (TOC) in waste water

C 2.5 0.00249 g L– 1 Working solution concentration in range 1


C5 0.00497 g L– 1 Working solution concentration in range 1
Range 2
V2 0.002 L Volume of pipette
V12.5 0.0125 L Volume of pipette
V25 0.025 Ll Volume of pipette
V50 0.05 Ll Volume of pipette
V100 0.1 L Volume of pipette
CTOC1 0.994 g L– 1 Working reference solution concentrations in range 2
C2 0.0019 g L– 1 Working solution concentration in range 2
C12.5 0.0124 g L – 1
Working solution concentration in range 2
C25 0.0249 g L – 1
Working solution concentration in range 2
C50 0.0497 g L– 1 Working solution concentration in range 2
C100 0.0994 g L– 1 Working solution concentration in range 2
Range 3
V20 0.02 Ll Volume of pipette
V25 0.025 L Volume of pipette
V33.3 0.0333 Ll Volume of pipette
V50 0.05 L Volume of pipette
V100 0.1 Ll Volume of pipette
CTOC1 0.497 g L– 1 Working reference solution concentrations in range 3
C100 0.0994 g L– 1 Working solution concentration in range 3
C125 0.1243 g L – 1
Working solution concentration in range 3
C166.5 0.1665 g L – 1
Working solution concentration in range 3
C250 0.2485 g L– 1 Working solution concentration in range 3
C500 0.497 g L– 1 Working solution concentration in range 3

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Table 5: Uncertainty components of TOC concentration measurement


Input
Value Units Remarks
quantity
Range 1

A 42.6 No unit Peak area of sample

A0.2 8.2 No unit Peak area of working solution concentration

A0.5 16.7 No unit Peak area of working solution concentration

A1 20.9 No unit Peak area of working solution concentration

A2.5 42.5 No unit Peak area of working solution concentration

A5 86.9 No unit Peak area of working solution concentration

C0.2‑5 a gL – 1
Working solution concentration

Fd* 1 No unit Dilution factor of the sample

Fr* 1 No unit Repeatability factor

Fh* 1 No unit Stability factor

FR* 1 No unit Recovery factor

Fs *
1 No unit Factor of sampling procedure

CTOCsample 0.00230 g L– 1 Concentration of TOC in sample

Range 2

A 127.1 No unit Peak area of sample

A2 11.7 No unit Peak area of working solution concentration

A12.5 24.3 No unit Peak area of working solution concentration

A25 49.1 No unit Peak area of working solution concentration

A50 126.3 No unit Peak area of working solution concentration

A100 256.0 No unit Peak area of working solution concentration

C2‑100 A g L– 1 Working solution concentration

CTOCsample 0.05062 g L– 1 Concentration of TOC in sample

Range 3

A 532.7 No unit Peak area of sample

A100 207.8 No unit Peak area of working solution concentration

A125 259.9 No unit Peak area of working solution concentration

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Measurement of the concentration of total organic carbon (TOC) in waste water

A166.5 350.9 No unit Peak area of working solution concentration

A250 530.6 No unit Peak area of working solution concentration

A500 1102 No unit Peak area of working solution concentration

C100‑500 A g L– 1 Working solution concentration

CTOCsample 0.24596 g L– 1 Concentration of TOC in sample

(a) Refer Table 4.


(*) The same for all ranges.

5. Evaluate the standard uncertainty of each input quantity


Table 6: Uncertainty components of the stock reference solution.
Input
Value Units Remarks
quantity
mPHP 0.00012 g Mass of potassium hydrogen phthalate, Eq. (13)
PPHP 0.00289 No unit Purity of potassium hydrogen phthalate, Eq. (15)
V1000 0.00061 L Volume of (one mark) volumetric flask, Eq. (14)
MPHP 0.00372 g mol– 1 Molecular weight of potassium hydrogen phthalate, Eq. (17)
MC 0.00005 g mol – 1
Molecular weight of carbon, Eq. (16)
CTOC 0.0295 gL – 1
Stock reference solution concentration, Eq. (18)

Table 7: Uncertainty components of the working reference solution (ranges 1, 2 and 3).
Input Standard
Units Remarks
quantity uncertainty
Range 1
CTOC 0.00295 g L– 1 Stock reference solution concentration, Eq. (18)
V0.5 0.000000683 L Volume of pipette, Eq. (14)
V100 0.0000609 Ll Volume of one mark volumetric flask, Eq. (14)
CTOC1 0.000017 gL – 1
Working reference solution concentrations in range 1, Eq. (19)
Range 2
CTOC 0.00295 g L– 1 Stock reference solution concentration, Eq. (18)
V10 0.00000629 Ll Volume of pipette, Eq. (14)
V100 0.0000609 Ll Volume of one mark volumetric flask, Eq. (14)
CTOC1 0.00031 gL – 1
Working reference solution concentrations in range 2, Eq. (19)

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Range 3
CTOC 0.00295 g L– 1 Stock reference solution concentration, Eq. (18)
V50 0.00003067 Ll Volume of pipette, Eq. (14)
V100 0.0000609 Ll Volume of one mark volumetric flask, Eq. (14)
CTOC1 0.00154 g L– 1 Working reference solution concentrations in range 3, Eq. (19)

Table 8: Uncertainty components of the working solutions


Input Standard
Units Remarks
quantity uncertainty
Range 1
V4 0.000003 L Volume of pipette, Eq. (14)
V10 0.000006 Ll Volume of pipette, Eq. (14)

V20 0.000012 L Volume of pipette, Eq. (14)

V50 0.000031 L Volume of pipette, Eq. (14)


V100 0.000061 L Volume of pipette, Eq. (14)
Working reference solution concentrations in range 1,
CTOC1 0.000017 g L– 1
Eq. (19)
V100(flask) 0.000061 L Volume of one mark volumetric flask (all 3 ranges)
C 0.2 0.0000007 g L – 1
Working solution concentration in range 1, Eq. (19)
C 0.5 0.0000017 g L– 1 Working solution concentration in range 1, Eq. (19)
C1 0.0000035 g L– 1 Working solution concentration in range 1, Eq. (19)
C 2.5 0.0000087 g L – 1
Working solution concentration in range 1, Eq. (19)
C5 0.0000174 g L – 1
Working solution concentration in range 1, Eq. (19)
Range 2
V2 0.000001 L Volume of pipette, Eq. (14)
V12.5 0.000008 L Volume of pipette, Eq. (14)
V25 0.000015 L Volume of pipette, Eq. (14)
V50 0.000031 L Volume of pipette, Eq. (14)
V100 0.000061 L Volume of pipette, Eq. (14)
Working reference solution concentrations in range 2,
CTOC1 0.000308 g L– 1
Eq. (19)
C2 0.000006 g L– 1 Working solution concentration in range 2, Eq. (19)
C12.5 0.00004 g L – 1
Working solution concentration in range 2, Eq. (19)

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Measurement of the concentration of total organic carbon (TOC) in waste water

C25 0.00008 g L– 1 Working solution concentration in range 2, Eq. (19)


C50 0.000159 g L– 1 Working solution concentration in range 2, Eq. (19)
C100 0.000318 g L – 1
Working solution concentration in range 2, Eq. (19)
Range 3
V20 0.000012 L Volume of pipette, Eq. (14)
V25 0.000015 L Volume of pipette, Eq. (14)
V33.3 0.000020 L Volume of pipette, Eq. (14)
V50 0.000031 L Volume of pipette, Eq. (14)
V100 0.000061 L Volume of pipette, Eq. (14)
Working reference solution concentrations in range 3,
CTOC1 0.001536 g L– 1
Eq. (19)
C100 0.0003 g L– 1 Working solution concentration in range 3, Eq. (19)
C125 0.0004 g L– 1 Working solution concentration in range 3, Eq. (19)
C166.5 0.0005 g L – 1
Working solution concentration in range 3, Eq. (19)
C250 0.0008 g L – 1
Working solution concentration in range 3, Eq. (19)
C500 0.0016 g L– 1 Working solution concentration in range 3, Eq. (19)

Table 9: The components of uncertainty for TOC concentration measurement


Input Standard
Units Remarks
quantity uncertainty
Range 1
A 0.21 No unit Peak area of sample, sd of the mean
A0.2 0.26 No unit Peak area of working solution concentration, sd of the mean
A0.5 0.21 No unit Peak area of working solution concentration, sd of the mean
A1 0.22 No unit Peak area of working solution concentration, sd of the mean
A2.5 0.14 No unit Peak area of working solution concentration, sd of the mean
A5 0.58 No unit Peak area of working solution concentration, sd of the mean
C0.2‑5 a gL  1
Working solution concentration, a
Fd
*
0.0000609 No unit Dilution factor of the sample, Eq. (14)
Fr *
0.0321 No unit Repeatability factor, Eq. (21)
Fh* 0.0069 No unit Stability factor, Eq. (25)
FR* 0.019 No unit Recovery factor, Eqs (22), (23), (24)
Fs* 0.033 No unit Factor of sampling procedure, Eq. (21)
CTOCsample 0.00012 gL – 1
Concentration of TOC in sample, Eqs (26) and (27)

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Range 2
A 0.56 No unit Peak area of sample, sd of the mean
A2 0.10 No unit Peak area of working solution concentration, sd of the mean
A12.5 0.15 No unit Peak area of working solution concentration, sd of the mean
A25 0.56 No unit Peak area of working solution concentration, sd of the mean
A50 2.54 No unit Peak area of working solution concentration, sd of the mean
A100 1.89 No unit Peak area of working solution concentration, sd of the mean
C2‑100 a g L– 1 Working solution concentration, a
CTOCsample 0.00258 g L– 1
Concentration of TOC in sample, Eqs (26) and (27)
Range 3
A 2.56 No unit Peak area of sample, sd of the mean
A100 3.18 No unit Peak area of working solution concentration, sd of the mean
A125 2.50 No unit Peak area of working solution concentration, sd of the mean
A166.5 3.74 No unit Peak area of working solution concentration, sd of the mean
A250 2.97 No unit Peak area of working solution concentration, sd of the mean
A500 9.60 No unit Peak area of working solution concentration, sd of the mean
C100‑500 a g L– 1 Working solution concentration, a
CTOCsample 0.01248 g L– 1 Concentration of TOC in sample, Eqs (26) and (27)

6. Calculate the value of the measurand, using the model equation


Stock reference solution:
mPHP × PPHP × M C × 8
CTOC = (5, yellow spreadsheet)
V1000 × M PHP

where:
mPHP = Mass of potassium hydrogen phthalate (g)
PPHP = Purity of potassium hydrogen phthalate
M PHP = Molecular weight of potassium hydrogen phthalate (g mol‑1)
MC = Molecular weight of carbon (g mol‑1)
V1000 = Volume of one mark volumetric flask (L
8 = Conversion factor

From the stock reference solution, working reference solutions ( CTOC ) and working
solutions ( CTOC ) were prepared by further dilutions in two dilution steps.
1

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Measurement of the concentration of total organic carbon (TOC) in waste water

First step: three different working reference solutions ( CTOCi ) in three different ranges
1

were prepared (calculated using Eq. (4)).


CTOC CTOC × Vp
CTOC1 = = (6, yellow spreadsheet)
FD V100

By further dilution (second dilution step), the working solutions ( CTOC ) for developing
2

of each curve were prepared (calculated using Eq. (5)).

CTOC1 CTOC1 × Vp
CTOC1 = = , (7, yellow spreadsheet)
FD V100

where:
FD = Dilution factor of working reference solutions and working solutions
V100 = Volume of one mark volumetric flask (L)
VP = Volume of pipettes (L)

Concentration of TOC in sample ( CTOC−sample ) was calculated using the following equation:
A − B0
CTOC−sample = × Fd × Fr × Fh × FR × Fs , (8, yellow spreadsheet)
B1

where:
CTOC−sample = Concentration of TOC in sample (mgL‑1)

A = Peak area of sample


B1 = Slope of calibration curve
B0 = Intercept of calibration curve

Fd = Dilution factor of the sample

Fr — Repeatability factor

Fh — Stability factor
FR — Recovery factor

Fs — Factor of sampling procedure

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7. Calculate the combined standard uncertainty (uc) of the result and specify units

Using: Spreadsheet approach Mathematical solution Commercial software

Table 10: Uncertainty components and combined uncertainty of the stock reference
solution.
Input Estimated Standard
Units Remarks
quantities (xi) value uncertainty u(xi)
mPHP g 2.125 0.00012 Eq. (13)
PPHP No unit 0.995 0.00289 Eq. (15)
V1000 L 1 0.00061 Eq. (14)
MPHP g mol– 1 204.2212 0.00372 Eq. (17)
MC g mol – 1
12.0107 0.00005 Eq. (16)
CTOC g L – 1
0.994 0.00295 Eq. (18)

u( mPHP )2 u( PPHP )2 u( M C )2 u(V1000 )2 u( M PHP )2


u(CTOC ) = CTOC × 2
+ 2
+ + +
mPHP PPHP M C2 2
V1000 2
M PHP

(18, yellow spreadsheet)

u(CTOC ) = CTOC × + + + +

Solution:

0.000122 0.002892 0.000052 0.000612 0.003722


u(CTOC ) = CTOC × + + + + = 0.994 × 0.00295
2.1252 12 12.0107 2 12 204.22122
= 0.00295 g L−1

(18, yellow spreadsheet)

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Measurement of the concentration of total organic carbon (TOC) in waste water

Table 11: Uncertainty components and combined uncertainty of the working reference
solution (ranges 1, 2 and 3).
Estimated Standard
Input quantities (xi) Units Remarks
value uncertainty u(xi)
Range 1
CTOC g L– 1 0.994 0.00295 Eq. (18)
V0.5 L 0.0005 0.000000683 Eq. (14)
V100 L 0.1 0.0000609 Eq. (14)
CTOC1 g L– 1 0.0497 0.000017 Eq. (19)
Range 2
CTOC g L– 1 0.994 0.00295 Eq. (18)
V10 L 0.01 0.00000629 Eq. (14)
V100 L 0.1 0.0000609 Eq. (14)
CTOC1 g L– 1 0.0994 0.00031 Eq. (19)
Range 3
CTOC g L– 1 0.994 0.00295 Eq. (18)
V50 L 0.05 0.00003067 Eq. (14)
V100 L 0.1 0.0000609 Eq. (14)
CTOC1 g L – 1
0.497 0.00154 Eq. (19)

2
u(CTOC )2 u(Vp ) u(V100 )2
u(CTOC1 ) = CTOC1 × 2
+ + , (19, yellow spreadsheet)
CTOC Vp2 2
V100

where Vp is the volume of pipette (mL)

u(CTOC1 ) = × + +

u(CTOC1 ) = × + +

u(CTOC1 ) = × + +

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Solution:
Range 1:

0.002952 0.0000006832 0.00006092


u(CTOC ) = 0.00497 × + + = 0.00497 × 0.0033
0.9942 0.00052 0.12
= 0.000017 g L−1

(19, yellow spreadsheet)

Range 2:

0.002952 0.000006292 0.00006092


u(CTOC1 ) = 0.0994 × + + = 0.0994 × 0.0031
0.9942 0.012 0.12
= 0.00031 g L−1

Range 3:

0.002952 0.00003067 2 0.00006092


u(CTOC1 ) = 0.497 × + + = 0.497 × 0.0031
0.9942 0.052 0.12
= 0.00154 g L−1

Table 12: Uncertainty components and combined uncertainties of the working solutions
Estimated Standard
Input quantities (xi) Unit Remarks
value uncertainty u(xi)
Range 1
V4 L 0.004 0.000003 Eq. (14)
V10 L 0.01 0.000006 Eq. (14)
V20 L 0.02 0.000012 Eq. (14)
V50 L 0.05 0.000031 Eq. (14)
V100 L 0.1 0.000061 Eq. (14)
CTOC1 g L– 1 0.00497 0.000017 Eq. (19)
V100(flask) L 0.1 0.000061 All 3 ranges
C 0.2 g L – 1
0.00019 0.0000007 Eq. (19)
C 0.5 g L– 1 0.00049 0.0000017 Eq. (19)
C1 g L– 1 0.00099 0.0000035 Eq. (19)
C 2.5 g L – 1
0.00249 0.0000087 Eq. (19)
C5 g L – 1
0.00497 0.0000174 Eq. (19)

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Measurement of the concentration of total organic carbon (TOC) in waste water

Range 2
V2 L 0.002 0.000001 Eq. (14)
V12.5 L 0.0125 0.000008 Eq. (14)
V25 L 0.025 0.000015 Eq. (14)
V50 L 0.05 0.000031 Eq. (14)
V100 L 0.1 0.000061 Eq. (14)
CTOC1 g L – 1
0.994 0.000308 Eq. (19)
C2 g L – 1
0.0019 0.000006 Eq. (19)
C12.5 g L– 1 0.0124 0.00004 Eq. (19)
C25 g L– 1 0.0249 0.00008 Eq. (19)
C50 g L – 1
0.0497 0.000159 Eq. (19)
C100 g L – 1
0.0994 0.000318 Eq. (19)
Range 3
V20 L 0.02 0.000012 Eq. (14)
V25 L 0.025 0.000015 Eq. (14)
V33.3 L 0.0333 0.000020 Eq. (14)
V50 L 0.05 0.000031 Eq. (14)
V100 L 0.1 0.000061 Eq. (14)
CTOC1 g L – 1
0.497 0.001536 Eq. (19)
C100 g L – 1
0.0994 0.0003 Eq. (19)
C125 g L– 1 0.1243 0.0004 Eq. (19)
C166.5 g L– 1 0.1665 0.0005 Eq. (19)
C250 g L – 1
0.2485 0.0008 Eq. (19)
C500 g L – 1
0.497 0.0016 Eq. (19)

Range 1:
u(CTOC1 )2 u(Vp )2 u(V100 )2
u(CTOC2 ) = CTOC2 × 2
+ 2
+ 2
(20, yellow spreadsheet)
C TOC1 V p V100

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Practical examples of traceability, measurement uncertainty and validation in chemistry

u(CTOC2 ) = × + +

u(CTOC2 ) = × + +

u(CTOC2 ) = × + +

u(CTOC2 ) = × + +

u(CTOC2 ) = × + +

Solution:
0.000017 2 0.00000332 0.00006092
u(CTOC2 ) = u(C0.2 ) = C0.2 × + + = 0.00019 × 0.00355
0.00497 2 0.0042 0.12
= 0.0000007

0.000017 2 0.000006 62 0.00006092


u(CTOC2 ) = u(C0.5 ) = C0.5 × + + = 0.00049 × 0.00342
0.00497 2 0.012 0.12
= 0.0000017

0.000017 2 0.0000122 0.00006092


u(CTOC2 ) = u(C1 ) = C1 × + + = 0.00099 × 0.00353
0.00497 2 0.022 0.12
= 0.0000035

0.000017 2 0.00003112 0.00006092


u(CTOC2 ) = u(C2.5 ) = C2.5 × + + = 0.00249 × 0.00353
0.00497 2 0.052 0.12
= 0.0000087

0.000017 2 0.0000612 0.00006092


u(CTOC2 ) = u(C5 ) = C5 × + + = 0.00497 × 0.00353
0.00497 2 0.12 0.12
= 0.0000174

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Measurement of the concentration of total organic carbon (TOC) in waste water

Ranges 2 and 3:

Procedure is same as for range 1.

Table 13: Uncertainty components and combined uncertainties of TOC concentration


measurement
Estimated Standard
Input quantities (xi) Units Remarks
Value uncertainty u(xi)
Range 1
A No unit 42.6 0.21 sd of the mean
A0.2 No unit 8.2 0.26 sd of the mean
A0.5 No unit 16.7 0.21 sd of the mean
A1 No unit 20.9 0.22 sd of the mean
A2.5 No unit 42.5 0.14 sd of the mean
A5 No unit 86.9 0.58 sd of the mean
C0.2‑5 g L– 1 a a a
Fd
*
No unit 1 0.0000609 Eq. (14)
Fr *
No unit 1 0.0321 Eq. (21)
Fh* No unit 1 0.0069 Eq. (25)
FR* No unit 1 0.019 Eqs (22)‑(24)
Fs *
No unit 1 0.033 Eq. (21)
CTOCsample gL – 1
0.00230 0.00012 Eqs (26) and (27)
Range 2
A No unit 127.1 0.56 sd of the mean
A2 No unit 11.7 0.10 sd of the mean
A12.5 No unit 24.3 0.15 sd of the mean
A25 No unit 49.1 0.56 sd of the mean
A50 No unit 126.3 2.54 sd of the mean
A100 No unit 256.0 1.89 sd of the mean
C2‑100 gL – 1
a a a
CTOCsample g L– 1 0.05062 0.00258 Eqs (26) and (27)
Range 3
A No unit 532.7 2.56 sd of the mean
A100 No unit 207.8 3.18 sd of the mean

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Practical examples of traceability, measurement uncertainty and validation in chemistry

A125 No unit 259.9 2.50 sd of the mean


A166.5 No unit 350.9 3.74 sd of the mean
A250 No unit 530.6 2.97 sd of the mean

A500 No unit 1102 9.60 sd of the mean


C100‑500 g L– 1 a a a
CTOCsample gL – 1
0.24596 0.01248 Eqs (26) and (27)

(a) Refer Table 4.


(*) The same for all ranges.

For the calculation of the curves, the method of least squares was used in an Excel
spreadsheet (point 10, last three tables).

8. Calculate expanded uncertainty (Uc) and specify the coverage factor k and the
units

range 1:U(C) = 2 × 0.12 mg L– 1 = 0.24 mg L– 1


(2.3 ± 0.24) mg L– 1 or 10.4 %, k = 2, 95 %

range 2:U(C) = 2 × 2.6 mg L– 1 = 5.20 mg L– 1


(50.6 ± 5.20) mg L– 1 or 10.2 %, k = 2, 95%

range 3:U(C) = 2 × 12.5 mg L– 1 = 24.9 mg L– 1


(246 ± 24.9) mg L– 1 or 10.1 %, k = 2, 95 %

9. Analyse the uncertainty contribution and specify the main three input quantities
contributing the most to Uc
Table 14: Uncertainty components

1 Repeatability factor and recovery factor (range 1)


2 Factor of sampling procedure (all three ranges)
3 Dilution factor of the sample (range 3) and stability factor (ranges 2 and 3)

10. Prepare your Uncertainty budget report


Range 1: (2.3 ± 0.24) mg L– 1 or 10.4 %, k = 2, 95 %
Range 2: (50.6 ± 5.20) mg L– 1 or 10.2 %, k = 2, 95 %
Range 3: (246 ± 24.9) mg L– 1 or 10.1 %, k = 2, 95 %

Conclusion: U(CTOC‑sample (0.3–500 mg L– 1)) = 10 %, k = 2, 95 %

216
Measurement of the concentration of total organic carbon (TOC) in waste water

Further reading

1. SIST ISO 8245:1999 Water quality — Guidelines for the determination of total
organic carbon (TOC) and dissolved organic carbon (DOC), International Organisation
for Standardisation, Geneva, pp. 1-11.

2. User Manual for TOC‑VCPH/CPN, Analytical & Measuring Instruments Division, Kyoto,
Schimadzu Corporation, Japan (2005).

3. Ellison, S. L. R., Rosslein, M., Williams, A., Quantifying Uncertainty in Analytical


Measurement, Eurachem/CITAC (2001), pp. 1-120.

4. Barwick, V. J., Ellison, S. L. R., Development and Harmonisation of Measurement


Uncertainty Principles, Part (d): Protocol for uncertainty evaluation from validation
data, VAM Project 3.2.1 (2000), pp. 1-40.

5. Measurement Uncertainty in Testing, Technical Report No 1/2002, Eurolab Technical


Secretariat, Germany (2002), pp. 1-27.

6. SIST ISO 5667‑14:1998 Water quality — Sampling — Part 14: Guidance on quality
assurance of environmental water sampling and handling, International Organisation for
Standardisation, Geneva, pp. 1-7.

7. Kessel, R. Kacker, R., Berglund, M., ‘Coefficient of contribution to the combined


standard uncertainty’, Metrologia, 43 (2006), pp. 189-195.

217
Practical examples of traceability, measurement uncertainty and validation in chemistry

addendum I: Measurement uncertainty calculation:


spreadsheet approach (excel)

Measurement uncertainty of stock reference solution:


Parameter CTOC m PHP P PHP V1000 M PHP MC
Value 2.125 0.995 1 204.2212 12.0107
Std.‑unc. 0.00012 0.00289 0.00061 0.003716 0.0000462
Units g No unit L g mol – 1
g mol– 1
m PHP 2.125 2.12512 2.125 2.125 2.125 2.125
P PHP 0.995 0.995 0.99789 0.995 0.995 0.995
V1000 1 1 1 1.00061 1 1
M PHP 204.221 204.2212 204.2212 204.2212 204.22492 204.2212
MC 12.0107 12.0107 12.0107 12.0107 12.0107 12.010746

C TOC g L– 1 0.99 0.99 1.00 0.99 0.99 0.99


u(y,xi) – 0.000056 – 0.002889 0.000606 0.000018 – 0.000004
u(y)2,
0.000009 0.000000 0.000008 0.000000 0.000000 0.000000
u(y,xi)2
u(CTOC) g L– 1 0.002953
Index 1.9024 97.8479 20.5372 0.6130 0.1296

Measurement uncertainty of working reference solutions:


Range 1
Parameter C PHP V 0.5 V 100
Value 0.994 0.0005 0.1
Std.‑unc. 0.00295 0.000000683 0.0000609
Units g L
– 1
L Ll
C PHP 0.994 0.99695 0.994 0.994
V 0.5 0.0005 0.0005 0.000500683 0.0005
V100 0.1 0.1 0.1 0.1000609

C ‑1 g L– 1 0.00497 0.00498 0.00498 0.00497


u(y,xi) – 0.000015 – 0.000007 0.000003
u(y)2, u(y,xi)2 2.73E‑10 2.18E‑10 4.61E‑11 9.15E‑12
u(C c) g L – 1
0.000017
index 0.4995 0.2299 0.1024

218
Measurement of the concentration of total organic carbon (TOC) in waste water

Range 2
parameter C PHP V 10 V 100
value 0.994 0.01 0.1
std.‑unc. 0.00295 0.00000629 0.0000609
units g L– 1
L Ll
C PHP 0.994 0.99695 0.994 0.994
V 10 0.01 0.01 0.01000629 0.01
V100 0.1 0.1 0.1 0.1000609

C ‑2 g L– 1 0.09940 0.09970 0.09946 0.09934


u(y,xi) – 0.000295 – 0.000063 0.000060
u(y)2, u(y,xi)2 9.46E‑08 8.70E‑08 3.91E‑09 3.66E‑09
u(C c) g L – 1
0.000308
Index 9.9894 2.1172 2.0486

Range 3
Parameter C PHP V 10 V 100
Value 0.994 0.05 0.1
Std.‑unc. 0.00295 0.00003067 0.0000609
Units g L– 1
Ll Ll
C PHP 0.994 0.99695 0.994 0.994
V 50 0.05 0.05 0.05003067 0.05
V100 0.1 0.1 0.1 0.1000609

C ‑3 g L– 1 0.49700 0.49848 0.49730 0.49670


u(y,xi) – 0.001475 – 0.000305 0.000302
u(y)2, u(y,xi)2 2.36E‑06 2.18E‑06 9.29E‑08 9.15E‑08
u(C c) g L – 1
0.001536

Index 49.9468 10.3232 10.2429

219
Practical examples of traceability, measurement uncertainty and validation in chemistry

Measurement uncertainty of working solutions:


In the same way as the reference working solution measurement uncertainties, the
working solutions measurement uncertainties were also evaluated for each point
of the curve for all three ranges. Results are in following tables for range 1 (first
calibration curve):

Parameter C TOC1 V4 V 100


Value 0.00497 0.004 0.1
Std.‑unc. 0.000017 0.000003 6.09E‑05
Units g L– 1 L Ll
C TOC1 0.00497 0.004987 0.00497 0.00497
V4 0.004 0.004 0.004003 0.004
V100 0.1 0.1 0.1 0.100061

C 0.2 g L– 1 0.00020 0.00020 0.00020 0.00020


u(y,xi) – 0.000001 – 1.49E‑07 1.21E‑07
u(y)2, u(y,xi)2 4.99E‑13 4.62E‑13 2.22E‑14 1.46E‑14
u(C0.2) g L– 1 7.07E‑07
Index 96.2367 21.1013 17.1238

Parameter C TOC1 V 10 V 100


Value 0.00497 0.01 0.1
Std.‑unc. 0.000017 0.000006 6.09E‑05
Units g L– 1
L L
C TOC1 0.00497 0.004987 0.00497 0.00497
V 10 0.01 0.01 0.010006 0.01
V100 0.1 0.1 0.1 0.100061

C 0.5 g L– 1 0.00050 0.00050 0.00050 0.00050


u(y,xi) – 0.000002 – 2.98E‑07 3.02E‑07
u(y)2, u(y,xi)2 3.07E‑12 2.89E‑12 8.89E‑14 9.15E‑14
u(C0.5) g L – 1
1.75E‑06
Index 97.0174 17.0180 17.2628

220
Measurement of the concentration of total organic carbon (TOC) in waste water

Parameter C TOC1 V 20 V 100


Value 0.00497 0.02 0.1
Std.‑unc. 0.000017 0.000012 6.09E‑05
Units g L– 1
Ll Ll
C TOC1 0.00497 0.004987 0.00497 0.00497
V 20 0.02 0.02 0.020012 0.02
V100 0.1 0.1 0.1 0.100061

C 1 g L– 1 0.00099 0.00100 0.00099 0.00099


u(y,xi) – 0.000003 – 0.000001 0.000001
u(y)2, u(y,xi)2 1.23E‑11 1.16E‑11 3.56E‑13 3.66E‑13
u(C 1) g L – 1
3.50E‑06
Index 97.0174 17.0180 17.2628

Parameter C TOC1 V 50 V 100


Value 0.00497 0.05 0.1
Std.‑unc. 0.000017 0.000031 6.09E‑05
Units g L– 1
L L
C TOC1 0.00497 0.004987 0.00497 0.00497
V 50 0.05 0.05 0.050031 0.05
V100 0.1 0.1 0.1 0.100061

C 2.5 g L– 1 0.00249 0.00249 0.00249 0.00248


u(y,xi) – 0.000009 – 0.000002 0.000002
u(y)2, u(y,xi)2 7.69E‑11 7.23E‑11 2.37E‑12 2.29E‑12
u(C2.5) g L – 1
8.77E‑06
Index 96.9224 17.5680 – 17.2458

221
Practical examples of traceability, measurement uncertainty and validation in chemistry

Parameter C TOC1 V 100 V 100


Value 0.00497 0.1 0.1
Std.‑unc. 0.000017 0.000061 6.09E‑05
Units g L– 1
L Ll
C TOC1 0.00497 0.004987 0.00497 0.00497
V 100 0.1 0.1 0.100061 0.1
V100 0.1 0.1 0.1 0.100061

C 5 g L– 1 0.00497 0.00499 0.00497 0.00497


u(y,xi) – 0.000017 – 0.000003 0.000003
u(y)2, u(y,xi)2 3.07E‑10 2.89E‑10 9.19E‑12 9.15E‑12
u(C 5) g L – 1
1.75E‑05
Index 96.9703 17.2932 17.2544

Ranges 2 and 3:
Procedure is same as for range 1.

222
Measurement uncertainty of sample (Range 1)
Parameter CTOC A A0.2 A0.5 A1 A2.5 A5 C0.2 C0.5 C1 C2.5 C5 Fd Fr Fh Fre Fs
Value 42.96 8.218 16.69 20.92 42.48 86.88 0.0002 0.0005 0.001 0.00249 0.00497 1 1 1 1 1
Std.‑unc. 0.2056 0.2598 0.2104 0.2155 0.1425 0.5831 7E‑07 1.7E‑06 4E‑06 8.7E‑06 1.7E‑05 6.1E‑05 0.0321 0.0069 0.019 0.033
Units No unit No unit No unit No unit No unit No unit g/L g/L g/L g/L g/L g/L g/l g/L g/L g/L
A 42.56 43.1656 42.53 42.53 42.53 42.53 42.53 42.53 42.53 42.53 42.53 42.53 42.53 42.53 42.53 42.53 42.53
A0.2 8.218 8.218 8.4778 8.218 8.218 8.218 8.218 8.218 8.218 8.218 8.218 8.218 8.218 8.218 8.218 8.218 8.218
A0.5 16.69 16.69 16.69 16.9004 16.69 16.69 16.69 16.69 16.69 16.69 16.69 16.69 16.69 16.69 16.69 16.69 16.69
A1 20.92 20.92 20.92 20.92 21.1355 20.92 20.92 20.92 20.92 20.92 20.92 20.92 20.92 20.92 20.92 20.92 20.92
A2.5 42.48 42.48 42.48 42.48 42.48 42.6225 42.48 42.48 42.48 42.48 42.48 42.48 42.48 42.48 42.48 42.48 42.48
A5 86.88 86.88 86.88 86.88 86.88 86.88 87.4631 86.88 86.88 86.88 86.88 86.88 86.88 86.88 86.88 86.88 86.88
C0.2 0.0002 0.000199 0.000199 0.000199 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.000199 0.0002 0.000199
C0.5 0.0005 0.000497 0.000497 0.000497 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.0005 0.000497 0.0005 0.000497
C1 0.00099 0.000994 0.000994 0.000994 0.00099 0.00099 0.00099 0.00099 0.00099 0.001 0.00099 0.00099 0.00099 0.00099 0.000994 0.00099 0.000994
C2.5 0.00249 0.002485 0.002485 0.002485 0.00249 0.00249 0.00249 0.00249 0.00249 0.0025 0.00249 0.00249 0.00249 0.00249 0.002485 0.00249 0.002485
C5 0.00497 0.00497 0.00497 0.00497 0.00497 0.00497 0.00497 0.00497 0.00497 0.005 0.00497 0.00499 0.00497 0.00497 0.00497 0.00497 0.00497
Fd 1 1 1 1 1 1 1 1 1 1 1 1 1.00006 1 1 1 1
Fr 1 1 1 1 1 1 1 1 1 1 1 1 1 1.0321 1 1 1
Fh 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.0069 1 1
FR 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.019 1
Fs 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.033

B1 16056 1 6056.03 1 6028.57 1 6037.86 1 6044.4 1 6062.1 1 6174.8 1 6057.2 1 6058.7 1 6059 1 6048.3 1 6000.9 1 6056 1 6056 1 6056.03 1 6056 1 6056.03
A— 35.0376 35.0376 35.08956 35.07968 35.0807 35.0661 35.1542 35.0376 35.0376 35.038 35.0376 35.0376 35.0376 35.0376 35.0376 35.0376 35.0376
Measurement of the concentration of total organic carbon (TOC) in waste water

Cp— 0.00183 0.001829 0.001829 0.001829 0.00183 0.00183 0.00183 0.00183 0.00183 0.0018 0.00183 0.00183 0.00183 0.00183 0.001829 0.00183 0.001829
Bo 5.67177 5.67177 5.773946 5.747079 5.73621 5.68919 5.57123 5.6674 5.66139 5.6553 5.65801 5.71697 5.67177 5.67177 5.67177 5.67177 5.67177
Cx 0.0023 0.002335 0.002293 0.002294 0.00229 0.00229 0.00228 0.0023 0.0023 0.0023 0.0023 0.0023 0.0023 0.0023 0.002296 0.0023 0.002296
CTOC 0.0023 0.002335 0.002293 0.002294 0.00229 0.00229 0.00228 0.0023 0.0023 0.0023 0.0023 0.0023 0.0023 0.00237 0.002311 0.00234 0.002371
u(y,xi) ‑3.77E‑05 4.31E‑06 3.96E‑06 4.2E‑06 3.8E‑06 1.3E‑05 1.8E‑06 1.6E‑06 1E‑06 ‑1E‑07 ‑3E‑06 1.7E‑06 ‑7E‑05 ‑1.4E‑05 ‑4.2E‑05 ‑7.4E‑05
u(y)2,
1.42E‑09 1.86E‑11 1.57E‑11 1.8E‑11 1.5E‑11 1.6E‑10 3.1E‑12 2.6E‑12 2E‑12 9.3E‑15 1E‑11 3E‑12 5.2E‑09 1.95E‑10 1.7E‑09 5.46E‑09
u(y,xi)2
u(CTOC)2 1.4E‑08
u(CTOC) 0.00012
index 100 31.62802 3.614814 3.323666 3.53486 3.2023 10.4851 1.47761 1.34722 1.0498 0.081 2.7003 1.44955 60.224 11.7154 35.0074 61.9567

223
224
Range 2
Parameter CTOC A A2 A12.5 A25 A50 A100 C2 C12.5 C25 C50 C100 Fd Fr Fh Fre Fs
Value 127.1 11.72 24.26 49.08 126.3 256 0.00199 0.01243 0.0249 0.0497 0.0994 1 1 1 1 1
Std.‑unc. 0.5624 0.1024 0.1465 0.5638 2.5371 1.8902 6E‑06 0.00004 8E‑05 0.00016 0.00032 6.1E‑05 0.0321 0.0069 0.019 0.033
Units No unit No unit No unit No unit No unit No unit g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1
A 127.1 127.6624 127.1 127.1 127.1 127.1 127.1 127.1 127.1 127.1 127.1 127.1 127.1 127.1 127.1 127.1 127.1
A2.5 11.72 11.72 11.8224 11.72 11.72 11.72 11.72 11.72 11.72 11.72 11.72 11.72 11.72 11.72 11.72 11.72 11.72
A12.5 24.26 24.26 24.26 24.4065 24.26 24.26 24.26 24.26 24.26 24.26 24.26 24.26 24.26 24.26 24.26 24.26 24.26
A25 49.08 49.08 49.08 49.08 49.6438 49.08 49.08 49.08 49.08 49.08 49.08 49.08 49.08 49.08 49.08 49.08 49.08
A50 126.3 126.3 126.3 126.3 126.3 128.837 126.3 126.3 126.3 126.3 126.3 126.3 126.3 126.3 126.3 126.3 126.3
A100 256 256 256 256 256 256 257.89 256 256 256 256 256 256 256 256 256 256
C2.5 0.00199 0.001988 0.001988 0.001988 0.00199 0.00199 0.00199 0.00199 0.00199 0.002 0.00199 0.00199 0.00199 0.00199 0.001988 0.00199 0.001988
C12.5 0.01243 0.012425 0.012425 0.012425 0.01243 0.01243 0.01243 0.01243 0.01247 0.0124 0.01243 0.01243 0.01243 0.01243 0.012425 0.01243 0.012425
C25 0.02485 0.02485 0.02485 0.02485 0.02485 0.02485 0.02485 0.02485 0.02485 0.0249 0.02485 0.02485 0.02485 0.02485 0.02485 0.02485 0.02485
C50 0.0497 0.0497 0.0497 0.0497 0.0497 0.0497 0.0497 0.0497 0.0497 0.0497 0.04986 0.0497 0.0497 0.0497 0.0497 0.0497 0.0497
C100 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.09972 0.0994 0.0994 0.0994 0.0994 0.0994
Fd 1 1 1 1 1 1 1 1 1 1 1 1 1.00006 1 1 1 1
Fr 1 1 1 1 1 1 1 1 1 1 1 1 1 1.0321 1 1 1
Fh 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.0069 1 1
FR 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.019 1
Fs 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.033

B1 2597.14 2597.138 2596.532 2596.524 2595.94 2602.2 2616.49 2597.24 2597.55 2597.4 2596.35 2588.82 2597.14 2597.14 2597.138 2597.14 2597.138
A— 93.472 93.472 93.49248 93.5013 93.5848 93.9794 93.85 93.472 93.472 93.472 93.472 93.472 93.472 93.472 93.472 93.472 93.472
Cp— 0.03767 0.037673 0.037673 0.037673 0.03767 0.03767 0.03767 0.03767 0.03768 0.0377 0.0377 0.03774 0.03767 0.03767 0.037673 0.03767 0.037673
Bo – 4.36893 – 4.368932 – 4.325623 – 4.316524 – 4.211 – 4.0521 – 4.7198 – 4.37593 – 4.40517 – 4.4215 – 4.4217 – 4.2202 – 4.36893 – 4.3689 – 4.36893 – 4.36893 – 4.36893
Cx 0.05062 0.050837 0.050616 0.050612 0.05058 0.0504 0.05038 0.05062 0.05063 0.0506 0.05066 0.05073 0.05062 0.05062 0.050621 0.05062 0.050621
CTOC 0.05062 0.050837 0.050616 0.050612 0.05058 0.0504 0.05038 0.05062 0.05063 0.0506 0.05066 0.05073 0.05062 0.05225 0.05097 0.05158 0.052291
u(y,xi) – 0.000217 4.87E‑06 8.23E‑06 3.7E‑05 0.00022 0.00024 – 6.9E‑07 – 6E‑06 – 1E‑05 – 4E‑05 – 0.0001 – 3.1E‑06 – 0.0016 – 0.00035 – 0.00096 – 0.00167
u(y)2, u(y,xi)2 4.69E‑08 2.37E‑11 6.77E‑11 1.4E‑09 4.8E‑08 5.8E‑08 4.7E‑13 3.5E‑11 2E‑10 1.3E‑09 1.1E‑08 9.5E‑12 2.6E‑06 1.22E‑07 9.3E‑07 2.79E‑06
u(CTOC)2 6.6E‑06
u(CTOC) 0.00258
Practical examples of traceability, measurement uncertainty and validation in chemistry

index 100 – 8.400382 0.188752 0.319092 1.45307 8.5412 9.31933 0.02662 0.23098 0.5641 1.3864 4.0811 0.11959 63.035 13.5496 37.3104 64.8024
Range 3
Parameter CTOC A A100 A125 A166.5 A250 A500 C100 C125 C166.5 C250 C500 Fd Fr Fh Fre Fs
Value 532.7 207.8 259.9 350.9 530.6 1102 0.0994 0.1245 0.1665 0.2485 0.497 1 1 1 1 1
Std.‑unc. 2.561 3.1836 2.5029 3.7423 2.9651 9.6021 0.0003 0.0004 0.0005 0.0008 0.0016 6.1E‑05 0.0321 0.0069 0.019 0.033
Units No unit No unit No unit No unit No unit No unit g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1 g L– 1
A 532.7 535.261 532.7 532.7 532.7 532.7 532.7 532.7 532.7 532.7 532.7 532.7 532.7 532.7 532.7 532.7 532.7
A100 207.8 207.8 210.9836 207.8 207.8 207.8 207.8 207.8 207.8 207.8 207.8 207.8 207.8 207.8 207.8 207.8 207.8
A125 259.9 259.9 259.9 262.4029 259.9 259.9 259.9 259.9 259.9 259.9 259.9 259.9 259.9 259.9 259.9 259.9 259.9
A166.5 350.9 350.9 350.9 350.9 354.642 350.9 350.9 350.9 350.9 350.9 350.9 350.9 350.9 350.9 350.9 350.9 350.9
A250 530.6 530.6 530.6 530.6 530.6 533.565 530.6 530.6 530.6 530.6 530.6 530.6 530.6 530.6 530.6 530.6 530.6
A500 1102 1102 1102 1102 1102 1102 1111.6 1102 1102 1102 1102 1102 1102 1102 1102 1102 1102
C100 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0997 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994 0.0994
C125 0.12425 0.12425 0.12425 0.12425 0.12425 0.12425 0.12425 0.12425 0.1249 0.1243 0.12425 0.12425 0.12425 0.12425 0.12425 0.12425 0.12425
C166.5 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 0.167 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665 0.1665
C250 0.2485 0.2485 0.2485 0.2485 0.2485 0.2485 0.2485 0.2485 0.2485 0.2485 0.2493 0.2485 0.2485 0.2485 0.2485 0.2485 0.2485
C500 0.497 0.497 0.497 0.497 0.497 0.497 0.497 0.497 0.497 0.497 0.497 0.4986 0.497 0.497 0.497 0.497 0.497
Fd 1 1 1 1 1 1 1 1 1 1 1 1 1.00006 1 1 1 1
Fr 1 1 1 1 1 1 1 1 1 1 1 1 1 1.0321 1 1 1
Fh 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.0069 1 1
FR 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.019 1
Fs 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.033

B1 2254.73 2254.726 2250.811 2252.247 2252.54 2255.34 2279.68 2255.57 2256.18 2255.4 2254.28 2245.44 2254.73 2254.73 2254.726 2254.73 2254.726
A— 490.24 490.24 490.8767 490.7406 490.988 490.833 492.16 490.24 490.24 490.24 490.24 490.24 490.24 490.24 490.24 490.24 490.24
Cp— 0.22713 0.22713 0.22713 0.22713 0.22713 0.22713 0.22713 0.22719 0.22726 0.2272 0.22729 0.22745 0.22713 0.22713 0.22713 0.22713 0.22713
Measurement of the concentration of total organic carbon (TOC) in waste water

Bo – 21.876 – 21.87598 – 20.34999 – 20.81229 – 20.631 – 21.422 – 25.622 – 22.2037 – 22.5001 – 22.247 – 22.136 – 20.484 – 21.876 – 21.876 – 21.876 – 21.876 – 21.876
Cx 0.24596 0.247097 0.245711 0.24576 0.24565 0.24569 0.24491 0.24601 0.24608 0.2461 0.24613 0.24636 0.24596 0.24596 0.245962 0.24596 0.245962
CTOC 0.24596 0.247097 0.245711 0.24576 0.24565 0.24569 0.24491 0.24601 0.24608 0.2461 0.24613 0.24636 0.24598 0.25386 0.247659 0.25063 0.254078
u(y,xi) —0.001136 0.00025 0.000202 0.00031 0.00027 0.00105 – 5.3E‑05 —0.00012 – 9E‑05 – 0.0002 – 0.0004 – 1.5E‑05 — 0.0079 – 0.0017 — 0.00467 — 0.00812
u(y)2, u(y,xi)2 1.29E‑06 6.26E‑08 4.06E‑08 9.9E‑08 7.2E‑08 1.1E‑06 2.8E‑09 1.4E‑08 9E‑09 2.7E‑08 1.6E‑07 2.2E‑10 6.2E‑05 2.88E‑06 2.2E‑05 6.59E‑05
u(CTOC)2 0.00016
u(CTOC) 0.01248
index 100 9.099454 2.003833 1.614493 2.51561 2.14729 8.39986 0.42402 0.94407 0.7582 1.3114 3.1878 0.12 63.252 13.5961 37.4387 65.0251

225
Appendix 1
how to use this book

227
How to use this book

From experience gained during several TrainMiC® courses in various European countries,
a standardised approach of the TrainMiC® example session has been developed to:

• facilitate the exchange of training material that has been prepared and
collected by the various trainers;
• facilitate the exchange of feedback from the participants as well as from
the trainers;
• improve the teaching impact of the course.

Therefore, a structure for a TrainMiC® example has been developed and some guidelines
on how to conduct a typical TrainMiC® example session have been drafted. As this is
crucial for the proper understanding and conducting of TrainMiC® example sessions: a
detailed description is given below.

What does a standard TrainMiC® example look like?


Each TrainMiC® example includes a section on:

(a) the input information (description of the analytical procedure, customer’s


requirement and measurement data);
(b) the questions regarding traceability, validation and measurement uncertainty
(this part is thus subdivided into three exercises, which are known as the
‘Traceability’, ‘Validation’ and ‘Measurement uncertainty’ exercises);
(c) the solutions to the exercises.

To easily distinguish between different parts of an example, colours have been assigned
to each part, as shown in Figure 1.

The input information files, which include a description of the analytical procedure, the
customer’s requirement and measurement data, which all needed for the three exercises,
are referred to as the ‘yellow pages’. During the TrainMiC® example session, they are
given to each participant, as well as a booklet of exercises on traceability, validation
and measurement uncertainty. The latter are referred to as the ‘white pages’ and the
questions that are to be answered by the trainees are fully aligned with the theoretical
presentations. On the other hand, they are complementary to them in a sense that by
presenting theory as well as doing the examples, each of the topics is appropriately
addressed and sufficiently covered.

229
Practical examples of traceability, measurement uncertainty and validation in chemistry

Figure 1 Harmonised TrainMiC® example

The so‑called green pages provide answers to the questions asked in all three exercises,
i.e. traceability, validation and measurement uncertainty. Ideally, for the measurement
uncertainty exercise, three different approaches to the measurement uncertainty
evaluation are presented: a simple arithmetic approach, a spreadsheet solution and result
obtained by using professional software. At present, the green pages are only for the
internal use amongst the trainers (they do not carry the TrainMiC® logo) and are not to
be handed over to the participants.

Furthermore, as a quality management tool, a summary form (blue page) wraps up each
example. It contains all the essential information about each example: the analytical
procedure; type of sample; analytes; measurement method; customer’s requirements;
other information which helps in managing and selecting the examples.

230
How to use this book

What is the recommended approach to conducting a TrainMiC® example session?


The TrainMiC® national team leader, who organises the TrainMiC® event, decides on the
exact format of the TrainMiC® example session taking into account the knowledge and
needs of the trainees as well as specific areas that are to be addressed during the training
course, for example environmental analysis, analysis of food or clinical analysis.

In practice, this means that a TrainMiC® example session at a certain TrainMiC®


event can be conducted in any of the following forms:

• one example, all three exercises;


• one example, one or two exercises only;
• more than one example, all exercises for each;
• more than one example, one or two exercises only;
• one example, Measurement uncertainty exercise: comparing different
tools for its evaluation.

When deciding on which format to choose, it is essential not to forget the time constraints
of the particular training event as it is crucial that the trainees have enough time to do
the exercises as well as enough time for a properly led discussion after completing the
exercises.

Based on our experience, we suggest dedicating about 60 minutes for each of the exercises
(group work) and about 30 minutes for a follow‑up discussion. The groups should not be
bigger than five participants and each group should, at the beginning of each exercise,
nominate a rapporteur who afterwards reports on the results and on the questions and
the discussions the group had during the exercise. Nominating a rapporteur improves the
reporting significantly, so it is highly recommended giving each group a card ‘rapporteur’
at the start. It is of vital importance that the trainees are properly briefed before starting
the example session. The slides, which can be used for this purpose, are in Appendix 3
and a dynamic process of conducting a TrainMiC® example session is schematically
shown in Figure 2.

About the structure of this handbook


In this handbook, five different analytical procedures are worked as TrainMiC® examples.
Following the above described standardised approach, each of them contains:

(a) a summary form (blue page);


(b) a short introduction to the analytical procedure (such as a PowerPoint
presentation) to be given by the trainer;
(c) all the input needed to do the three exercises (yellow pages);
(d) the solved exercises (green pages).

Nineta Majcen and Philip Taylor

231
232
Introduction and Brief the participants Form the Nomination Distribution Participants work Participants
traceability exercise about how the TrainMiC® groups — of rapporteur of the yellow on the exercise. report back
example session is and white
max. five for each group A trainer(s) is (a guided
organised and about the
analytical procedure participants (hand out the pages by the present to help discussion)
they will be working on. per group ‘rapporteur’card) trainer when needed.

Approx. 60 minutes Approx. 30 minutes

Validation exercise Nomination of Participants Participants


rapporteur for work on the
each group exercise. report back (a
(can be the A trainer(s) is guided
same as for the present to help discussion)
previous exercise) when needed.

Approx. 60 minutes

Measurement uncertainty exercise


Nomination Participants Participants
of rapporteur work on the report back (a
for each exercise. guided
group (can be A trainer(s) is discussion)
the same as present to
for the help when
previous needed.
exercise)

Approx. 60 minutes
Practical examples of traceability, measurement uncertainty and validation in chemistry
Appendix 2
TrainMiC exercises (white pages)

233
TrainMiC Exercises (white pages)

TrainMiC® ExErCisEs

Analytical procedure:

exerCIse 1:
establishing traceability in analytical chemistry

exerCIse 2:
single laboratory validation of measurement procedures
Part I: General issues
Part II: Parameters to be validated
Part III: Some calculations and conclusions

exerCIse 3:
building an uncertainty budget
Addendum I: By spreadsheet approach
Addendum II: By dedicated software

235
Practical examples of traceability, measurement uncertainty and validation in chemistry

Filename: 03‑TEMPLATE‑White‑T‑V‑MU‑A4
Version: 01‑EN
Prepared by: TrainMiC® 2005/2006
Editors: Nineta Majcen, Philip Taylor
Issued: March 2007
For use at the TrainMiC® courses only.

236
TrainMiC Exercises (white pages)

ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

1. Specifying the analyte and measurand


Analyte

Measurand

Units

2. Choosing a suitable measurement procedure with associated model equation

Measurement
procedure

Type of calibration Standard curve Standard addition Internal standard

Model equation:

237
Practical examples of traceability, measurement uncertainty and validation in chemistry

3. List the input quantities according to their influence on the uncertainty of the
result of the measurement (first the most important ones): at this point, your
judgement should be based on your previous experience only.
1
2
3
4
5

4. List the reference standards needed and state the information regarding
traceability of the reference value

For the analyte


1 Name/chemical formula/producer
2 Name/chemical formula/producer

For the other input quantities


Quantity/equipment/calibration
1 (e.g. mass/balance/calibrated by NMI, U  = xx
(k = 2))
2 Quantity/equipment/calibration
3 Quantity/equipment/calibration
4 Quantity/equipment/calibration

5. Estimating uncertainty associated with the measurement


Are all important parameters included in the model
Yes No
equation?
Other important parameters:

6. How would you prove traceability of your result?


1
2
3

238
TrainMiC Exercises (white pages)

7. Any other comments, questions …

239
Practical examples of traceability, measurement uncertainty and validation in chemistry

SINGLE LABORATORY VALIDATION


OF MEASUREMENT PROCEDURES

parT I: General IssUes

1. Specify the measurement procedure, analyte, measurand and units


The measurement procedure
Analyte
The measurand
Units

2. Specify the scope


Matrix
Measuring range

3. Requirements of the measurement procedure


Intended use of the results
Parameters to be validated Value requested by the customer
LOD
LoQ
Mark the customer’s Repeatability
requirements and give their
values Within‑lab reproducibility
Trueness
Measurement uncertainty
Other — state

4. Origin of the measurement procedure


VALIDATION
New in‑house method Full
Modified validated method Partial
Official standard method Confirmation/verification

240
TrainMiC Exercises (white pages)

parT II: paraMeTers To be ValIdaTed

5. Selectivity/interference/recovery

Where yes, give further information, for example which CRM, reference method.
CRM/RM: analysis of available CRM or RM
Further information
Spike of pure substance

Compare with a reference method

Selectivity, interferences

Test with different matrices

Other — specify

6. Measuring range
Linearity
Upper limit
LOD
LoQ

7. Spread — precision
Repeatability
Reproducibility (within lab)
Reproducibility (between labs)

8. Robustness
Variation of parameters

241
Practical examples of traceability, measurement uncertainty and validation in chemistry

9. Quality control
Control charts
Participation in proficiency testing schemes

10. Other parameters to be tested


Working range and testing of homogeneity of variances
R square
Residual standard deviation
Standard deviation of the analytical procedure
Coefficient of variation of the analytical procedure
Measurement uncertainty

242
TrainMiC Exercises (white pages)

parT III: soMe CalCUlaTIons and ConClUsIons

11. Calculation of parameters requested by the customer


Parameters requested to be
Calculations
validated
LOD
LoQ
Repeatability
Within‑lab reproducibility
Trueness
Measurement uncertainty
Other — state

12. Does the analytical procedure fulfil the requirement(s) for the intended use?
Value requested by the Is the requirement
Value obtained
Parameter customer fulfilled?
during validation
(the same as stated in Question 3) Yes/No
LOD
LoQ
Repeatability
Within‑lab
reproducibility
Trueness
Measurement
uncertainty
Other

The analytical procedure is fit for the intended use:


Yes No

For measurement uncertainty and traceability, refer to the corresponding pages.

243
Practical examples of traceability, measurement uncertainty and validation in chemistry

BUILDING AN UNCERTAINTY BUDGET

1. Specify the measurand and units


Measurand
Units

2. Describe the measurement procedure and provide the associated model


equation

Measurement procedure:

Model equation:

3. Identify (all possible) sources of uncertainty


Uncertainty of concentration of reference solutions
Uncertainty of measurements of peak area
Method bias
Matrix effect
Other:
Other:
Other:

244
TrainMiC Exercises (white pages)

4. Evaluate values of each input quantity


Input quantity Value Units Remarks

5. Evaluate the standard uncertainty of each input quantity


Standard
Input quantity Units Remarks
uncertainty

6. Calculate the value of the measurand, using the model equation

7. Calculate the combined standard uncertainty (uc) of the result and specify units
using:

Mathematical solution Spreadsheet approach Commercial software

Input Standard
Value Units Remarks
quantity uncertainty

245
Practical examples of traceability, measurement uncertainty and validation in chemistry

8. Calculate expanded uncertainty (Uc) and specify the coverage factor k and the
units

9. Analyse the uncertainty contribution and specify the main three input quantities
contributing the most to Uc
1
2
3

10. Prepare your Uncertainty budget report

246
TrainMiC Exercises (white pages)

addendum I: Measurement uncertainty calculation:


spreadsheet approach (excel)

addendum II: Measurement uncertainty calculation

247
Appendix 3
briefing of the trainees on the example session

249
Practical examples of traceability, measurement uncertainty and validation in chemistry

250
Briefing of the trainees on the example session

251
Practical examples of traceability, measurement uncertainty and validation in chemistry

252
Briefing of the trainees on the example session

253
Practical examples of traceability, measurement uncertainty and validation in chemistry

254
Briefing of the trainees on the example session

Notes

255
Practical examples of traceability, measurement uncertainty and validation in chemistry

Notes

256
European Commission

EUR 24688 — Joint Research Centre — Institute for Reference Materials and
Measurements
Title: Practical examples of traceability, measurement uncertainty and validation in
chemistry, Volume 2
Author(s): Ilaria Altieri, Sabrina Barbizzi, Jelena Bebić, Maria Belli, Elena Amico di
Meane, Gordana Horvat, Nada L. Lazić, Snježana Marinčić, Munir Mehović, Mustafa
Memić, Antonio Menditto, Tidža Muhić‑Šarac, Marina Patriarca, Giancarlo Pistone,
Michela Sega, Antonella Semeraro, Marjana Simonič, Brigita Tepuš

Luxembourg: Publications Office of the European Union


2011 — 260 pp. — 18.2 x 25.7 cm
EUR — Scientific and Technical Research series — ISSN 1018‑5593
ISBN 978‑92‑79‑18998‑2
doi:10.2787/36024

Price (excluding VAT) in Luxembourg: EUR 25

Abstract
Examples on traceability, measurement uncertainty and validation for measurements of
retinol and α‑tocopherol in human serum, cyclamate in soft drinks, arsenic in groundwater,
sodium chloride in milk products and total organic carbon in waste water are presented
in this book. Additionally, the idea and structure of the TrainMiC® examples, which
complement the TrainMiC® theoretical presentations, are described in detail to give a
complete overview of the TrainMiC® teaching material.

257
HOW TO OBTAIN EU PUBLICATIONS
Free publications:
• via EU Bookshop (http://bookshop.europa.eu);
• at the European Union’s representations or delegations. You can
obtain their contact details on the Internet (http://ec.europa.eu) or
by sending a fax to +352 2929-42758.
Priced publications:
• via EU Bookshop (http://bookshop.europa.eu).
Priced subscriptions (e.g. annual series of the Official Journal
of the European Union and reports of cases before the Court of
Justice of the European Union):
• via one of the sales agents of the Publications Office of the European
Union (http://publications.europa.eu/others/agents/index_en.htm).
The mission of the JRC is to provide customer-driven scientific and technical support for the

LA-NA-24688-EN-C
conception, development, implementation and monitoring of EU policies. As a service of the
European Commission, the JRC functions as a reference centre of science and technology for
the Union. Close to the policymaking process, it serves the common interest of the Member
States, while being independent of special interests, whether private or national.

Comparability of measurement results is of the utmost importance in all sectors of human activity.
In today’s world, important decisions regarding the environment, food safety, public and individual
health, consumer protection and the conformity of products to regulations and specifications rely on
the results of analytical measurements. Hence, the need to assure comparability of analytical results,
to remove barriers to trade, movement and international relationships, based on the principle ‘once
tested, accepted everywhere’.
However, the application of the principles of metrology in analytical sciences faces many challenges,
starting from the availability of suitable references to the complexity of the measurement procedures
and the wide range of demands. For this reason, the availability of practical examples, developed
within the framework of the European Lifelong learning programme TrainMiC®, provides the means
to show how the metrological concepts of traceability, validation and measurement uncertainty can
be put into practice in real-life situations. The examples in this book, created with contributions from
the TrainMiC® national teams in Bosnia and Herzegovina, Croatia, Italy, Serbia and Slovenia, are
taken from the fields of environmental analysis, control of food and drinking water and laboratory
medicine.
For this second volume, the fairy-tale character is the worldwide known Pinocchio, a wooden pup-
pet, who faces various misadventures on the road to becoming a real boy. Initially, reluctant to listen
to a talking cricket who advises him for the best, Pinocchio ends up in prison, but he eventually
learns his lesson and succeeds, with the help of the Blue Fairy. This book is also intended to bring
help and advice to those committed to producing reliable results in analytical chemistry. As in Pinoc-
chio’s story, success will depend on listening to advice, learning from one’s mistakes and a commit-
ment to achievement.
With our best wishes for success,
Antonio Menditto
Marina Patriarca

Price (excluding VAT)


in Luxembourg: EUR 25

doi:10.2787/36024

9 7 8 9 2 7 9 1 8 9 9 8 2
ISBN 978-92-79-18998-2

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