Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
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
http://irmm.jrc.ec.europa.eu/
http://www.jrc.ec.europa.eu/
Legal Notice
Neither the European Commission nor any person acting on behalf of the Commission
is responsible for the use which might be made of this publication.
(*) Certain mobile telephone operators do not allow access to 00 800 numbers or these calls may be billed.
JRC 65988
EUR 24688
ISBN 978-92-79-18998-2
ISSN 1018-5593
doi: 10.2787/36024
Printed in Belgium
Table oF ConTenTs
InTrodUCTIon ..................................................................................................................5
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
3
appendIx 1 ..................................................................................................................... 227
how to use this book
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)
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).
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!’
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®!”
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.
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).
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 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.
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 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.
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.
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
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.
20
About the authors
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
Example No Ex‑22
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
25
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
analytical procedure
parT I ...................................................................................................................................31
description of the analytical procedure
parT II .................................................................................................................................34
The customer’s requirements concerning the quality of the measurement result
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…
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.
31
Practical examples of traceability, measurement uncertainty and validation in chemistry
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.
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
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:
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).
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…
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.
The results of the 11 measurements under repeatability conditions are shown in Table 1.
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
n 10 10 10 10
n 24 26 25 26
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
37
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
α‑tocopherol
Relevant equations
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…
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
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
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
40
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…
Units mg L– 1
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
4. List the reference standards needed and state the information regarding
traceability of the reference value
42
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…
43
Practical examples of traceability, measurement uncertainty and validation in chemistry
Units mg L– 1
Retinol: 0.05–1.0 mg L– 1
Measuring range
α‑tocopherol: 0.95–23.0 mg L– 1
44
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…
45
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
Selectivity, interferences
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
R square
Measurement uncertainty
47
Practical examples of traceability, measurement uncertainty and validation in chemistry
SD
RSD % = × 100
mean
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
49
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
50
Simultaneous measurement of the concentration of retinol and α‑tocopherol in human serum…
α‑tocopherol:
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
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)
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
3. Other contributions
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…
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
3. Other contributions
7. Calculate expanded uncertainty (U) and specify the coverage factor k and the
units
Retinol:
K = 2, confidence level of 95 %
U(C)/C % = 9.2 %
53
Practical examples of traceability, measurement uncertainty and validation in chemistry
α‑tocopherol:
k = 2, confidence level of 95 %
U(C)/C % = 8.2 %
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
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).
5. BIMP, IEC, IFCC, ISO, IUPAC, IUPAP, OIML, Guide to the Expression of Uncertainty
in Measurement, ISO, Geneva (1995).
55
Chapter 2
Measurement of the concentration of cyclamate
concentration in soft drinks by a high-performance
liquid chromatographic method
Gordana Horvat, Snježana Marinčić
57
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
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
59
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
analytical procedure
parT I ...................................................................................................................................65
description of the analytical procedure
parT II .................................................................................................................................68
The customer’s requirements concerning the quality of the measurement result
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…
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.
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
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
∑ (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:
67
Practical examples of traceability, measurement uncertainty and validation in chemistry
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)
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…
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.
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
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.
70
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…
Input quantities:
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
71
Practical examples of traceability, measurement uncertainty and validation in chemistry
Relevant equations
* 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
Recovery R — 1 0.0032
73
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
74
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…
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
75
Practical examples of traceability, measurement uncertainty and validation in chemistry
Units mg L– 1
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.
76
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
77
Practical examples of traceability, measurement uncertainty and validation in chemistry
78
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…
Units mg L– 1
79
Practical examples of traceability, measurement uncertainty and validation in chemistry
5. Selectivity/interference/recovery
Where yes, give further information, for example which CRM, reference method.
Further information
Selectivity, interferences
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
80
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…
9. Quality control
Control charts
Participation in PT schemes
81
Practical examples of traceability, measurement uncertainty and validation in chemistry
( )
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
82
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…
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.
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
83
Practical examples of traceability, measurement uncertainty and validation in chemistry
B1 4395 mAU mg L– 1
B0 314.126 mAU
R 1 —
RW 48.21 mg L– 1
A 5820 mAU
84
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
Standard
Input quantity Value Units Remarks
uncertainty
As 212 217 701 mAU Average As of three replicates
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
85
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
86
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…
Recovery R — 1 0.0032
Intermediate precision (Within lab
Rw mg L– 1 48.2 1.98
reproducibility)
87
Practical examples of traceability, measurement uncertainty and validation in chemistry
Further reading
88
Measurement of the concentration of cyclamate concentration in soft drinks by a high‑performance…
Calibration solution C1
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., %
89
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., %
90
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., %
91
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…
93
Practical examples of traceability, measurement uncertainty and validation in chemistry
ccyc
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
Example No Ex‑25
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
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
97
Practical examples of traceability, measurement uncertainty and validation in chemistry
• 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
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)
U = k u (CAs )
101
Practical examples of traceability, measurement uncertainty and validation in chemistry
analytical procedure
102
Measurement of the concentration of arsenic…
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.
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.
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
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.
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.
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
Containers of glass, PE, PP and PTFE are suitable for sample collection. Clean all
containers with HNO3 (φHNO3 = 10 %) and rinse with water.
Procedure
Instrument set‑up
As is measured by Flame atomic absorption spectrometry (hydride technique) — GBC
Avanta AAS Version 2.0.
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.
106
Measurement of the concentration of arsenic…
Calculation
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
107
Practical examples of traceability, measurement uncertainty and validation in chemistry
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.
• 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.
108
Measurement of the concentration of arsenic…
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
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
Result
1
You may also want to include the relevant certificates here.
110
Measurement of the concentration of arsenic…
111
Practical examples of traceability, measurement uncertainty and validation in chemistry
Equations:
The input quantities in the model equation:
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
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
112
Measurement of the concentration of arsenic…
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
113
Practical examples of traceability, measurement uncertainty and validation in chemistry
Units µg L– 1
Model equation:
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
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
115
Practical examples of traceability, measurement uncertainty and validation in chemistry
116
Measurement of the concentration of arsenic…
117
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
Selectivity, interferences
Other — specify
6. Measuring range
Linearity
Upper limit
LOD
LoQ
7. Spread — precision
Repeatability
118
Measurement of the concentration of arsenic…
8. Robustness
Variation of parameters
9. Quality control
Control charts
Participation in PT schemes
119
Practical examples of traceability, measurement uncertainty and validation in chemistry
LoQ = 5 µg L– 1
Calculations
Ablank (aver) = – 0.00081
LoQ
s(A) 0.002895
A(LoQ) 0.028136
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
121
Practical examples of traceability, measurement uncertainty and validation in chemistry
Measurement procedure:
ISO 11969:1996 — Water quality — Determination of arsenic — Atomic absorption
spectrometric method (hydride technique)
Model equation:
A − b0 1
CAs = ×
b1 R
122
Measurement of the concentration of arsenic…
7. Calculate the combined standard uncertainty (uc) of the result and specify units
using:
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
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
124
Measurement of the concentration of arsenic…
Further reading
6. Kratgen, J., ‘Calculating standard deviation and confidence intervals with a universally
applicable spreadsheet technique’, Analyst, 119 (1994), pp. 2161-2165.
125
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
131
Practical examples of traceability, measurement uncertainty and validation in chemistry
Example No Ex‑16
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
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
133
Practical examples of traceability, measurement uncertainty and validation in chemistry
134
Measurement of the mass fraction of sodium chloride…
chloride in milk products by Volhard’s method
• Add 10.00 mL (25.00 mL) of 0.1 mol L-1 AgNO3 standard solution
and 10 mL of HNO3 solution, respectively.
• 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
c NaCl V2
cAgNO3 = (3)
V3
136
Measurement of the mass fraction of sodium chloride…
chloride in milk products by Volhard’s method
mNaCl PNaCl V2
cAgNO3 =
M NaCl V1 V3
mNaCl PNaCl V2 V4
cKSCN =
M NaCl V1 V3 V5
cAgNO3 V6 = n1 cKSCN V7 = n2
137
Practical examples of traceability, measurement uncertainty and validation in chemistry
u (WNaCl )
2 2
u ( n) u (msample)
+ (rep )
2
= +
WNaCl n msample
138
Measurement of the mass fraction of sodium chloride…
analytical procedure
139
Practical examples of traceability, measurement uncertainty and validation in chemistry
Task description
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.
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.
140
Measurement of the mass fraction of sodium chloride…
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
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.
141
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
mNaCl × PNaCl × V2
cAgNO3 = (5)
M NaCl × V1 × V3
142
Measurement of the mass fraction of sodium chloride…
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
( 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)
143
Practical examples of traceability, measurement uncertainty and validation in chemistry
START
144
Measurement of the mass fraction of sodium chloride…
145
Practical examples of traceability, measurement uncertainty and validation in chemistry
146
Measurement of the mass fraction of sodium chloride…
Comparison of results:
( 0.0529 )
2
s12
F ( v1 , v2 ) = F= = 2.44
s22
( 0.0216 )
2
147
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
148
Measurement of the mass fraction of sodium chloride…
( n1 − n2 ) × M NaCl × 100
WNaCl =
msample
as well as the values of combined standard and relative standard uncertainties are shown
in Table 1.
n1 = 0.001 mol
149
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
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.
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
u ( ∆n ) = u ( n1 ) + u ( n2 )
2 2
150
Measurement of the mass fraction of sodium chloride…
( n1 − n2 ) × M NaCl × 100
WNaCl =
msample
as well as the values of combined standard and relative uncertainties are shown in
Table 3.
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
151
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
152
Measurement of the mass fraction of sodium chloride…
Measurand Chloride mass fraction expressed as total sodium chloride in milk cream
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
cNaCl × V2
cAgNO3 = (3)
V3
cAgNO3 × V4
cKSCN = (4)
V5
mNaCl × PNaCl × V2
cAgNO3 = (5)
M NaCl × V1 × V3
153
Practical examples of traceability, measurement uncertainty and validation in chemistry
mNaCl × PNaCl × V2 × V4
cKSCN = (6)
M NaCl × V1 × V3 × V5
2. Combined model equation for the calculation of mass fraction WNaCl (in %):
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
155
Practical examples of traceability, measurement uncertainty and validation in chemistry
156
Measurement of the mass fraction of sodium chloride…
5. Selectivity/interference/recovery
Where yes, give further information, for example which CRM, reference method.
Selectivity, interferences
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)
157
Practical examples of traceability, measurement uncertainty and validation in chemistry
8. Robustness
Variation of parameters
9. Quality control
Control charts
Participation in PT schemes
158
Measurement of the mass fraction of sodium chloride…
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
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
159
Practical examples of traceability, measurement uncertainty and validation in chemistry
160
Measurement of the mass fraction of sodium chloride…
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
cNaCl × V2
cAgNO3 = (3)
V3
161
Practical examples of traceability, measurement uncertainty and validation in chemistry
mNaCl × PNaCl × V2
cAgNO3 = (5)
M NaCl × V1 × V3
mNaCl × PNaCl × V2 × V4
cKSCN = (6)
M NaCl × V1 × V3 × V5
2. Combined model equation for the calculation of mass fraction wNaCl (in %):
( n1 − n2 ) × M NaCl × 100
WNaCl =
msample
3. Model equation for calculation combined uncertainty (with all sources uncertainty)
162
Measurement of the mass fraction of sodium chloride…
163
Practical examples of traceability, measurement uncertainty and validation in chemistry
7. Calculate the combined standard uncertainty (uc) of the result and specify units
using:
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
164
Measurement of the mass fraction of sodium chloride…
Further reading
10. EN ISO/IEC 17025:2005 General requirements for the competence of testing and
calibration laboratories.
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
167
Practical examples of traceability, measurement uncertainty and validation in chemistry
Example No Ex‑09
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
169
Practical examples of traceability, measurement uncertainty and validation in chemistry
170
Measurement of the concentration of total organic carbon (TOC) in waste water
Measurement procedure
Addition of acid
171
Practical examples of traceability, measurement uncertainty and validation in chemistry
Model equation
A B0
CTOC sample = Fd Fr Fh FR Fs
B1
172
Measurement of the concentration of total organic carbon (TOC) in waste water
173
Practical examples of traceability, measurement uncertainty and validation in chemistry
analytical procedure
174
Measurement of the concentration of total organic carbon (TOC) in waste water
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.
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).
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
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
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
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
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.
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Practical examples of traceability, measurement uncertainty and validation in chemistry
Weighting of PHP
Stock reference
solution (CTOC1)
Working solutions
Sample dilution (CTOC2)
Addition of acid
Sparging of the
sample
Measuring concentration of
CTOC-sample, mg L-1
178
Measurement of the concentration of total organic carbon (TOC) in waste water
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).
179
Practical examples of traceability, measurement uncertainty and validation in chemistry
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).
The limit for quantification (LoQ) regarding carbonate was determined at 0.3 mg L– 1.
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.
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 %.
180
Measurement of the concentration of total organic carbon (TOC) in waste water
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
By further dilution (second dilution step), the working solutions ( CTOC ), used for the
2
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.
181
Practical examples of traceability, measurement uncertainty and validation in chemistry
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.
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)
182
Measurement of the concentration of total organic carbon (TOC) in waste water
∑ (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)
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Practical examples of traceability, measurement uncertainty and validation in chemistry
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
184
Measurement of the concentration of total organic carbon (TOC) in waste water
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
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):
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Practical examples of traceability, measurement uncertainty and validation in chemistry
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
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):
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Measurement of the concentration of total organic carbon (TOC) in waste water
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.
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:
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 :
188
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
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
189
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
190
Measurement of the concentration of total organic carbon (TOC) in waste water
Units mg L– 1
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
Quantity/equipment/calibration
1 Class A for pipettes and glassware
(e.g. mass/balance/calibrated by NMI, U = xx (k = 2))
192
Measurement of the concentration of total organic carbon (TOC) in waste water
2
3
193
Practical examples of traceability, measurement uncertainty and validation in chemistry
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.
194
Measurement of the concentration of total organic carbon (TOC) in waste water
195
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
Selectivity, interferences
Other — specify
6. Measuring range
Linearity
Upper limit
LOD
LoQ
7. Spread — precision
Repeatability
Reproducibility (in‑lab)
Reproducibility (between labs)
196
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
197
Practical examples of traceability, measurement uncertainty and validation in chemistry
LoQ calculation:
198
Measurement of the concentration of total organic carbon (TOC) in waste water
12
10
8
RSD (%)
4
0.3 mg L-1
2
0
0.0 0.5 1.0 1.5 2.0 2.5
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
199
Practical examples of traceability, measurement uncertainty and validation in chemistry
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 (—)
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Measurement of the concentration of total organic carbon (TOC) in waste water
u( Fd ) — Dilution of sample
u( Ft ) — Repeatability
u( FR ) — Recovery
u( Fh ) — Store conditions
u( Fn ) — Sampling procedure
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Practical examples of traceability, measurement uncertainty and validation in chemistry
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
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Measurement of the concentration of total organic carbon (TOC) in waste water
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Practical examples of traceability, measurement uncertainty and validation in chemistry
C0.2‑5 a gL – 1
Working solution concentration
Fs *
1 No unit Factor of sampling procedure
Range 2
Range 3
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Measurement of the concentration of total organic carbon (TOC) in waste water
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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Practical examples of traceability, measurement uncertainty and validation in chemistry
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)
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Measurement of the concentration of total organic carbon (TOC) in waste water
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Practical examples of traceability, measurement uncertainty and validation in chemistry
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)
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
By further dilution (second dilution step), the working solutions ( CTOC ) for developing
2
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)
Fr — Repeatability factor
Fh — Stability factor
FR — Recovery factor
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Practical examples of traceability, measurement uncertainty and validation in chemistry
7. Calculate the combined standard uncertainty (uc) of the result and specify units
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(CTOC ) = CTOC × + + + +
Solution:
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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
u(CTOC1 ) = × + +
u(CTOC1 ) = × + +
u(CTOC1 ) = × + +
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Practical examples of traceability, measurement uncertainty and validation in chemistry
Solution:
Range 1:
Range 2:
Range 3:
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
213
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
214
Measurement of the concentration of total organic carbon (TOC) in waste water
Ranges 2 and 3:
215
Practical examples of traceability, measurement uncertainty and validation in chemistry
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
9. Analyse the uncertainty contribution and specify the main three input quantities
contributing the most to Uc
Table 14: Uncertainty components
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).
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.
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Practical examples of traceability, measurement uncertainty and validation in chemistry
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
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
219
Practical examples of traceability, measurement uncertainty and validation in chemistry
220
Measurement of the concentration of total organic carbon (TOC) in waste water
221
Practical examples of traceability, measurement uncertainty and validation in chemistry
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.
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
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
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.
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. 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)
Measurand
Units
Measurement
procedure
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
238
TrainMiC Exercises (white pages)
239
Practical examples of traceability, measurement uncertainty and validation in chemistry
240
TrainMiC Exercises (white pages)
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
Selectivity, interferences
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
242
TrainMiC Exercises (white pages)
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
243
Practical examples of traceability, measurement uncertainty and validation in chemistry
Measurement procedure:
Model equation:
244
TrainMiC Exercises (white pages)
7. Calculate the combined standard uncertainty (uc) of the result and specify units
using:
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
246
TrainMiC Exercises (white pages)
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
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
doi:10.2787/36024
9 7 8 9 2 7 9 1 8 9 9 8 2
ISBN 978-92-79-18998-2