Practical Examples On Traceablity, Measurement Uncertainty and Validation in Chemistry Vol 1 by Nineta Majcen & Philip Taylor

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ISBN 978-92-79-12021-3
Practical Examples on
Traceability,
Measurement Uncertainty
and Validation
in Chemistry
Volume 1
Edited by
Nineta Majcen, Philip Taylor
Authors:
Ljudmila Benedik
Steluta Duta
Koit Herodes
Monika Inkret
Veselin Kmetov
Allan Knnapas
Ivo Leito
Bertil Magnusson
Urka Repinc
Philip Taylor
Emilia Vassileva
Te mission of the JRC is to provide customer-driven scientifc and technical support for the
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 policy-making process, it serves the common interest of the Member
States, while being independent of special interests, whether private or national.
Producing reliable measurements in analytical chemistry can be rather demanding.
Some would say an uphill struggle. Comparable to mountain walking. Hard work, but then
the satisfaction of reaching the top is absolutely great. And so is the view.
As with all human endeavour, it always helps to know what you are doing, thus theoretical
knowledge forms the basis. Likewise in analytical chemistry. Understanding the measurement
science, the metrology, is important. Tat is why in the international standard ISO/IEC-17025
General requirements for the competence of testing and calibration laboratories section fve deals
with technical requirements such as traceability, validation and uncertainty. Te European Life
Long Learning Programme TrainMiC, created in 2001, produced material for teaching the
theory.
As excellence in theory does not necessarily mean mastering practice, a need for developing
practical examples later arose. Tis is what you can fnd in this book, which is intended as a frst
of a series of such compilations.
Inspired by the NORDTEST Trollbook, we also decided to have a mascot. For each volume,
a diferent one, which would be taken from the treasure of European fairy tales and legends.
For this frst volume, the fairy tale character of Kekec (pronounced as Kekets) was chosen. Kekec
is a brave, clever and cheerful shepherd boy who lives in Slovenian mountains. He always brings
good to the people that surround him and he helps those that are in trouble. And in that sense,
that is what is the intention of this book.
We hope it succeeds in doing so.
Nineta Majcen
Philip Taylor
EUR22791/2 EN - 2010
Practical Examples on
Traceability,
Measurement Uncertainty
and Validation
in Chemistry
Volume 1
Second edition
Edited by
Nineta Majcen, Philip Taylor
Authors:
Ljudmila Benedik
Steluta Duta
Koit Herodes
Monika Inkret
Veselin Kmetov
Allan Knnapas
Ivo Leito
Bertil Magnusson
Urka Repinc
Philip Taylor
Emilia Vassileva
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: Retieseweg 111, B-2440 Geel, Belgium
E-mail: philip.taylor@ec.europa.eu
Tel.: +32 (0)14 571 605
Fax: +32 (0)14 571 863
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.
Europe Direct is a service to help you hnd answers
to your questions about the European Union
Freephone number (*):
00 800 6 7 8 9 10 11
(*) Certain mobile telephone operators do not allow access to 00 800 numbers or these calls may be billed.
More information on the European Union is available on the Internet (http://europa.eu).
Cataloguing data can be found at the end of this publication.
Luxembourg: Publications Offce of the European Union, 2010
JRC 59026
EUR 22791/2 EN
ISBN 978-92-79-12021-3
ISSN 1018-5593
doi: 10.2787/10402
European Union, 2010
Reproduction is authorised provided the source is acknowledged
Printed in
3
INTRODUCTION ..................................................................................................................5
HOW TO USE THE BOOK ...................................................................................................6
ABOUT THE AUTHORS .....................................................................................................11
CHAPTER 1 ..........................................................................................................................17
Analysis of Gold Alloys by Flame Atomic Absorption Spectrometry
Veselin Kmetov, Emilia Vassileva
CHAPTER 2 ..........................................................................................................................51
Determination of Calcium in Serum by Spectrophotometry
Steluta Duta, Philip Taylor
CHAPTER 3 ..........................................................................................................................81
Determination of Radium in Water by -Spectrometry
Ljudmila Benedik, Urka Repinc, Monika Inkret
CHAPTER 4 ....................................................................................................................... 121
Determination of Polar Pesticides by Liquid Chromatography Mass Spectrometry
Allan Knnapas, Koit Herodes, Ivo Leito
CHAPTER 5 ....................................................................................................................... 157
Determination of Ammonium in Water by Continuous Flow Analysis (CFA) and
Spectrometric Detection
Bertil Magnusson
APPENDIX 1 ..................................................................................................................... 193
TrainMiC Exercises (white pages)
APPENDIX 2 ..................................................................................................................... 209
Briefng of the trainees on the example session
TABLE OF CONTENTS
4
Practical examples on traceability, measurement uncertainty and validation in chemistry
Abbreviations
CRM Certifed reIerence materials
RM ReIerence materials
QC Quality control
PT Profciency testing
ILC Inter-laboratory comparisons
5
If you will tell it to me, I will forget
If you will show it to me, I will forget
If you involve me, I will remember.
Xun Zi
Chinese philosopher
310-237 BC
Chemical and bio-analytical measurements are omnipresent and oIten very important
in our society. Just think oI the quality oI the Iood we eat, the air we breathe, the role
oI these measurements in healthcare, in trade and in research. In all these cases, people
strive to get reliable data. There is an international standard Ior assuring the quality oI
measurement data, namely EN ISO/IEC-17025. It contains particular management as well
as technical requirements. These technical requirements are linked to the science behind
the measurements, meaning that metrological issues such as traceability, uncertainty and
validation are at the heart oI this.
This book will help you moving Irom the theory oI EN ISO/IEC-17025 to the practice,
by sharing practical examples in a detailed way. Reading the book might give you
inspiration on how to demonstrate the traceability oI your results to a technical assessor
in an accreditation audit. Or you can maybe use this material to explain to students what
is an appropriate way to tackle validation oI a certain measurement procedure? Perhaps
you will fnd in the book the confrmation oI your approach oI uncertainty estimation?
In short, this book is intended Ior analytical chemists perIorming measurements in
laboratories or Ior those responsible Ior such measurements. Those lecturing analytical
chemistry might also fnd it useIul as well as the students themselves.
The book is a product oI the TrainMiC
programme |www.trainmic.org|. This programme

was conceived in 2001 by the Institute Ior ReIerence Materials and Measurements oI
the European Commission Joint Research Centre, to address a need arising with those
countries wanting to enter the EU at that time. Rather than approaching this training
in an anecdotal way and organizing ad hoc events, a programme was set up called
TrainMiC
to create harmonised training material as well as to disseminate knowledge

in the various countries via a network oI authorised TrainMiC
trainers. AIterwards, the

TrainMiC
programme was spread also in the rest oI the EU and Europe`s largest LiIe
Long Learning programme in this area was created. Up to now 20 national TrainMiC

teams have been set up and more than 4000 people have been trained all across Europe.
By sharing the resources in a pre-defned manner at the European level, a better training
to the national communities can be provided than iI they would only rely on themselves.
Introduction
6
From experience gained during several TrainMiC
courses in various European countries,

a standardised approach oI the TrainMiC
example session has been developed to

Iacilitate exchange oI training material that has been prepared and collected by
the various trainers
Iacilitate exchange oI Ieedback Irom the participants as well as Irom the trainers
improve teaching impact oI the course.
ThereIore, a structure Ior a TrainMiC
example has been developed and some guidelines

on how to conduct a typical TrainMiC
example session have been draIted. As this is

crucial Ior proper understanding and conducting oI the TrainMiC
example sessions, a
detailed description is given below.
How does a standardised TrainMiC example look like?
Each TrainMiC
example includes a part on

a) the input inIormation (description oI the analytical procedure, customer`s
requirement and measurement data)
b) the questions regarding traceability, validation and measurement uncertainty
(this part is thus sub-divided in three exercises, which are known as Traceability
exercise`, Validation exercise` and Measurement uncertainty exercise`)
c) the solutions Ior the exercises.
To easily distinguish between diIIerent parts oI an example, colours have been assigned
to each part, as shown in Figure 1.
The input inIormation fles, which include a description oI the analytical procedure, the
customer`s requirement and measurement data, all needed Ior the three exercises, are
reIerred to as yellow pages. During the TrainMiC
example session they are given

to each participant, as well as a booklet oI exercises on traceability, validation and
measurement uncertainty. The latter are reIerred to as white pages and the questions
that are to be answered by the trainees are Iully aligned with the theoretical presentations.
On the other side they are complementary to them in a sense that by presenting theory as
well as doing the examples, each oI the topics is appropriately addressed and suIfciently
covered.
How to use the Book
7
How to use the Book
Data sheet
Extract from the
analytical
procedure
Customer's
requirements
Relevant
equations and
measurement data
Exercises
Traceability
Validation
Measurement
uncertainty
Solutions
Traceability
Validation
Measurement
uncertainty
Summary
fiche
General information
about the example
Check list on what
exactly is included for
the particular
example
Structure of a TrainMiC example
lntroductory
slides
About the analytical
procedure
Figure 1. Harmonised TrainMiC
example
The so called green pages provide answers to the questions asked in all three exercises
i.e. traceability, validation and measurement uncertainty. Ideally, Ior the measurement
uncertainty exercise three diIIerent approaches to the measurement uncertainty evaluation
are presented: a simple arithmetic approach, a spreadsheet solution and result obtained
by using dedicated proIessional soItware.
On top, as a quality management tool, a summary Iorm (blue page) is wrapping up
each example. It contains all the essential inIormation about each example e.g. analytical
procedure, type oI the sample, analytes, measurement method, customer`s requirements
and some other, which help in managing and selecting the examples.
What is a recommended approach of conducting a TrainMiC
example session?
The TrainMiC
national team leader, who is organising a TrainMiC
event, decides on
the exact Iormat oI the TrainMiC
example session, taking into account the knowledge

and needs oI the trainees as well as specifc areas that are addressed during the training
course e.g. environmental analysis, analysis oI Iood or clinical analysis.
8
In practice, this means that a TrainMiC
example session at a certain TrainMiC
event
can be conducted in one oI the Iollowing Iorms:
One example, all three exercises
One example, one or two exercises only
More than one example, all exercises Ior each
More than one example, one or two exercises only
One example, Measurement uncertainty exercise: comparing diIIerent tools Ior
its evaluation
When deciding on which Iormat to choose it is essential not to Iorget about the time
constraints oI a certain training event, as it is crucial that the trainees have enough time
to do the exercises as well as to dedicate enough time to a properly led discussion aIter
completing the exercises. Based on our experience, we suggest to dedicate about 60 minutes
Ior each oI the exercises (group work) and about 30 minutes Ior a Iollow-up discussion. The
groups should not be bigger than fve participants and each group should in the beginning
oI each exercise nominate a rapporteur who aIterwards reports on the results and on the
questions and the discussion the group had during the exercise. Nominating a rapporteur
improves the reporting signifcantly, so it is higly recommended to give each group a card
rapporteur` at the start. It is oI a vital importance that the trainees are properly brieIed
beIore starting with the example session. The slides, which can be used Ior this purpose
are in Appendix 2 and a dynamic process oI conducting a TrainMiC
example session is
schematically shown in Figure 2.
About the structure of this handbook
In this handbook, fve diIIerent analytical procedures are worked out as TrainMiC

examples. Following the above described standardised approach, each oI them contains
a) A summary Iorm (blue page`)
b) A short introduction to the analytical procedure (as a Power Point presentation)
that is given by the trainer
c) All input needed to do the three exercises (yellow pages`) and
d) The solved exercises (green pages`).
Nineta Majcen and Philip Taylor
Acknowledgment
The Metrology Institute oI the Republic oI Slovenia is grateIully acknowledged Ior its contribution to the
graphic illustrations in this book.
9
How to use the Book
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11
Introduction
Philip Taylor
ProI. Philip Taylor has been in analytical chemistry since 1982. He made his PhD at the
University oI Gent (Belgium) and started his career in R&D in industry beIore moving
to the metrology institute oI the European Commission, the Institute Ior ReIerence
Materials and Measurements which is part oI the Joint Research Centre.
There he is heading a unit dealing with reIerence isotope measurements (including
the Avogadro project) and is involved in activities oI the Consultative Committee oI
Chemistry (CCQM), applied isotope measurements in the Iood and environmental area,
inter-laboratory comparisons and training related to metrology in chemistry (TrainMiC
)
to support the European measurement inIrastructure. He has about 200 research papers
to his name.
He is keen in ensuring that metrology is relevant Ior today`s needs in society, Ior
instance to help implementing European legislation. This involves training and education
activities. He has also been very involved in technical assistance projects related to the
enlargement oI the EU.
He initiated the TrainMiC
programme and is chairing the TrainMiC
Management
Board.
Nineta Majcen
Nineta Majcen started as a researcher at the University oI Ljubljana (Slovenia), where
she obtained her PhD on validation oI newly developed methods and chemometrics. She
has continued her analytical work in the quality control laboratories in industry beIore
stepping into metrology activities at the national and European level. In metrology, she
has been mainly involved in topics related to metrology in chemistry and in issues related
to measurement inIrastructure. She is also closely collaborating with accreditation bodies
and she is lecturing at the University oI Maribor.
She is the Slovenian TrainMiC
team leader, member oI the TrainMiC
Management
Board and is chairing the TrainMiC
Editorial Board. She is currently working at the

Metrology institute oI the Republic oI Slovenia.
Chapter 1
Veselin Kmetov
He obtained his master degree in chemistry at the University oI Plovdiv (Bulgaria) and
there he took the position oI a research chemist at the Center oI Analytical Chemistry
and Applied Spectroscopy. He moved to assistant proIessor position in the Department
oI Analytical Chemistry where he received his PhD degree. He become a head oI the
research group oI atomic spectrometry, that accomplished a number oI research and
applied projects in the feld oI the instrumental analysis with atomic spectrometry methods
About the Authors
12
(AAS, ICP-OES and ICP-MS) Ior trace elements determination in environmental,
clinical and industrial materials. The group maintains excellent collaboration with a
research team Irom the University oI Alicante, Spain.
His research interests are: sample preparation; use oI microwave radiation; analysis
oI heavy matrices by discrete sample introduction, fow injection, electrothermal
vaporisation; metrology in chemistry; data acquisition and treatment; methodology oI
teaching.
At the moment he is lecturing 'Instrumental analysis, 'Statistics and metrology
and 'Computer Qualimetry.
He joined the TrainMiC
program in 2002 as a member oI Bulgarian team oI

authorised TrainMiC

trainers.
Emilia Vassileva
Emilia Vassileva is a research scientist and inorganic chemistry group leader in the
UN-IAEA-Environmental Laboratories in Monaco. She received her master degree at
the University oI Geneva (Switzerland) and her PhD degree at the University oI Sofa
(Bulgaria), where she started as assistant proIessor. Her main research interests are in the
area oI trace and ultra trace isotope and elemental analysis using ICP-Mass Spectrometry
and other advanced instrumental techniques. She is author and co-author oI about 90
scientifc papers and reports. In the context oI metrology in chemistry she has been
working Ior seven years at the at Institute Ior ReIerence Materials and Measurements,
where she was involved in reIerence isotope measurements Ior certifcation purposes.
Important part oI her present activities is the training Ior UN Member States analytical
laboratories in QA/QC.
She is TrainMiC
coordinator, member oI the TrainMiC
Editorial and Management

Boards and National TrainMiC
team leader Ior Bulgaria.

Chapter 2
Steluta Duta
Steluta Duta works at National Institute oI Metrology Romanian Bureau oI Legal
Metrology in Bucharest, Romania (INM-BRML) in the feld oI Spectrometry and
ReIerence Materials. Her PhD on Chemical Metrology topic was deIended at the
Politechnic Institute Bucharet in Romania. She is involved in development/coordination
oI INM-BRML research programmes and her personal research interests are related
to the development oI primary/reIerence measurement methods, mainly UV-VIS-IR,
FAAS, ET-AAS, ICP-MS and measurement standards in the feld oI physico-chemical
quantities. She is active in international research networks and in the dissemination
oI knowledge on issues related to metrology in chemistry (uncertainty, traceability,
validation, CRMs, etc.).
She is a member oI the TrainMiC
Management Board and National TrainMiC

team leader Ior Romania.
About the Authors
13
Philip Taylor
(see above)
Chapter 3
Ljudmila Benedik
Ljudmila Benedik started her proIessional career in the feld oI radiochemistry in
1986 when she joined the Department Ior Nuclear Chemistry at JozeI SteIan Institute,
Ljubljana (Slovenia). She fnished PhD study in 1994 at Faculty Ior Chemistry and
Chemical Technology, University oI Ljubljana with thesis Development oI methods Ior
determination oI low-level uranium and thorium by radiochemical neutron activation
analysis.
Her scientifc research work was mainly devoted to research in the feld oI
determination oI trace elements by neutron activation analysis as well as determination
oI natural and man-made radionuclides using alpha, beta and gamma spectrometry
and liquid scintillation technique. She published results oI her research work in over
35 scientifc papers and presented it at numerous international conIerences. She was
involved in various projects connected to measurement oI environmental radioactivity.
She was acting as a mentor Ior undergraduate and postgraduate students.
She has been working at Institute Ior ReIerence Materials and Measurements
oI the Joint Research Centre oI the European Commission where she was involved,
amongst over, in the project NUSIMEP (Nuclear Signature Interlaboratory Measurement
Programme) and in project related to EU drinking water directive.
At the present, she works as a senior research associate at the Department oI
Environmental Sciences, JozeI SteIan Institute.
She is a member oI Slovenian TrainMiC
team.
Urka Repinc
Urska Repinc started her proIessional career as radiochemist at the Department oI
Environmental Sciences, JozeI SteIan Institute, Ljubljana (Slovenia) in 1999. Her
diploma work was devoted to determination oI Ra-226 by liquid scintillation counting.
She continued the work on developing new methods based on alpha, beta spectrometry or
radiochemical neutron activation analysis. In 2006 she fnished her PhD, which consists
oI developed procedures Ior determination oI vanadium, uranium, manganese and cobalt
via induced their short lived nuclides 48V, 239U, 56Mn and 60mCo, respectively. Her
research work was not Iocused only on a single element determination but improved
procedures Ior their simultaneous determination.
In February 2007 she joined the Institute Ior Transuranium Elements oI the Joint
Research Centre oI the European Commission, Karlsruhe, Germany, as a postdoc Iellow.
14
Monika Inkret
Monika Inkret started her career at the Metrology Institute oI the Republic oI Slovenia
in Laboratory Ior Precious Metals.
For six years she has been working aIterwards at the Institute Ior ReIerence Materials
and Measurements oI the Joint Research Centre oI the European Commission. There she
was working on the Avogadro project Ior the redetermination oI the Avogadro constant.
Her main research topic was silicon isotope ratio measurements and silicon molar mass
determination using diIIerent mass spectrometric techniques (thermal ionization and
electron impact).
AIter deIending her doctoral dissertation in March 2008 at the University oI
Ljubljana, mass spectrometry and metrology being the central topic, she worked as
a postdoc at University oI Singapore, Faculty oI Science, in the Laboratory Ior trace
metals determination in Iood and biological samples.
Since May 2009 she has been working in Cinkarna Celje, Celje, Slovenia in R&D
Unit with the research Iocused on production oI nano-titanium dioxide white pigment.
Currently she is employed at Krka, Novo mesto, where she is dealing with the
quality issues related to pharmaceuticals.
She is a member oI Slovenian TrainMiC
team.
Chapter 4
Allan Knnapas
Allan Knnapas received his MSc in 2007 at University oI Tartu, Estonia. In Masters
thesis he studied the implementation oI QuEChERS method in LC-MS in Finnish
Customs Lab (Espoo, Finland). The example 'Determination oI Polar Pesticides by
Liquid Chromatography Mass Spectrometry was compiled in conjunction with his BSc
work involving the developing a pesticide residues analytical method.
He is currently a doctoral student at University oI Tartu and in his doctoral thesis
he will study isotope dilution, matrix eIIect and suppression in LC-MS analysis. He
has been involved in projects concerning Iurther widening pesticide residues analytical
method, implementing QuEChERS in governmental laboratories, analysis oI antibiotics
in sewage sludge. His research interests include chromatographic separation methods,
especially LC and LC-MS, mass spectrometry residues analytical methods, IDMS,
method development, sample preparation.
Koit Herodes
Koit Herodes received his PhD in 2002 at University oI Tartu, Estonia. He has
investigated binary properties oI solvent mixtures by means oI ionic equilibria (at
University oI Barcelona, Spain) and ET(30) parameters. At University oI Freiburg
(Germany) he acquired skills oI synthesizing phosphazene superbases in strictly non-
aqueous conditions.
About the Authors
15
He is currently working as associate proIessor oI analytical chemistry at University
oI Tartu. Together with his students he is developing and validating sample preparation
and liquid chromatographic (LC-MS) analysis methods Ior small molecules in diIIerent
matrices, e.g. pesticide residues in Iood, pharmaceutical residues in sewage sludge,
amino acids in honey, pharmaceuticals in biological fuids etc. More Iundamental
research topics include electrospray ionization process and eIfciency studies and novel
buIIer compounds Ior LC-MS.
At University oI Tartu he is head oI University Testing Centre, a unit that provides
analytical services to industry and other laboratories.
Ivo Leito
ProI. Ivo Leito received his PhD in 1998 at University oI Tartu, Estonia. He has worked
at several universities and research institutes in France, Spain, Japan and Belgium and
is currently working as proIessor oI analytical chemistry at University oI Tartu. His
research interests include experimental and theoretical studies oI acid-base equilibria
and solvent eIIects in non-aqueous solvents and in the gas phase; metrology in chemistry,
measurement uncertainty in chemistry; analytical chemistry (instrumental analytical
methods IR spectroscopy, GC-MS, LC-MS, SEM EDS, etc) applied to analysis oI
materials and objects oI cultural heritage and methodical aspects oI teaching analytical
chemistry and metrology in chemistry (http://tera.chem.ut.ee/~ivo/RD.html). Number
oI international scientifc publications: 65 (all in journals indexed by ISI). He has
supervised 5 PhD students more than 10 MSc and more than 20 BSc students. He is
currently supervising 4 PhD students, 3 MSc students and 1 BSc student. He has set up
and is the program director oI the international Master Curriculum Applied Measurement
Science (http://www.ut.ee/ams) and is the coordinator oI the Euromaster consortium
Measurement Science in Chemistry (http://www.msc-euromaster.eu/).
Chapter 5
Bertil Magnusson
Bertil Magnusson started as a marine chemist looking Ior traces oI metals in the oceans,
rivers, lakes and rain in the 70`s. AIter PhD he joined a chemical company, Eka Chemicals
within AKZO-NOBEL and worked there as a specialist in analytical chemistry mainly
with spectroscopy (XRF, XRD and ICP) and wet chemistry.
In 2002 he joined the SP Technical Research Institute oI Sweden and is currently
working with quality in measurements, Metrology in Chemistry, a research area on
international comparability and traceability oI chemical measurement results. A major
part is teaching and writing guidelines and research papers regarding measurement
quality. Important part oI his work is education Ior analytical laboratories in QA/QC.
In Nordic cooperation he has written a Handbook on Measurement Uncertainty Ior
Environmental Laboratories (Nordtest technical report 537), and a Handbook Ior Internal
16
Quality Control Ior Environmental Laboratories (report 569). He is also working with
primary measurements using Isotope-Dilution ICP-MS, XRF and conductivity.
He is a member oI the TrainMiC
Editorial Board and a team leader oI the Nord/

Baltic TrainMiC
team.
Chapter 1
Analysis of Gold Alloys by Flame Atomic
Absorption Spectrometry
Veselin Kmetov, Emilia Vassileva
TrainMiC example summary form (blue page)
A short introduction to the analytical procedure (slides)
All input needed to do the three exercises (yellow pages)
The solved exercises (green pages)
17
18
TrainMiC example summary form
I. General information about the example
Measurand Mass fraction of Au in gold alloys ()
Example number Ex-06
Authors of the example Veselin Kmetov, Emilia Vassileva
Analytical procedure
Determination of gold in jewellery gold alloys by fame atomic
absorption spectrometry
Customers requirement U = 9 (k = 3)
19
II. Attached fles
File number, type
and name
Content of the fle
File is
attached
Remark
Yes No
1

-

I
Ex-06-1-I-Au-
alloys-FAAS-2006-
Ver1.ppt
About the analytical procedure: short introduction

Given by the
lecturer
2

-

Y
e
l
l
o
w
Ex-06-2-Y-Au-
alloys-FAAS-2006-
Ver1.doc
PART I Description of the analytical procedure

Each
participant
receives own
copy and
may keep it
PART II
The customers requirements
concerning the quality of the
measurement result
PART III
Validation of the measurement
procedure relevant equations and
measurement data
PART IV
Measurement uncertainty of the result
relevant equations and measurement
data
3

-

G
r
e
e
n
EX-06-3-G-Au-
alloys-FAAS-2006-
Ver1.doc
PART I
Establishing traceability in analytical
chemistry

PART II
Single laboratory validation of
measurement procedures

PART III
Building an uncertainty budget

Addendum 1: By spreadsheet approach

Addendum 2: By dedicated software

III. History of the example
Version Uploaded on the webhotel Short description of the change
0 April 2007 -
1
2
20
Practical examples on traceability, measurement uncertainty and validation in chemistry Practical examples on traceability, measurement uncertainty and validation in chemistry
A short introduction to the analytical procedure
Analysis of Gold Alloys by FAAS Analysis of Gold Alloys by FAAS
Scope oI the presentation
The analytical procedure and the customer`s requirements
About the chemistry` and the measurement method
Model equation
The analytical procedure and the
customer`s requirements
The traditional internationally recognised method is based on cupellation (Iire
assay) ISO Standard 1142.
The Iineness oI precious metal alloys are speciIied in ISO 9202:1991,
according to the purity oI gold as : 375, 585, 750 and 916 ( 9, 14, 18 and
22 karats respectively). One karat is equal to 41.667 .
The Au analysis has to keep the expanded uncertainty (k 3) less than 9 .
21
22
Model equation
R
C
G
G
m
J
o W
x
P
vials
1
1000
1

4 . 0
12
1 . 0
50

GoId content (/) in jeweIIery goId aIIoys
23
Some more equations (2)

ref
W
W
R
N
u
u
set one A
A

Calculation of signal standard uncertainty

Recovery correction
Bracketing calibration
Two calibration standard solutions used are
with Au concentration 37 and 43 mg kg
respectively in 5 NH
4
Cl
The selected calibration range corresponds to
the range 13.514.5 kt recalculated Ior
analyzed alloy.
It is proved that the S/N ration is minimum
and the calibration interval belongs to the
linear range
y = 0,014x + 0,0036
R
2
= 1
0,500
0,520
0,540
0,560
0,580
0,600
0,620
36 37 38 39 40 41 42 43 44
Au mg/kg
A
b
s
o
r
b
a
n
c
e
1 2
1 2 2 1
) ( ) (
St St
St x st X St st
A A
A A C A A C
Cx

24
Determination of gold in jewellery gold alloys by Flame Atomic
Absorption Spectrometry
PART I ...................................................................................................................................25
Description of the analytical procedure
PART II .................................................................................................................................33
The customers requirements concerning the quality of the measurement result
PART III ................................................................................................................................34
Validation of the measurement procedure relevant equations and
measurement data
PART IV ................................................................................................................................35
Measurement uncertainty of the result relevant equations and measurement
data
All input needed to do the three exercises yellow pages
25
Task description
The fneness oI jewellery gold alloys depends Irom the Au contend in the used material
that must be strictly marked. This mark is given by the Ministry oI Finances aIter testing
in accredited Ior the scope laboratories. The set oI marks oI precious metal alloys are
specifed in ISO 9202:1991, and are nominated according to the purity Ior gold m/m
as : 375, 585, 750 and 916, which correspond to 9, 14, 18 and 22 carats respectively.
One carat is equal to 41.667 . In a common practice 585 (14 carats) gold
alloys are used Ior jewellery, thereIore such samples are more oIten a subject oI analyses.
Important conclusions and decisions linked to the customer interest protection sphere
derive Irom measurement results that have to be based on reliable data oI good quality
(e.g. suIfciently small uncertainty). The legislation require the actual fneness oI
jewellery alloys shall not be less by more than three one-thousandth parts than the
fneness indicated by the mark stamped. ThereIore the testing method should be able
to provide results with high accuracy and low uncertainty (less than 3 ; k 1) oI
analytical measurements. The calculated expanded uncertainty Ior Au mass Iraction
should be less than 9 (k 3).
The traditional and internationally recognised method Ior gold alloy analysis is based
on the cupellation (fre assay) (ISO Standard 11426). Recently other alternative methods
based on atomic spectrometry have been suggested.
1. ISO/TC 174. rev.N71. Gouda 1992 Determination of gold in gold jewelry
allows ICP solution spectrometric method using yttrium as internal
standard
2. CNR-PRO Art Project (1998) Tecniche spettrometriche alternative alla
copellazione per il saggio delle leghe dioro
Scope
This example describes a laboratory developed method Ior determination oI gold aIter aqua
regia solubilisation and measurements by fame atomic absorption spectrometry (FAAS).
The range oI application is jewellery alloys containing gold 585 + 21 (14 + 0.5) carats.
The procedure is optimised to ft Ior purpose by means oI a system Ior air-segmented
discrete introduction (ASDI) that allows signals to be accumulated without driIt. External
procedure Ior pseudo steady state signal smoothing and ensemble summation is used Ior
bettering the repeatability oI the instrumental perIormance.
The experimental protocol is shown in the Figure 3.
PART I. Description of the analytical procedure
26
Figure 3. Flow chart of the analytical procedure for determination of gold in gold
alloys
27
Reagents
- E. Merck class p.a. 5 v/v HNO
3
; Ireshly prepared aqua regia; NH
4
Cl salt
- Pure Au 99.99 certifed Irom Non-Ferrous Metallurgical Plant Plovdiv
Apparatus
- Flame Atomic Absorption Spectrometer equipped with hollow cathode lamp Ior
gold
- Discrete sample introduction system (ASDI)
- Balance d 0.0001 g (certifed BDS EN 45501)
- Hot plate
- Pipette variable 2001000 L gravimetrically checked (certifed d 0.4 in 200
500 L range)
- Volumetric fask 50 mL (laboratory glassware class A; certifed d 0.02mL Ior
20 C)
- Volumetric fask 100 mL (laboratory glassware class A; certifed d 0.08 mL Ior
20 C)
- Polypropylene vials 12 g (ASDI autosampler kit)
Sample preparation procedure
Gold alloy samples are stretched to Iolio with 0.30.4 mm thickness. The surIace is
washed by 5 v/v HNO
3
. A dry piece oI 0.1 g accurately weighed to +0.0001 g is
directly dissolved into a volumetric fask oI 50 mL by 5 mL Ireshly prepared aqua regia.
The fask is heated on ceramic hot plate Ior 20 min. During this process Ag precipitates
as AgCl. AgCl is dissolved by adding oI 10 g NH
4
Cl to the cooled solution and volume
is made up to the mark (50 mL) with ultrapure water at (20 C).
The solution is diluted additionally by transIerring 0.400 g with micro-pipette to a conical
vial adding 5 NH
4
Cl in order to keep the solution homogeneous with fnal weight oI
12.000 g gravimetrically controlled.
Procedural blank is subject to exactly the same sample preparation procedure as the
analysed sample.
Calibration
Stock standard solution was made in laboratory by dissolution oI 0.1 g Au with purity
99.99 with 5 mL aqua regia and flled up to 100 g with 5 NH
4
Cl. Two calibration
28
standards are obtained in polypropylene vials aIter Iurther dilution oI 0.370 g and 0.430 g
Irom stock standard solution with procedural blank solution to 10.000 g (gravimetrically
controlled).
The selected calibration range, recalculated Ior analysed alloy, corresponds to the range
13.514.5 carats.
The calibration standards are subject to the same sample preparation procedure as the
analysed sample. The exact matching oI sample and solution used Ior calibration allows
to avoid the infuence oI matrix eIIect on obtained signals.
Atomic absorption measurement
Gold is determined by air segmented discrete introduction fame atomic absorption
spectrometry (ASDI-FAAS) using brackets calibration. In order to improve the
repeatability oI absorption measurements, the Iollowing experimental conditions are
respected:
- Working with the best SIGNAL/NOISE ratio according the scedastic curves
(signals near 0.6 absorbance units) and in very narrow concentration interval (37
43 g g
-1
) with linear response according the Beer`s low.
- Removing the driIt by aspiration washing solution between injections and
application oI standard-sample-standard sequence (St
1
sample St
2
)
- Auto zero perIormance beIore every injection
- Applying signal smoothing and ensemble summation.
Instrumental parameters are described in Table 1.
Signals are accumulated in the sampling set (St
1
sample St
2
) by precise time control
(0.1 s) and are smoothed by means oI external data treatment soItware. Signal profles
are summated as ensembles Irom N replicates oI the sampling set Ior the St1, sample and
St2 respectively and Ior each oI them an ensemble pseudo plateau profle is obtained.
The stable plateau part (3 s) oI summated ensembles is used Ior calibration and quantitative
calculations. Standard uncertainty oI the signals repeatability was calculated as standard
deviation oI absorbance measured in the plateau part (3 s).
29
Table 1. Instrumental parameters for ASDI-FAAS determination of Au
FAAS parameters Values ASDI parameters
Au spectral line [nm]
Au spectral slit [nm]
242.8
0.7
Q
l-
aspiration rate 6.4 mL min
-1
checked by BDW
Injection time 5 s; Injection volume 0.530 L
Au hollow cathode lamp current [mA] 10 Washing time 10 s; Total replicate time 15 s
Air/C
2
H
2
units
Observation high [mm]
50/18
6
Smoothing Savitzky-Golay 24 points
Ensemble summation N signal profles
Working range g g
-1
Deuterium BG corrector
3743
OFF
Pseudo plateau 3 s
Sampling mode (St
1
_ sample _ St
2
) N
Readings points [s] 50 Total time for one set 66 s
30
Calculations
Concentration of initial standard solution made up from pure gold
C
m Au
G
Au
pureAu purity
_ .
_ _
_
9
100
99 9
4
10

C
Au _ 999.9 concentration oI initial standard solution made up Irom pure gold |g g
-1
|
m
pureAu _ mass weighed oI pure gold |g|
G
100
mass oI the solution in the volumetric fask made up to 100 g with 5
NH
4
Cl |g|
Au
purity the purity oI gold stated in the certifcate ||
10
4

conversion Iactor Irom to g g
-1
; p - 1 equalised Ior standard and
samples in 5 NH
4
Cl
Concentration of calibration standard solutions
C C
G
G
St Au
_
_
1

_999.9
_0.37
100

C C
G
G
St Au
_
_
2

_999.9
_0.43
100
C
St
_
1
C
St
_
2
Concentration oI Au working standard solutions |g g
-1
|:
C
St1
Ior low (37 g g
-1
) and C
St2
Ior high (43 g g
-1
)
C
Au_999.9
Concentration oI Au standard solution Au 999.9 |g g
-1
| prepared Irom
pure gold
G
_0.37
G
_0.43
Masses oI the initial Au standard solution transIerred Ior the preparation
oI calibration solutions C
St1
(37 g g
-1
) or C
St2
(43 g g
-1
) |g|: 0.370 g or
0.430 g respectively
G
_100
Mass oI gravimetrically controlled calibration standard solutions aIter
adding 5 NH
4
Cl in polypropylene vials |g|
31
Bracketing calibration
Cx
C A A C A A
A A
st St X St x St
St St

1 2 2 1
2 1
( _ ) ( )
_
Cx
Concentration oI Au in the analysed solution |g g
-1
|
C
St 1
Concentration oI the lower calibration standard solution used Ior
bracketing calibration |g g
-1
|
C
St 2
Concentration oI the higher calibration standard solution used Ior
bracketing calibration |g g
-1
|
A
St 1
Absorbance measured Ior the lower calibration standard solution C
St1
A
St _ 2
Absorbance measured Ior the higher calibration standard solution C
St2
A
X _
Absorbance measured Ior the analysed sample solution
Calculation of Au mass fraction (W_) in analysed sample
W

V
m R
G
G
C
vials
P
x
_
_
_ . _ ,

1
1000
1
50
0 1
12
0 4
W fnal concentration oI Au in tested jewellery gold alloy w/w ||
V
_ 50
volume oI the solution in the volumetric fask |mL|
m
_ . 0 1
mass oI analysed alloy sample |g|
G
vials
_
12
weight oI fnal sample solution prepared in vials |g|
G
P
_
. 0 4
mass oI Au sample solution taken Irom V
50
|g|
R correction Ior recovery
Combined model equation for calculation of Au content ()
W
V
m
G
G
C
G
G
vials
P
P _
_ _
.
_
.
Au_999.9
_10
_

_
,

1
1000
50
0 1
12
0 4
00.37 _0.43
( _ ) ( )
_
A A G A A
A A R
St X P X St
St St
2 1
2 1
1
+
( )

32
Calculation of signal standard uncertainty estimated as standard deviation
u
u
N
A
A one set
_
_ _
_
u
A _
calculated standard uncertainty Irom the plateau part (3 s) oI
the absorbance signal aIter ensemble averaging oI N sets oI sampling
(St
1
sample St
2
)
u
A one set _ _
_
calculated standard uncertainty Irom the plateau part (3 s) oI the
absorbance signal obtained Irom one set oI sampling (St
1
sample
St
2
)
N number oI sets perIormed and summated as ensemble
33
Expanded measurement uncertainty: 9 (k 3)
PART II. The customers requirements concerning quality of the
measurement result
34
The procedure has been developed in the laboratory, thus a Iull validation must be
perIormed.
However, Ior the purposes oI this exercise, recovery (R) and repeatability will be
calculated only.
1. Equations
See Part I
2. Measurement data
Recovery:
Cupellation method: 585.1
ADI-FAAS: 583.5
Repeatability:
586.48
582.32
581.68
583.82
585.88
580.56
PART III. Validation of the measurement procedure relevant
equations and measurement data
35
Calculate combined and expanded uncertainty (k 3) Irom the Iollowing
measurement data:
Input
quantity
Value Unit
Standard
uncertainty
Remark
V
_ 50
50 mL 0.0379 Volume of analysed solution
V
_100
100 mL 0.0697 Volume of stock standard solution
m
_0.1
0.1001 g 0.0002 Mass of analysed alloy sample
G
vials
_
12
12.0030 g 0.0008 Mass of sample solution prepared in vials
G
P_ . 0 4
0.4015 g 0.0009
Mass of Au sample solution taken from V
_50

fask
m
pureAu _
0.1004 g 0.0002 Mass weighed of pure gold
Au
purity _
99.99 % 0.0058 The purity of gold stated in the certifcate
G
p_0.37
G
p_0.43
0.3701
0.4302
g 0.0006
Masses of the stock Au standard solution
transferred for the preparation of calibration
solutions C
_St1
and C
_St2

G
_10
10.0321 g 0.0008 Mass of calibration standard solutions
A
St 1
A
St _ 2
0.5203
0.6041
AU
0.0010
0.0011
Absorbance measured for calibration
standard solutions
A
X
0.5488 AU 0.0011
Absorbance measured for the analysed
sample solution
R 1.002 - 0.0025 Recovery
PART IV. Measurement uncertainty of the result relevant
36
TrainMiC Exercises
Determination of gold in jewellery gold alloys by fame atomic
absorption spectrometry
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:
Addendum I: By spreadsheet approach
Addendum II: By dedicated software
The solved exercises green pages
37
1. Specifying the analyte and measurand
Analyte Gold
Measurand Gold mass fraction in jewellery alloys after aqua regia dissolution
Units (g/1000 g)
2. Choosing a suitable measurement procedure with associated model equation
Measurement
procedure
Type of calibration standard curve standard addition internal standard
Model equation
1. Standard solutions
1.1. Stock standard solution - prepared from pure gold
C
m Au
G
Au
pureAu purity
_999.9
100
_
_
10
4
1.2. Calibration standard solutions
C C
G
G
St Au
p
_
_
1
100

_999.9
_0.37

C C
G
G
St Au
p
_
_
2
100

_999.9
_0.43
2. Bracketing calibration
Cx
C A A C A A
A A
St St X St x St
St St

1 2 2 1
2 1
( ) ( )
_ _
3. Calculation of Au content (W_) in analysed sample
W
V
m
G
G
C
R
vials
P
x
_
_
_
_ . _ .
0
1
1000
1
50
0 1
12
0 4
4. Calculation of signal standard uncertainty
u
u
N
A
A one set
_
_ _
_
ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

EXERCISE
38
5. Calculation of recovery
R
W
W
observed
ref
6. Combined model equation for calculation of Au mass fraction ()

W
V
m
G
G
m Au
vials
P
pureAu purity _
_ .
_
_ .

_
,
1
1000
50
0 1
12
0 4
GG V
_100 100

_
10
4

G A A G A A
A
P St X P X St _0.37 _0.43
+
( )

( _ ) ( )
_ 2 1
SSt St
A R
2 1
1
V _
50
volume oI analysed solution |mL|
V _
100
volume oI stock standard solution |mL|
m
_ . 0 1 mass oI analysed alloy sample |g|
G
vials
_
12
mass oI sample solution diluted in vials |g|
G
P
_
. 0 4
mass oI Au sample solution taken Irom V50 fask |g|
m
pureAu _
mass weighed oI pure gold |g|
Au
purity _
the purity oI gold stated in the certifcate ||
G or G
p p _0.37 _0.43
masses oI the stock Au standard solution transIerred Ior the
preparation oI calibration solutions St
1
and St
2
|g|
G
_100
mass oI calibration standard solutions |g|
A
St 1

and
A
St _ 2
absorbance measured Ior calibration standard solutions 1 and 2
A
X
absorbance measured Ior the analysed sample solution
R recovery
3. List the input quantities according to their infuence on the uncertainty of the
result of the measurement (frst the most important ones). At this point, your
judgement should be based on your previous experience only.
1 Recovery 28.5 % to the expanded uncertainty
2 Absorption of analysed gold sample contributing 19.8 % to the expanded uncertainty
3 Mass of analysed gold sample contributing 11.8 % to the expanded uncertainty
39
4
Mass of stock solution taken for the preparation of frst standard solution contributing 12.1 % to the
expanded uncertainty
5 Volume of the analysed solution contributing 3.4 % to the expanded uncertainty
4. List the reference standards needed and give also the information regarding
traceability of the reference value
For the analyte
1 Name/Chemical Formula/Producer:
Pure Gold certifed by Non-Ferrous Metallurgical
Plant Plovdiv Bulgaria
For the other input quantities
1
Quantity/Equipment/Calibration:
e.g. mass/balance/calibrated by NMI, U = xx
(k = 2), see also data yellow sheet
Balance calibrated by NMI
2 Quantity/Equipment/Calibration: Volumetric fask class A quality
3 Quantity/Equipment/Calibration:
Absorbance relative measurement. Not direct part of
the traceability chain.
5. Estimating uncertainty associated with the measurement
Are all important parameters included in the
measurement equation?
Yes No
Other important parameters are: Within-lab reproducibility
6. How would you prove traceability of your result?
1 Comparing the results with independent method (cupellation)
40
7. Any other comments, questions
41
PART I: GENERAL ISSUES
1. Specify the measurement procedure, analyte, measurand and units
The measurement procedure Analysis of gold alloys by AAS
Analyte Gold
The measurand
Gold in jewellery alloys containing gold 14 0.5 carats after aqua
regia dissolution
Unit
2. Specify the Scope
Matrix Gold in 5 % NH4Cl
Measuring range 37-43 g g
-1
3. Requirement on the measurement procedure
Intended use of the results: Quality of products from precious metals alloys
Mark the customers requirements
and give their values
LOD
LOQ
Repeatability
Within-lab reproducibility
Measurement uncertainty 9
Trueness
Other-state
4. Origin of the measurement procedure
VALIDATION
New in-house method Full
Modifed validated method Partial
Ofcial standard method Confrmation/Verifcation
SINGLE LABORATORY VALIDATION
OF MEASUREMENT PROCEDURES
EXERCISE
42
PART II: PARAMETERS TO BE VALIDATED
5. Selectivity/Interference/Recovery
Where yes, please give further information e.g. which CRM, reference method
CRM/RM: analysis of available CRM or RM
Further information:
Spike of pure substance
Pure gold 99.99 % certifed from non-ferrous metallurgical plant Plovdiv, Bulgaria
Compare with a reference method
Comparison with cupellation method
Selectivity, interferences
Test with diferent matrices
Other please specify
Test for recovery with RM jewellery gold alloy marked 585
6. Measuring range
Linearity
Upper limit
LOD
LOQ
7. Spread precision
Repeatability
Reproducibility (within lab)
Reproducibility (between lab)
8. Robustness
Variation of parameters
43
9. Quality control
Control charts
Participation in PT schemes
10. Other parameters to be tested
Working range and testing of homogeneity of variances
Recovery
Residual standard deviation
Standard deviation of the method
Coefcient of variation of the method
44
11. Calculation of parameters requested by the customer
Parameters requested to be
validated
Calculations
LOD
LOQ
Repeatability 2.4
Within-lab reproducibilty
Trueness
Measurement uncertainty 8.3 (k = 3)
Other - please state
Recovery
1.0002 0.0025
12. Does the analytical procedure fulfl the requirement(s) for the intended use?
Parameter
Value requested by the
customer
(the same as stated in question3)
Value obtained
during
validation
The requirement
is fulflled
Yes/No
LOD
LOQ
Repeatability
Within-lab
reproducibility
Trueness
Measurement
uncertainty
9 (k = 3) 8.3 (k = 3) yes
Other
The analytical procedure is ft for the intended use:
Yes No
For measurement uncertainty and traceability refer to the corresponding report-
sheets
PART III: SOME CALCULATIONS AND CONCLUSIONS
45
BUILDING AN UNCERTAINTY BUDGET
EXERCISE
1. Specify the measurand and units
Measurand Gold mass fraction in jewellery alloys after aqua regia dissolution
Unit (g/1000 g)
2. Describe the measurement procedure and provide the associated model
equation
Measurement procedure:
Gold alloy samples are stretched to Iolio with 0.30.4 mm thickness. The surIace is
washed by 5 v/v HNO
3
. A dry piece oI 0.1 g accurately weighed to 0.0001 g is
directly dissolved into a volumetric fask oI 50 mL by 5mL Ireshly prepared aqua regia.
The fask is heated on ceramic hot plate Ior 20 min. During this process Ag precipitates
as AgCl. AgCl is dissolved by adding oI 10 g NH
4
Cl to the cooled solution and volume
is made up to the mark (50 mL) with BDW at (20 C).
The solution is diluted additionally by transIerring 0.400 mL with micro-pipette to a
conical vial adding 5 NH
4
Cl in order to keep the solution homogeneous with fnal
weight oI 12.000 g gravimetrically controlled.
Procedural blank and gold reIerence material are subject to exactly the same sample
preparation and measurement procedures as the analysed sample.
Model equation:
1. Concentration of initial standard solution made up from pure gold
C
m Au
G
Au
pureAu purity
9
100
99 9
4
10
.
_

C C
G
G
St Au
_
_
2

_999.9
_0.43
100
2. Concentration of calibration standard solutions
C C
G
G
St Au
_
_
1

_999.9
_0.37
100
3. Bracketing calibration
C
C A A C A A
A A
x
St St X St x St
St St

1 2 2 1
2 1
( ) ( )
_ _
4. Calculation of Au mass fraction (W_) in analysed sample
W
V
m R
G
G
C
vials
P
x
_
_ .
_
_ .

1
1000
1
50
0 1
12
0 4
46
5. Calculation of signal standard uncertainty
u
u
N
A
A one set
_
_ _
_
6. Calculation of recovery
R
W
W
observed
ref
7. Combined model equation for calculation of Au mass fraction ()

W

V
m
G
G
C
G
G
vials
P
P _
_ _
.
_
.
Au_999.9
_100

_
,

1
1000
50
0 1
12
0 4
__0.37 _0.43
( _ ) ( )
_
A A G A A
A A R
St X P X St
St St
2 1
2 1
1
+
( )

3. Identify (all possible) sources of uncertainty
Uncertainty of concentration of reference solutions
Uncertainty of measurements of absorption of standard and sample solutions
Mass of analysed gold sample
Volume of the analysed solution
Recovery
Other:
Other:
4. Evaluate values of each input quantity
Input quantity Value Unit Remark
V _
50
50 mL Volume of analysed solution
V _
100
100 mL Volume of stock standard solution
m
_ . 0 1
0.1001 g Mass of analysed alloy sample
G
vials
_
12
12.0030 g Mass of sample solution prepared in vials
G
P _ . 0 4
0.4015 g Mass of Au sample solution taken from V
_50
fask
m
pureAu
0.1004 g Mass weighed of pure gold
Au
purity _
99.99 % The purity of gold stated in the certifcate
G G
p p _0.37 _0.43
;
0.3701; 0.4302 g
solutions C
_St1
and C
_St2

47
G
_100
10.0321 AU Mass of calibration standard solutions
A
St 1
;
A
St _ 2
0.5203; 0.6041 AU
Absorbance measured for calibration standard
solutions
A
X
0.5488 AU
Absorbance measured for the analysed sample
solution
R 1.002 - Recovery
5. Evaluate the standard uncertainty of each input quantity
Input quantity
Standard
uncertainty
Unit Remark
V _
50
0.0379 mL Volume of analysed solution
V _
100
0.0697 mL Volume of stock standard solution
m
_ . 0 1
0.0002 g Mass of analysed alloy sample
G
vials
_
12
0.0008 g Mass of sample solution prepared in vials
G
P _ . 0 4
0.0009 g Mass of Au sample solution taken from V
_50
fask
m
pureAu
0.0002 g Mass weighed of pure gold
Au
purity _
0.0058 % The purity of gold stated in the certifcate
G G
p p _0.37 _0.43
;
0.0006; 0.0006 g
solutions C
_St1
and C
_St2

G
_10
0.0008 g Mass of calibration standard solutions
A
St 1
;
A
St _ 2
0.0010; 0.0011 AU
Absorbance measured for calibration standard
solutions
A
X
0.0011 AU
Absorbance measured for the analysed sample
solution
R 0.0025 Recovery
6. Calculate the value of the measurand, using the model equation
583.5
7. Calculate the combined standard uncertainty (u
c
) of the result and specify units
Using: Mathematical solution; Spreadsheet approach; Commercial soItware
48
Input
quantity
Value
Standard
uncertainty
Unit Remark
W_ 583.5 2.8 Au mass fraction in jewellery alloys
8. Calculate expanded uncertainty (U
c
) and specify the coverage factor k and the
units
8.4 (k 3)
9. Analyse the uncertainty contribution and specify the main three input quantities
contributing the most to U
c
1 Recovery contributing 37.6 % to the expanded uncertainty
2 Absorption of analysed gold sample contributing 26.1 % to the expanded uncertainty
3 Mass of analysed gold sample contributing 14.9 % to the expanded uncertainty
10. Prepare your uncertainty budget report
(583.5 + 8.4) (k 3)*
(*) the reported uncertainty is an expanded uncertainty calculated using a coverage Iactor oI
k 3, which gives a level oI confdence oI approximately 99.7
49
Further readings
1. ISO 9202:1991/Jewellery Fineness oI precious metal alloys.
2. CIBJO/Precious metals commission, The Precious Metals Book (2008-4-22).
3. ISO 11426:1997/Determination oI gold in gold jewellery alloys. Cupellation method
(fre assay).
4. ISO 15093:2008/Jewellery Determination oI precious metals in 999
o
gold,
platinum and palladium jewellery alloys. DiIIerence method using inductively coupled
plasma optical emission spectroscopy (ICP-OES).
5. R. Mauri, E. Huerta and M. de la Guardia, Flame atomic absorption analysis oI gold
in jewelry samples, Fresenius J. Anal. Chem. 338 (1990), 699702.
6. M. Mooiman, D.J. Kinneberg, L.W. Simpson, P. Cettou and P. Ramoni, High purity
precious metals, TMS Annual Meeting (1998), 141163.
7. I. Erusalimshik and S. Moiseev, Electrochemical and corrosion properties oI gold and
its alloys: VI. Jewelry gold alloys oI 375 purity standard in hydrochloric acid solutions,
Protect. Metals 32(6) (1996), 612613.
8. G. Galbacs, N. Jedlinszki, G. Cseh, Z. Galbacs and L. Turi, Accurate quantitative
analysis oI gold alloys using multi-pulse laser induced breakdown spectroscopy and
a correlation-based calibration method, Spectrochim. Acta B Atom. Spectrosc. 63(5)
(2008), 591597.
9. A. Marucco, C. Marcolli and R. Magarini, ICP-OES analysis oI gold alloys using
yttrium or indium as internal standard, Atom. Spectrosc. 20(4) (1999), 134141.
10. V. SteIanova, V. Kmetov and L. Futekov, Air segmented discrete introduction in
inductively coupled plasma mass spectrometry, J. Anal. Atom. Spectrom. 12 (1997),
1271 1276.
11. V. Kmetov, D. Hristozov, V. SteIanova, S. Tenev and L. Futekov, Computer automated
system Ior micro-sampling in FAAS, soItware Ior signal acquisition and treatment,
University oI Plovdiv 'P. Hilendarski, Scientic Works-Chem. 30(5) (2001), 5762.
12. V. Kmetov, V. SteIanova, D. Georgieva and L. Futekov, Analysis oI jewellery gold
alloys by micro-sampling oI complex solutions into FAAS and ICP-MS, in: Forth
National Conference of Chemistry Soa (2729 September, 2001), 4P9.
13. J. Kragten, Calculating standard deviations and confdence intervals with a universally
applicable spreadsheet technique, Analyst 119 (1994), 21612165.
50
Addendum I. Measurement uncertainty calculation:
spreadsheet approach (Excel)
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7
G
p
_
0
,
4
3
A
_
S
t
2
A
_
x
A
_
S
t
1
1
0
0
0
R
5
6
2
.
5

6
0
4
.
2

4
1
,
6
/
2
.
6
%
1
1
.
8
%
0
.
0
%
2
2
.
2
%
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.
1
%
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.
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4
.
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%
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.
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.
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%
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9
.
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%
8
.
0
%
0
.
0
%
2
8
.
5
%
R
e
s
u
I
t
k
=
3
T
a
r
g
e
t

U
S
U
M
U
=
8
.
3
/
9

7
.
6
5
3
2
M
a
i
n

c
o
n
t
r
u
b
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r
s

t
o

c
o
m
b
a
i
n
e
d

u
n
c
e
r
t
a
i
n
t
y
,
%
R
A
A
A
A
G
A
A
G
G
C
G
G
m J
o
W
S
t
S
t
S
t
X
P
X
S
t
P
P
v
i
a
l
s
1
)
(
)
(
*
*
4
,
0
1
2
*
1
,
0
5
0
1
0
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,
4
3
2
0
,
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7
1
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A
u
9
9
9
,
9

G
p
_
0
,
3
7
4
%
G
p
_
0
,
4
3
1
%
A
_
x
2
0
%
A
_
S
t
1
8
%
1
0
0
0
0
%
R
2
8
%
A
_
S
t
2
2
% m
_
0
,
1
1
2
%
V
_
5
0
3
%
V
_
5
0
m
_
0
,
1
G
v
i
a
l
s
_
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p
_
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_
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_
9
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_
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p
_
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7
G
p
_
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,
4
3
A
_
S
t
2
A
_
x
A
_
S
t
1
1
0
0
0
R
51
Chapter 2
Determination of Calcium in Serum by
Spectrophotometry
Steluta Duta, Philip Taylor
52
Measurand Concentration of calcium in human serum (mg dL
-1
)
Authors of the example Steluta Duta, Philip Taylor
Analytical procedure Standard WHO procedure
Customers requirement Standard WHO procedure
53
II. Attached fles
File number, type
and name
Content of the fle
File is
attached
Remark
Yes No
1

-

I
Ex-10-1-I-
Ca-serum-
Photometry-
2006-Ver1.ppt

Given by the
lecturer
2

-

Y
e
l
l
o
w
Ex-10-2-Y-
Ca-serum-
Photometry-
2006-Ver1.doc

Each
participant
receives own
copy and
may keep it
PART II
The customers requirements concerning
the quality of the measurement result

PART
III
Validation of the measurement procedure
relevant equations and measurement data

PART
IV
relevant equations and measurement data

3

-

G
r
e
e
nEx-10-3-G-
Ca-serum-
Photometry-
2006-Ver1.doc
PART I
chemistry

PART II

PART
III
Bulding an uncertainty budget



0 April 2007
1
54
Determination of calcium in serum
by (spectro)photometry
Scope of the presentation
The analytical procedure and the customer's requirements
About 'the chemistry' and the measurement aspects
Model equation and more about traceability, validation and
uncertainty exercises
Analytical procedure:
Guideline on Standard Operating Procedure for Clinical Chemistry:
Calcium-O-Cresolphtalein complexone method (Standard Methods)
http://w3.whosea.org
Customer's requirements:
Analytical reproducibility (CV, %): 8 % (WHO); 2 % (actual
state-of-art)
ntended use: clinical interpretation
55
prepare buffer reagents (pH = 10.7) and
colour reagents
Experimental protocol
prepare Ca stock solution calibrants
Sample = serum (no sample treatment)
MX: water+std+colour reag (+sample)
incubate at 25 C for 15 min
Calibration, check linearity
blank to zero
interpolation One point calibration
check QC & between days precision
56
Determination of concentration of calcium in serum by
molecular absorption spectrometry.
The quality of the results should comply with the requirements
in the WHO procedure
PART I ...................................................................................................................................57
PART II .................................................................................................................................60
PART III ................................................................................................................................61
measurement data
PART IV ................................................................................................................................62
data
57
Laboratory task
Clinical laboratory has to determine calcium concentration in human serum sample
ariving in their laboratory. It is the case when only the analitical part is considered, the
laboratory has not responsibility how sample is taken, prepared, transported.
The laboratory should evaluate the analytical procedure reliability (within laboratory
reproducibility). The reported results should comply with the clinical interpretation: the
expected normal range oI calcium concentration in serum is 8.510.4 mg dL
-1
.
Principle of the measurement method
Text extract Irom World Health Organization (WHO) Standard Operating Procedures
Ior Clinical Chemistry: Determination oI calcium concentration by calcium-O-
cresolphthalein complexone method (http://w3.whosea.org).
Determination oI calcium in human serum is perIormed by molecular absorption (spectro)
photometry method. Calcium Iorms a purple-coloured complex with orto-cresolphthalein
complexone in an alkaline medium. The inclusion oI HCl helps to release calcium
bound to proteins, and 8-hydroxy-quinoline eliminates the interIerence by magnesium.
Additional reagents as 2-amino,2-methyl,1-propanol (AMP) provides the proper alkaline
medium Ior the colour reaction. The intensity oI the colour is measured at 540 nm.
Serum sample preparation and storage
No serum preparation is perIormed by the laboratory in charge with the analytical work.
Serum sample arives in the laboratory aIter separation Irom the blood cells during
the preanalytical step perIormed by another department. Haemolysed and heparinised
samples are unsuitable Ior this method.
Calcium in serum is stable Ior 12 h at room temperature (2535 C), one week at 28 C
and Ior a longer period up to 3 months at 20 C.
Reagents
AMP BuIIer pH 10.7
In 37.8 mL oI AMP reagent add 150 mL oI distilled water and mix. Adjust the pH to 10.7
with HCl 6N and make up to 250 mL with distilled water. Store in the reIrigerator in a
brown coloured glass bottle. Stable Ior three weeks.
58
Colour reagents
Add 15 mL concentrated HCl to a 250 mL volumetric fask containing about 25 mL
oI distilled water. TransIer 25 mg O-cresolphthalein complexone power into it, mix to
dissolve. Then add 250 mg oI 8-hydroxy-quinoline, dissolve and than make up to 250
mL with distilled water. Store in a brown coloured glass bottle at room temperature (25
35 C). Stable Ior about one month.
Calcium standard solutions
Stock calcium standard solution
Calcium carbonate is dried at 100 C Ior 2 h. Allow to cool in a desicator. Dissolve 625
mg oI dried calcium carbonate in 50 mL oI distilled water taken in a 500 mL volumetric
fask and add 3.5 mL HCl conc. Mix to dissolve and make up to 500 mL with distilled
water. Store in brown bottle at room temperature (2535 C). Stable Ior 6 months. The
calcium concentration in this solution is 50 mg dL
-1
.
Calibration calcium standard solutions
The calibration calcium standard solutions are prepared by dilution oI stock calcium
standard solution: into Iour 100 mL volumetric fasks transIer 10, 15, 20 and 25 mL oI
stock calcium standard solution and dilute each to 100 mL with benzoic acid. The working
standards contain S5/5, S7.5/7.5, S10/10 and S12.5/12.5 mg dL
-1
calcium, respectively.
Store in brown bottles at room temperature (2535 C). Stable Ior 2 months.
Instrumentation
A photometer or spectrophotometer is used in the visible range, in text it is called (spectro)
photometer. The instrumental perIormances: spectral range: 190850 nm; absorbance
accuracy: 0.003 at 0.1 A. The instrument has the absorbance scale, as a concequence Ior
concentration measurements the calibration graph should be established by laboratory
itselI.
Experimental protocol
The experimental steps oI the measurement procedure are described in the table bellow:
a defned volumes oI calibration solutions and serum sample (0.1 mL) are mixed with
2.0 mL oI colour reagent. Mixed than with 2 mL oI buIIer solution.
Blank S5 S7.5 S10 S12.5 Serum QC
Distilled water (mL) 0.1 - - - - - -
Standard (mL) - 0.1 0.1 0.1 0.1 - -
Serum/QC (mL) - - - - - 0.1 0.1
59
Colour reagent (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Mix well
Bufer solution (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Mix well
AIter 15 min incubation at room temperature (2535 C), the absorbance is measured at
540 nm against distilated water as procedural blank. By plotting the absorbance oI the
standards against their respective concentrations, the calibration graph is plotted. Once
linearity is proved, it is just enough iI a single standard as S10 (10 mg dL
-1
) is used to
determine the calcium concentration in the sample.
The measurable range with this graph is Irom 1.0 to 12.0 mg dL
-1
. It is advisable to plot
a calibration graph whenever the reagents are Ireshly prepared.
Calculation of result
The Iollowing equation is indicated in WHO procedure:
c A A
Ca x
( )
10
10 |mg dL
-1
|
where: c
Ca
concentation oI calcium in serum sample
A
x
absorbance oI serum sample
A
10
absorbance oI the calibration solution (10 mg dL
-1
)
Analytical reliability
Include one internal quality control sample (QC) in every batch oI samples analysed
each day irrespective oI the number oI samples in a batch. Since calcium is analysed
single batch in a day in an intermediate laboratory, it will not be possible to analyse
several QC samples and calculate within-day precision. However even iI only a single
QC sample is analysed in a day, this value can be pooled with the preceding 10 or 20
values obtained in the previous days and between-day precision can be calculated and
express as CV. Ensure that this is well within the acceptable limit (i.e. 8 , actual
perIormance even 2 ).
At least once a week analyse another QC serum Irom either a low or high QC pool.
60
Clinical interpretation:
1
Calcium concentration in serum: 8.510.4 mg dL
-1
normal range
Calcium concentration in serum: 12.516.1 mg dL
-1
pathological range

* World Health Organization
measurement result according to WHO
*

61
Within-laboratory reproducibility (between day precision)
Model equation
CoeIfcient oI variation (CV)
CV
c c
n n
c
i obs QC
QC
( )
( )
,
2
1
5
1
100
CV coeIfcient as variation ||
c
obs
observed calcium concentration in QC serum in ith day (i 15) |mg dL
-1
|
c
QC
target calcium concentration in QC serum |mg dL
-1
|
n number oI reproducible measurements
Measurement data
Input quantity
Value standard deviation
(3 replicates)
Mean value
standard deviation Unit
c
i,obs
(i = 15) day
3 replicates/day
1
st
day: 9.280 0.021
9.16 0.05 mg dL
-1
2
nd
day: 8.990 0.057
3
rd
day: 9.210 0.105
4
th
day: 9.230 0.086
5
th
day: 9.110 0.120
c
QC
8.2410.52
9.38 0.38
mg dL
-1
n 5 no units
CV = 1.27
62
IV.1. Preparation of standard solutions
2
IV.1.1 Preparation of calcium stock standard solution, c
stock
c m M P V M
stock Ca CaCO
( )
( )
100
500
3
/
c
stock
concentration oI calcium stock solution |mg dL
-1
|
m mass oI CaCO
3
|mg|
M
Ca
molar mass oI calcium |g mol
-1
|
P purity oI CaCO
3
|mass Iraction|
V
500
fnal volume oI calcium stock solution |mL|
M
CaCO3
molar mass oI CaCO
3
|g mol
-1
|
Measurement data
Input quantity Value Standard uncertainty Unit
m 625.0 0.2 mg
M
Ca
40.078 0.002 g mol
-1
P 0.9999 0.0058 mass fraction
V
500
500.00 0.15 mL
M
CaCO3
100.0869 0.0024 g mol
-1
IV.1.2 Preparation of calibration standard solutions, c
i
:
c c
V
V
i stock
i

_
,
100
c
stock
Concentration oI calcium stock solution |mg dL
-1
|
V
i
Intake stock solution Ior dilution (V
i
V
-20
corresp. to c
-10
) |mL|
V
100
Volume oI calibration solution |mL|
where V
i
V
-10
, V
-15
, V
-20
, V
-25
corresponding to c
i
= c
-5
, c
-7.5
, c
-10
, c
-12.5
2
Here you may also want to include the relevant certifcates.
PART IV. Measurement uncertainty of the result: relevant
2
63
Measurement data:
c
stock
50.05 0.02 mg dL
-1
V
i
20.000 0.043 mL
V
100
100.000 0.058 mL
IV.2 Calibration one point calibration
Model equation (c
-10
the point oI calibration standard solution):
c c A A A A
x x blank blank

( )
( )
10 10
/
c
x
Concentration oI serum sample Irom calibration data |mg dL
-1
|
c
-10
Concentration oI calcium calibration solution (10 mg dL
-1
) |mg dL
-1
|
A
x
Absorbance corresponding to serum sample
A
-10
Absorbance corresponding to calibration solution (10 mg dL
-1
)
A
blank
Absorbance corresponding to blank solution
Measurement data:
c_
10
10.000 0.023 mg dL
-1
A
x
0.323 0.004 no units
A
-10
0.338 0.002 no units
A
blank
0.052 0.004 no units
IV.3 Calculation of calcium concentration in serum sample
c c
V
V
Ca x
f

_
,
int
c
Ca
calcium concentration in serum sample |mg dL
-1
|
c
x
concentration oI serum sample Irom calibration data |mg dL
-1
|
V
I
volume oI serum sample under investigation |mL|
V
int
intake volume Irom serum sample |mL|
Measurement data:
c
x
9.486 0.303 mg dL
-1
V
f
0.100 0.002 mL
V
int
0.100 0.002 mL
64
TrainMiC Exercises
Determination of calcium concentration in human serum by
molecular absorbtion (spectro)photometry
The quality of results should comply with WHO procedure
requirements
EXERCISE 1:
EXERCISE 2:
EXERCISE 3:
65
Analyte Calcium
Measurand Total concentration of calcium in human serum
Units mg dL-1
Measurement
procedure
To determine the calcium concentration in human serum, a serum sub-sample is
mixed with reagent colour and bufer solution, according to WHO standard operation
procedure. The absorbance of calcium calibration solutions and serum sample are
measured by visible spectrophotometry at 540 nm. From the calibration data the
concentration of calcium in human serum is calculated.
Model equation: calcium concentration in serum
c m M P V M V V A A
Ca Ca CaCO i x blank
( )
( )
1
]
( ) ( ) 100
500 100
3
/ / / AA A
V
V
blank
f
( )
1
]

_
,
10
int
c
Ca
total calcium concentration in serum sample |mg dL
-1
|
M mass oI CaCO
3
|mg|
M
Ca
-1
|
P purity oI CaCO
3
|mass Iraction|
V
500
fnal volume oI calcium stock standard solution |mL|
M
CaCO3
molar mass oI CaCO
3
|g mol
-1
|
V
i
intake stock solution Ior dilution (V
i
= V
-20
corresp. to c
-10
) |mL|
V
100
fnal volume oI calibration solution |mL|
A
x
absorbance corresponding to serum sample
A
-10
absorbance corresponding to calibration solution (10 mg dL
-1
)
A
blank
absorbance corresponding to blank
V
I
V
int
66
1 Matrix efect - recovery
2 Instrumental signal (absorbance)
3 Concentration of standard solutions - purity of CaCO
3
4 Volume of the glassware (pipettes, volumetric fasks)
5 Mass
4. List the reference standards needed and state the information regarding
For the analyte
1 Name/Chemical Formula/Producer: CaCO
3
purity, Merck, min. 99.99 %
2 Name/Chemical Formula/Producer: CaCO
3
molar masses/IUPAC
1
e.g. mass/balance/calibrated by NMI, U=xx
Absorbance/(Spectro)photometer/Calibrated against
traceable optical standard (i.e. PTB)
Volume/Laboratory glassware (pipettes, volumetric
fasks/calibrated by manufacturer (i.e. Hirschmann
Laborgerate )
Mass/Analytical balance/calibrated by manufacturer
against traceable mass standards
Are all important parameters included
in the model equation?
Yes No
Other important parameters are: Matrix efect
67
1 Via traceable calibration data
2 Via traceable volumetric measurements
3 Via traceable mass measurements
68
The measurement procedure
To determine the calcium concentration in human serum, a serum
sub-sample is mixed with reagent colour and bufer solution,
according to WHO standard operation procedure. The absorbance
of calcium calibration solutions and serum sample are measured by
visible spectrophotometry at 540 nm. From the calibration data the
concentration of calcium in human serum is calculated.
Analyte Calcium
The measurand Total calcium concentration in human serum
Unit mg dL
-1
2. Specify the scope
Matrix Human serum
Measuring range 1.012.0 mg dL
-1
Intended use of the results
Calcium concentration in serum result is intended to be used for clinical
interpretation
Mark the customers
requirements and give
their values
Parameters to be validated Value requested by the customer
LOD
LOQ
Repeatability
8 % as CV, by WHO procedure
2 % as CV, the actual state-of-art
Trueness
Measurement
uncertainty
Other-state
69
VALIDATION
70
Further information: ROCHE-Control serum type Precipath U
6. Measuring range
Linearity
Upper limit
LOD
LOQ
7. Spread precision
Repeatability
8. Robustness
71
9. Quality control
Control charts
R squared
Standard deviation of the analytical procedure
Coefcient of variation of the analytical procedure
Measurement uncertainty
72
validated
Calculations
LOD
LOQ
Repeatability
CV
c c
n n
c
i obs QC
QC
( )
( )
,
2
1
5
1
100 = 1.27 %
Trueness
Parameter
Value requested by
the customer
(the same as stated in question 3)
Value obtained
during validation
The requirement
is fulflled
Yes/No
LOD
LOQ
Repeatability
Within-lab
reproducibility
8 % as CV, by WHO procedure
2% as CV, the actual state-of-art
1.27 % YES
Trueness
Measurement
uncertainty
Other
Yes No
For measurement uncertainty and traceability refer to the corresponding sheets
73
Measurand Total calcium concentration in human serum
Unit mg dL
-1
equation
Measurement procedure
To determine the calcium concentration in human serum, a serum sub-sample is mixed
with reagent colour and buIIer solution, according to WHO standard operation procedure.
The absorbance oI calcium calibration solutions and serum sample are measured by
visible spectrophotometry at 540 nm. From the calibration data the concentration oI
calcium in human serum is calculated.
Model equation: calcium concentration in serum
c m M P V M V V A A
( )
( )
1
]
( ) ( ) 100
500 100
3
/ / / AA A
V
V
blank
f
( )
1
]

_
,
10
int
c
Ca
total calcium concentration in serum sample |mg dL
-1
|
m mass oI CaCO
3
|mg|
M
Ca
-1
|
P purity oI CaCO
3
|mass Iraction|
V
500
fnal volume oI calcium stock standard solution |mL|
M
CaCO3
molar mass oI CaCO
3
|g mol
-1
|
V
i
intake stock solution Ior dilution (V
i
= V
-20
corresp. to c
-10
) |mL|
V
100
fnal volume oI calibration solution |mL|
A
x
absorbance corresponding to serum sample
A
-10
absorbance corresponding to calibration solution (10 mg dL
-1
)
A
blank
absorbance corresponding to blank
V
I
V
int
74
Uncertainty of measurements of peak area
Method bias
Matrix efect
Other: Uncertainty of absorbance measurements
Other: Uncertainty of volume measurements
m 625.0 mg
M
Ca
40.078 g mol
-1
P 0.9999 mass fraction
V
500
500.00 mL
M
CaCO3
100.0869 g mol
-1
V
i
20.000 mL
V
100
100.000 mL
A
x
0.323 no units
A
-10
0.338 no units
A
blank
0.052 no units
V
f
0.100 mL
V
int
0.100 mL
Input quantity
Standard
uncertainty
Unit Remark
m 0.2 mg
M
Ca
0.002 g mol
-1
P 0.0058 mass fraction
V
500
0.15 mL
M
CaCO3
0.0024 g mol
-1
75
V
i
0.043 mL
V
100
0.058 mL
A
x
0.004 no units
A
-10
0.002 no units
A
blank
0.004 no units
V
f
0.002 mL
V
int
0.002 mL
c m M P V M V V A A
( )
( )
1
]
( ) ( ) 100
500 100
3
/ / / AA A
V
V
blank
f
( )
1
]

_
,
10
int
c
Ca
9.486 mg dL
-1
c
Input quantity Value
Standard
uncertainty
Unit Remark
m 625.0 0.2 mg
M
Ca
40.078 0.002 g mol
-1
P 0.9999 0.0058 mass fraction
V
500
500.00 0.15 mL
M
CaCO3
100.0869 0.0024 g mol
-1
V
i
20.000 0.043 mL
V
100
100.000 0.058 mL
A
x
0.323 0.004 no units
A
-10
0.338 0.002 no units
A
blank
0.052 0.004 no units
V
f
0.100 0.002 mL
V
int
0.100 0.002 mL
u(c
Ca
) 0.303 mg mol
-1
76
c
units
U(c
Ca
)

= k u (c
Ca
) = 0.606 [mg dL
-1
], k = 2
c
1 Volume serum measurements
2 Concentration of serum sample from calibration data
77
1. Guide to the Expression of Uncertainty in Measurement (GUM), 1st ed. (1995),
Geneve, Switzerland.
2. Eurachem/Citac Guide CG4: Qualtifying Uncertainty in Analytical Measurement,
2nd ed. (2000).
3. Standard operation procedure Ior Clinical chemistry: Determination oI calcium by
calcium-o-cresolphtalein complexone method, http://w3.whose.org.
4. S. Linko, U. rnemark and R. Kessel, Evaluation oI measurement uncertainty in
clinical chemistry, GE/R/IM/34/01, IRMM (2001).
5. J. Kragten, Calculating standard deviations and confdence intervals with a universally
applicable spreadsheet technique, Analyst 119 (1994), 21612165.
Further readings
78
Preparation of the standard solution
Addendum I: Measurement uncertainty calculation:
79
value std-unc cert
10 V_10 10 0.029 0.05
15 V_15 15 0.035 0.06
20 V_20 20 0.043 0.075
25 V_25 25 0.058 0.1
V_100 100 0.058 0.1
[B, rect]
Preparation of caIibration caIcium standard soIution
c_i = c_stock * V_i / V_100
value std-unc Rsu c-stock V1 V_100
c_stock 50.050 0.020 0.04% 50.070 50.050 50.050
20 V_20 20 0.043 0.22% 20.000 20.043 20.000
V_100 100.000 0.058 0.06% 100.000 100.000 100.058
c_10 10.01 0.0228 10.014 10.031672 10.004224 function
(k=1) 0.004 0.0216723 -0.005776 diff
1.6E-05 0.000469688 3.33615E-05 diff^2
0.000519 sum(diff^2)
C_stock V1 V_100
C, mg/dI value std-unc
c_5 5.005 0.015
c_7.5 7.510 0.018
c_10 10.010 0.023
c_12.5 12.512 0.030
80
Calibration
Calculation of calcium concentration in serum sample
c_Ca = c_x*V_f/V_int
value std-unc RSU c_x V_f V_int
c_x 9.486 0.146 1.5% 9.632 9.486 9.486
V_f 0.1 0.002 2.0% 0.1 0.102 0.1
V_int 0.1 0.002 2.0% 0.1 0.1 0.102
c_Ca 9.486 0.303 3.2% 9.632 9.676 9.300 function
mg/dL (k=1) -0.146 -0.190 0.186 diff
0.0213 0.0360 0.0346 diff^2
0.0919 sum(diff^2)
23.2% 39.2% 37.6% index
100.0% sum(index) s
Chapter 3
Determination of Radium in Water
by a-Spectrometry
Ljudmila Benedik, Urka Repinc, Monika Inkret
81
82
Measurand Activity concentration of Ra-226 in water (Bq L
-1
) (by -spectrometry)
Authors of the example Ljudmila Benedik, Urka Repinc, Monika Inkret
Determination of radium isotopes by BaSO
4
coprecipitation for the
preparation of alpha-spectrometric sources
J.C. Lozano, F. Fernandez and J.M.G. Gomez, Journal of Radioanalytical and
Nuclear Chemistry 223 (1997) 12, 133137
Customers requirement
Directive 98/83/EC on the quality of water intended for human
consumption
83
II. Attached fles
File number, type and
name
Content of the fle
File is
attached Remark
Yes No
1
-
IEX-08-1-I-Ra226-water-
AS-2006-Ver1.ppt
About the analytical procedure: short
introduction

Given
by the
lecturer
2

-

Y
e
l
l
o
w
EX-08-2-Y-Ra226-water-
AS-2006-Ver1.doc
PART I
Description of the analytical
procedure

Each
participant
receives
own copy
and may
keep it
PART II
measurement result
PART III
procedure relevant equations
and measurement data
PART IV
Measurement uncertainty of the
result relevant equations and
measurement data
3

-

G
r
e
e
n
EX-08-3-G-Ra226-water-
AS-2006-Ver1.doc
PART I
Establishing traceability in
analytical chemistry

PART II

PART III

Addendum 1: By spreadsheet
approach

Addendum 2: By dedicated
software

0 April 2007
1
84
Determination of Ra-226 in water
by o-spectrometry
Model equation
4
coprecipitation for the preparation of alpha-
spectrometric sources
J.C. Lozano, F. Fernandez and J.M.G. Gomez
Journal oI Radioanalytical and Nuclear Chemistry, 223,
12 (1997), 133137.
1he quality of the results should comply with the
requirement in the revised directive 98/83/EC on the
quality of water intended for human consumption
(in preparation)
85
Ra-226
Similar chemical properties as Ca
20 known isotopes with mass
206234
4 are Iound in nature
Emitted alpha particles have high
potential Ior causing biological
damage
86
Preparation of
standards and samples
Ra-226 standard disc
For determination oI
eIIiciency oI alpha
spectrometer
Ba-133 standard disc
For determination
recovery oI
radiochemical
separation
Sample
Addition oI Ba-133 tracer
Pb(Ra)(Ba)SO
4
precipitation
Washing the precipitate
Dissolution
Re-precipitation
MicroIiltration
Gamma counting
Ior determination oI
recovery
Alpha counting Ior
determination oI Ra-226
-spectrometry
-particIe
2 4
2
He
A very large mass
A high charge
-particles are easily
stopped by a few
microns of air.
A very thin source
allows adequate
transmission of the
-particles to the
detector surface
87
Model equation
Measurand: Activity concentration of Ra-226 in water (
Ra-226
)
Unit: Bq L
-1
chem sample det 226 - Ra
226 - Ra
226 - Ra
1
c R J t
P
A

=
o
133Std - Ba
133Std - Ba 133Std - Ba
sample 133 - Ba 133sample - Ba
133sample - Ba
chem
P
m t
m t
P
R

=
226Std - Ra 226SS - Ra 226SS - Ra 226Std - Ra
226Std - Ra
det u
c
R A m t
P

=
Model equation and equation for
measurement uncertainty calculation
( ) ( )
2
chem
chem
2
2
udet
det
2
226 - Ra
226 - Ra
226 - Ra
226 - Ra
) ( ) (
c
c ) (
|
|
.
|
\
|
+
|
.
|
\
|
+
|
|
.
|
\
|
+
|
|
.
|
\
|
=
R
R u
J
J u u
P
P u
A
A u
o
chem sample det 226 - Ra
226 - Ra
226 - Ra
1
c R J t
P
A

=
o
( )
226 - Ra
A u k U =
88
Determination of activity concentration of Ra-226 in drinking
water.
in the revised Directive 98/83/EC on the quality of water
intended for human consumption
PART I ..................................................................................................................... 89
PART II .................................................................................................................... 96
PART III ................................................................................................................... 97
measurement data
PART IV ................................................................................................................... 98
data
89
For the determination oI Ra-226 in water the Iollowing published procedure is used:
4
coprecipitation for the preparation of
alpha-spectrometric sources
Journal oI Radioanalytical and Nuclear Chemistry 223 (1997) 12, 133137.
1. Scope
1.1 General
The procedure is specifed Ior the determination oI Ra-226 in water with activity
concentration (drinking water, rain water, ground water and surIace water) in range oI
0.0110 Bq L
-1
.
In certain cases, the range oI application may be changed by variations in the working
conditions (e.g. sample volume, pre-concentration techniques, sensitivity ranges oI
detectors, etc.).
1.2 Interferences
In the case oI Ra-226 measurement by u-spectrometry extensive chemical separation
prior to counting to remove peak interIerences Irom other alpha emitters is required.
2. Principle
A coprecipitation procedure Ior the preparation oI alpha spectrometric source Ior
radium, using BaSO
4
as carrier Ior determination oI Ra-226 in water, is used. The use oI
Ba-133 as a suitable tracer Ior determination oI recovery oI the radiochemical procedure
by gamma spectrometry is applied. Experimental protocol is schematically shown in
Figure 4.
90
Figure 4. Experimental protocol for determination Ra-226 in water
3. Apparatus
- Alpha spectrometer with low background silicon surIace detector
- HP Ge gamma detector
- Analytical balance: d = 0.001
- CentriIuge and 50 mL centriIuge tubs
- Fume hood
- Hot plate
- Magnetic stirrer plate, bars and retriever
- Filter apparatus
- Glass beakers, volumetric fask and graduated cylinders, assorted sizes
- Pipettes, assorted sizes
- 0.1 m, 25 mm diameter polypropylene flters
- Stainless steel disks
- Petrislides, watch glasses
91
4. Reagents
- Mixed u standard source Ior detector calibration: 398 dpm + 3 (k 2)
- Ra-226 standard solution: NIST SRM 4967
- Activity: 2729 Bq g
-1
+ 1.18 (overall, k 3)
- Ba-133 tracer
- Activity: 124.9 kBq g
-1
+ 0.4 (k 2)
- Working solution: 100 Bq g
-1
- H
2
SO
4
concentrated
- Pb
2
solution (50 mg mL
-1
)
- Ba carrier solution (0.3 mg mL
-1
)
- 0.1 M EDTA/0.5 M NaOH
- Na
2
SO
4
solution (saturated)
- Indicator pH 35
- Acetic acid 1:1
- BaSO
4
seeding suspension
5. Sample preparation procedure
The radiochemical separation procedure of Ra-226 with lead coprecipitation
1. Measure 1000 mL oI the water into a beaker
Graduated cylinder 1000 mL 5 mL (BLAUBRAND
tolerance)
2. Add 0.3 g oI Ba-133 tracer (working solution)
3. Add 0.3 mL oI Ba
2
carrier solution
4. Add 10 mL oI conc. H
2
SO
4
5. Precipitate Pb(Ra)(Ba)SO
4
by adding 1 mL Pb
2
solution through a dripper while
stirring. Keep stirring Ior 12 hours
6. Remove stirrer bar, cover beaker with watch glass and allow to settle overnight
7. Decant supernatant liquor to as low volume as possible. Discard decanted
supernatant
8. Wash precipitate into 50 mL centriIuge tube and centriIuge at 3500 rpm Ior fve
minutes
9. Pour out supernatant
10. Repeat steps 7 and 8 once
11. Wash sides and the walls oI the centriIuge tube with Mili-Q and centriIuge at
3500 rpm Ior 5 min
12. Pour out supernatant (take care not to disturb the precipitate)
13. Add 4 mL 0.1 M EDTA/0.5 M NaOH
14. Vortex to dissolve the precipitate
15. Add 1:1 acetic acid to adjust pH (45): Pb
2
ions remain in solution
16. Add 4 mL oI saturated Na
2
SO
4
17. Add 0.3 mL oI Ba seeding solution
92
18. Allow to sit at least 30 min
19. Filter the colloidal suspension oI (Ra)(Ba)SO
4
through a pre-wetted 0.1 m pore
size, 25 mm polypropylene flter
20. AIter the sample has fltered rinse the centriIuge walls and the flter holder with
ultrapure water
21. Remove flter and allow to air dry
22. Mount the flter on a stainless steel disc, using double-sided tape or glue stick or
cover the source with 6 VYNS Ioil
6. Preparation of standard discs
6.1 Preparation of a Ba-133 standard disc
A Ba-133 standard disc is prepared to determine the recovery oI prepared source. It is
made by adding a known amount oI Ba-133 as a sulphate in the same manner as samples
are prepared.
1. Measure into 50 mL centriIuge tube approximatelly the same amount oI Ba-133
standard as used per sample
2. Add 0.3 mL oI Ba carier solution
3. Add appropriate volume oI conc. H
2
SO
4
4. Add 0.5 mL oI Pb
2
solution
5. CentriIuge at 3500 rpm Ior 5 min
6. Pour out supernatant
7. Wash sides and the walls oI the centriIuge tube with ultrapure water and
centriIuge at 3500 rpm Ior 5 min
8. Pour out supernatant liquid (take care not to disturb the precipitate)
10. Disolve the precipitate
11. Add 1:1 acetic acid to adjust pH (45)
2
SO
4
14. Micro-fltration oI suspension
15. Washing flter with water, Iollowed by drying
The fltrate and washing Irom the Ba-133 standard source preparation are collected in a
bottle, counted on a gamma detector and compared to an equivalent, known activity oI
Ba-133 in the same geometry. The losses incured in mounting the source are calculated
Irom these measurements, allowing the eIfciency oI mounting, and thereIore the
Iractional recovery oI the Ba-133 standard disc, to be calculated.
93
6.2 Preparation of a Ra-226 standard disc
A Ra-226 standard disc is prepared to determine the eIfciency and perIorm an energy
calibration oI each alpha spectrometer. The disc is made by mounting a known amount
oI Ra-226 calibration solution and Ba-133 solution, as a sulphate in the same manner as
samples are prepared.
Disc should be prepared with an activity oI 2550 Bq.
1. Measure into 50 mL centriIuge tube 0.01 g oI Ra-226 standard solution
2. Add approximatelly the same amount oI Ba-133 standard as used per sample
3. Add 0.3 mL oI Ba carier solution
4. Add appropriate volume oI concentrated H
2
SO
4
5. Add 0.5 mL oI Pb
2
solution
6. CentriIuge at 3500 rpm Ior fve minutes
7. Pour out supernatant liquid
8. Wash sides and the walls oI the centriIuge tube with ultrapure water and
centriIuge at 3500 rpm Ior 5 min
9. Pour out supernatant (take care not to disturb the precipitate)
11. Disolve the precipitate
12. Add 1:1 acetic acid to adjust pH (45)
2
SO
4
15. Micro-fltration oI suspension
16. Washing flter with water, Iollowed by drying
7. Preparation of blank flters
7.1 Preparation of blank flter
A blank flter is prepared to check iI 0.1 m pore size, 25 mm polypropylene flter
contains any traces oI Ra-226.
- Mount the flter on a stainless steel disc, using double-sided tape or glue stick or
7.2 Making a reagent blank flter
A reagent blank flter is prepared to determine iI there are any traces oI Ra-226 in used
chemical reagents.
- A reagent blank flter is made in the same manner as samples (steps 122, Irom
point 5). Ultrapure water is used instead oI sample water.
94
8. Gamma and alpha counting
Gamma
1. Measure the background oI gamma detector
2. Measure Ba-133 (Ba-133 standard disc) on a gamma spectrometer
3. Measure Ba-133 (sample) on a gamma detector
Alpha
4. Measure the background oI alpha detector
5. Measure the blank flter
6. Measure the reagent blank flter
7. Measure the Ra-226 standard disc Ior eIfciency determination
8. Measure the flter with Ra-226 (sample) on alpha spectrometer
9. Calculation
9.1 Sample recovery calculation
Determination oI recovery by gamma spectrometry is calculated as Iollows:
R
P
t m
t
chem
Ba-133sample
Ba-133sample Ba-133sample
Ba-133St

dd Ba-133Std
Ba-133Std
m
P
R
chem
radiochemical yield (recovery)
P
Ba-133 sample
peak area oI Ba-133 in the sample
t
Ba-133 sample
time oI the sample measurement |s|
m
Ba-133 sample
mass oI added Ba-133 in the sample |g|
P
Ba-133Std
peak area oI Ba-133 in barium standard disc
t
Ba-133Std
time oI measurement oI Ba-133 in barium standard disc |s|
m
Ba-133Std
mass oI added Ba-133 in barium standard disc |g|
u det

P
t m A R
Ra-226Std
Ra-226Std Ra-226SS Ra-226SS Ra-226Std

9.2 Alpha spectrometer efciency determination
u det
eIfciency oI alpha detector
R
Ra-226Std
radium standard disc recovery
P
Ra-226Std
peak area oI Ra-226 in standard disc
t
Ra-226Std
time oI measurement oI standard disc |s|
m
Ra-226SS
mass oI added Ra-226 in standard solution |g|
A
Ra-226SS
activity concentration oI Ra-226 in standard solution |Bq g
-1
|
95
9.3 Activity concentration of Ra-226 in the sample (Bq L
-1
)
A
P
t e V R
Ra-226
Ra-226
Ra-226 det sample chem
=

A
Ra-226
Activity concentration oI Ra-226 in the sample in |Bq L
-1
|
P
Ra-226
peak area oI Ra-226
t
Ra-226
time oI measurement |s|
V
sample
volume oI the sample |L|
u det
corrected eIfciency oI alpha detector
R
chem
96
Extract from the Directive 98/83/EC, Draft annex 2005/04/20 on the
quality of water intended for human consumption
Reference concentration for radioactivity in drinking water
*
Origin Nuclide Reference concentration
Natural Ra-226 0.5 Bq L
-1
* This table includes the most common natural and articial radionuclide Reference concentrations for other radionuclides can be
calculated using the dose coefcients for adults laid down in Annex III Table A of Directive 96/29/Euratom or more recent information
recognised by the competent authorities in the Member State, and by assuming an intake of 730 litres per year.
Performance characteristics and methods of analysis
For the Iollowing radioactivity parameters, the specifed perIormance characteristics
are that the method oI analysis used must, as a minimum, be capable oI measuring
concentrations equal to the parametric value with a limit oI detection specifed.
Parameter Limit of detection Notes
Ra-226 0.04 Bq L-1
Note 1
Note 2
Note 1: the limit of detection should be calculated according to ISO 11929-7, Determination of the detection limit and decision thresholds
for ionizing radiation measurements - Part 7: Fundamentals and general applications, with probabilities of errors of 1
st
and 2
nd
kind of
0.05 each
Note 2: measurement uncertainties should be calculated and reported as complete standard uncertainties, or as expanded standard
uncertainties with an expansion factor of 1.96, according to the ISO Guide for the Expression of Uncertainty in Measurement (ISO, Geneva
1993, corrected reprint Geneva, 1995)
measurement result
97
In the present case study, methodology Ior validation oI measurement procedure Ior
determination oI Ra-226 in water by u-spectrometry is presented. For the calculation
part, the emphasis is on the parameters that are required by the customer. In this particular
case, these parameters are:
- LOD
- within-laboratory reproducibility.
For the purpose oI this exercise, LOD (LLD) will be calculated only.
Equation
LLD
Bkg
Bkg chem sample
=
+

2 71 4 65 . .
det
t R V
Measurement data
Input quantity Unit Value
R
chem
radiochemical yield (recovery) - 0.803
det
efciency of alpha detector - 0.2453
Bkg peak area of background of alpha detector at the Ra-226 alpha energy -
t
Bkg
time of measurement of background s
V
sample
volume of the sample L
98
In the present case study, methodology Ior evaluation oI measurement uncertainty oI
result oI Ra-226 determination in drinking water is presented. Ra-226 was determined
using u-spectrometry. The necessary relevant inIormation was obtained Irom the method
validation data, the quality control data and equipment calibration certifcates. The
method oI measurement is described together with the measurement equation, selected
traceable reIerence standards and the associated measurement uncertainty. The major
sources oI uncertainty oI the result oI measurement were identifed and the combined
uncertainty was calculated. Identifcation oI the main uncertainty sources represent basis
Ior target operation Ior reducing the measurement uncertainty oI this determination.
Equations
( ) u A
A
u P
P
u e
e
Ra-226
Ra-226
Ra-226
Ra-226
2
det
det
( ) ( )
=

2
sample
sample
2
chem
chem
2
( )
( )
u V
V
u R
R
u R
R
u P
P
u m
chem
chem
Ba-133Std
Ba-133Std
2
Ba-133Std
( ) ( )
( )
_
,
+
mm
u P
P
Ba-133Std
2
Ba-133sample
Ba-133sample
2
_
,
+
( )
_
,
+
uu m
m
Ba-133sample
Ba-133sample
2
( )
_
,
u e
e
u P
P
u m
m
det
det
Ra-226Std
Ra-226Std
2
Ra-226SS
( ) ( ) ( )
=

+
RRa-226SS
2
Ra-226SS
Ra-226SS
2
Ra-226Std
( )
+
u A
A
u R ( ))
R
Ra-226Std
2
u A k A ( ) ( )
Ra-226 Ra-226

99
M
e
a
s
u
r
e
m
e
n
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d
a
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a

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100
TrainMiC Exercises
Determination of activity concentration of Ra-226 in drinking
water.
The quality of the results should comply with the requirement
in the revised Directive 98/83/EC on the quality of water
EXERCISE 1:
EXERCISE 2:
EXERCISE 3:
101
Analyte Ra-226
Measurand Activity concentration of Ra-226 in water (drinking, surface, waste, )
Units Bq L
-1
Measurement
procedure
4
alpha-spectrometrical sources
Lozano et al., Journal of Radioanalytical and Nuclear Chemistry
Type of calibration mixed standard source standard addition internal standard
Model equation
The activity concentration oI Ra-226 in sample (Bq L
-1
), is calculated by
A
P
t e V R
Ra-226
Ra-226
=

A
Ra-226
activity concentration oI Ra-226 in the sample |Bq L
-1
|
P
Ra-226
peak area oI Ra-226
t
Ra-226
V volume oI the sample |L|
u det
R
chem
R
P
t m
t
chem
Ba-133sample
Ba-133Std

m
P
Ba-133Std
Ba-133Std
Recovery obtained by gamma spectrometry is calculated as Iollows:
R
chem
P
Ba-133 sample
peak area oI Ba-133 in the sample
t
Ba-133 sample
m
Ba-133 sample
P
Ba-133Stda
102
t
Ba-133Std
time oI measurement oI Ba-133 standard disc |s|
m
Ba-133Std
Alpha spectrometer eIfciency is calculated as Iollows:
u det
P
t m A R
Ra-226Std
u det
R
Ra-226Std
P
Ra-226Std
peak area oI Ra-226 oI standard disc
t
Ra-226Std
time oI measurement oI Ra-226 standard disc |s|
m
Ra-226SS
mass oI added Ra-226 standard solution |g|
A
Ra-226SS
Ra-226 activity concentration in standard solution |Bq g
-1
|
1 Uncertainty of concentration of reference solutions
2 Uncertainty of volumes
3 Uncertainty of weighing
4 Uncertainty of measurement, using alpha and gamma detectors
For the analyte
1 Name/Chemical Formula/Producer: Standard Radionuclide Source, Analytics, SRS 67978-121
Ba-133 standard solution, Czech Metrological Institute,
Cert. No: 931-OL-137/99
2 Name/Chemical Formula/Producer: Ra-226 standard solution, NIST SRM 4967
103
1
(k = 2) see also data yellow sheet
Graduated and mixing cylinders, volumetric fask/with
established traceability
BLAUBRAND

tolerance
Mass/calibrated balance/with established traceability
Sartorius
model equation?
Yes No
Other important parameters are:
Uncertainty of measured background of detector,
uncertainty of measured blank reagents (minor
contributions)
1 Analysis of matrix CRM
2 Participation in a profciency testing scheme
3 -
104
The measurement
procedure
4
alpha-spectrometric sources
Journal of Radioanalytical and Nuclear Chemistry 223 (1997) 12, 133137.
Analyte Ra-226
The measurand Activity concentration of Ra-226 in drinking water
Unit Bq L
-1
Matrix Drinking water
Measuring range 0.0110 Bq L
-1
Compliance to the requirements in the revised water directive 98/83/EC on
the quality of water intended for human consumption
Mark the customers
their values
LOD 0.04 Bq L
-1
LOQ
Repeatability
Trueness
Other-state
VALIDATION
105
spiking of samples with pure substances and calculation of recovery
6. Measuring range
Linearity
Upper limit
LOD
LOQ
7. Spread precision
Repeatability
106
8. Robustness
9. Quality control
Control charts
R square
107
Parameters requested to
be validated
Calculations
LOD
LLD
+

2 71 4 65 14 26092744
420 730 0 2453 0 803 1
0 000245
. . .
. .
.

Bq L
-1
LOQ
Repeatability
Trueness
Parameter
customer
Value obtained
during validation
The requirement
is fulflled
Yes/No
LOD 0.04 Bq L
-1
0.00025 Bq L
-1
YES
LOQ -
Repeatability -
Within-lab
reproducibility
Trueness -
Measurement
uncertainty
Other -
Yes No
108
Measurand Activity concentration of Ra-226 in water (drinking, surface, waste, )
Unit Bq L
-1
equation
Determination oI radium isotopes by BaSO
4
coprecipitation Ior the preparation oI alpha-
spectrometric sources
Journal oI Radioanalytical and Nuclear Chemistry 223 (1997) 12, 133137.
Model equation:
The activity concentration oI Ra-226 in the sample (Bq L
-1
), is calculated by
A
P
t V R
Ra-226
Ra-226
=

A
Ra-226
Activity concentration oI Ra-226 in the sample |Bq L
-1
|
P
Ra-226
peak area oI Ra-226
t
Ra-226
V volume oI the sample |L|
u det
R
chem
109
Recovery measured by gamma spectrometry is calculated as Iollows:
R
P
t m
t
chem
Ba-133sample
Ba-133Std

mm
P
Ba-133Std
Ba-133Std
R
chem
P
Ba-133 sample
Peak area oI Ba-133 in the sample
t
Ba-133 sample
m
Ba-133 sample
P
Ba-133Std
t
Ba-133Std
time oI measurement oI Ba-133 in barium standard disc |s|
m
Ba-133Std
Alpha spectrometer eIfciency determination is calculated as Iollows:
u det
P
t m A R
Ra-226Std
u det
R
Ra-226Std
P
Ra-226Std
peak area oI Ra-226 in standard disc
t
Ra-226Std
time oI measurement oI Ra-226 standard disc |s|
m
Ra-226SS
mass oI added Ra-226 standard solution |g|
A
Ra-226SS
Ra-226 activity concentration in standard solution |Bq g
-1
|
Uncertainty of measurements of peak area (alpha and gamma detectors)
Method bias
Matrix efect
Other: Uncertainty of volume measurements
Other: Uncertainty of weighing
Other: Uncertainty of measured background of alpha and gamma detectors
Other: Uncertainty of measured blank reagents, flters, discs
110
P
Ra-226
7516 -
t
Ra-226
300 000 s
det
0.2453 -
V
sample
1.0 L
R
chem
0.803 -
Input quantity
Standard
uncertainty
Unit Remark
P
Ra-226
87 -
t
Ra-226
0 s Constant
det
0.01392 -
V
sample
0.0020 L
R
chem
0.0142 -
A
P
t e V R
Ra-226
Ra-226
Ra 226 det sample chem
1
=

A
Ra-226
-1
7516
300 000 0.2453 1
1
0.803
Bq L

0 127 .
c
Using: Mathematical solution; Spreadsheet Approach; Commercial SoItware
Input quantity Value
Standard
uncertainty
Unit Remark
P
Ra-226
7516 87 -
t
Ra-226
300 000 0 s
det
0.2453 0.01392 -
111
V
sample
1.0 0.0020 L
R
chem
0.803 0.0142 -
( ) u A
A
u P
P
u e
e
Ra-226
Ra-226
Ra-226
Ra-226
2
det
det
( ) ( )
=

2
sample
sample
2
chem
chem
2
(
( )
u V
V
u R
R
)
u A
A
Ra-226
Ra-226
2 2
87
7516
0.01392
0
0.002 ( )
_
,
_
,
+
.2453
00
1
0.0142
0.803
2 2
_
,
_
,
0 00806 .
c
units
u A k A ( ) ( )
Ra-226 Ra-226

U 2 0.00806 0.016 Bq L
-1
contributing the most to u
c
1 Mass of Ra-226 standard solution
2 Peak area of Ba-133 in the standard disc
3 Peak area of Ra-226 of the sample
See the attached Excel calculations and calculations done using the software
GumWorkbench
112
1. EC 1980, Council Directive oI 15 July 1980 relating to the quality oI water intended
Ior human consumption, Ofc. J. Eur. Commun. L229 (1980), 30.8.
2. EC 1998, Council Directive 98/83/EC oI 3 November. The quality oI water intended
Ior human consumption, Ofc. J. Eur. Commun. L330 (1998), 5.12.
3. EURATOM 1989, Council Regulation (Euratom) No. 2218/89 oI 18 July 1989
amending Regulation (Euratom) No. 3954/87 laying down maximum permitted levels
oI radioactive contamination oI IoodstuIIs and oI IeedingstuIIs Iollowing a nuclear
accident or any other case oI radiological emergency, Ofc. J. Eur. Commun. L211
(1989), 22.07.
4. EURATOM 2001, European Commission Recommendation 2001/928/EURATOM,
Ofc. J. Eur. Commun. L344 (2001), 28.12.
5. Guidelines for Drinking Water Quality, Recommendation, vol. 1, 2nd ed. (1993),
WHO, Geneva.
6. Guidelines for Drinking Water Quality, Recommendation, vol. 1, 3rd (current) ed.
including the frst addendum (2006), WHO, Geneva. Available Irom: http://www.who.int/
watersanitationhealth/.
7. J.C. Lozano, F. Fernandez and J.M.G. Gomez, Determination oI radium isotopes by
BaSO
4
coprecipitation Ior the preparation oI alpha-spectrometric sources, J. Radioanalyt.
Nucl. Chem. 223(12) (1997), 133137.
8. Y. Spasova, S. Pomme, L. Benedik and U. Wtjen, Uncertainty Budget Ior
226
Ra
Activity concentration in Water by Alpha Spectrometry, Acta. Chim. Slov. 54 (2007),
854858.
9. L. Benedik, Y. Spasova, M. Vasile, and U. Wtjen, Determination oI
234
U,
238
U and
226
Ra in bottled drinking water by alpha spectrometry, in: CD Proceedings of the 7th
International Conference on Nuclear and Radiochemistry (NRC-7) (2429 August
2008), Budapest, Hungary.
Further readings
113
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a
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td
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m
B
a
-
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td
0
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1
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8
0
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1
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1
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117
Model equation:
A
Ra-266
= (P
Ra-266
/(t
Ra-266

det
V
sample
)) (1/R
chem
);
R
chem
(P
Ba-133sample
/(t
Ba-133sample
m
Ba-133sample
)) ((t
Ba-133Std
m
Ba-133Std
)/P
Ba-133Std
);
det
= P
Ra-226Std
/(t
Ra-226Std
m
Ra-226SS
A
Ra-226SS
R
Ra-226Std
)
List of quantities:
Quantity Unit Defnition
A
Ra-266
Bq L
-1
Activity of Ra-266 in sample
P
Ra-266
Area of Ra-266
t
Ra-266
s Time of measurement
e
det
Efciency for alfa detector
V
sample
L Volume of the sample
R
chem
Radiochemical yield (recovery)
P
Ba-133sample
Area of Ba-133 in sample
t
Ba-133sample
s Time of measurement of the sample
m
Ba-133sample
g Mass of Ba-133 in the sample
t
Ba-133Std
s Time of measurement of Ba-133 standard disc
m
Ba-133Std
g Mass of Ba-133 standard disc
P
Ba-133Std
Area of Ba-133 in standard disc
P
Ra-226Std
Area of Ra-266 in standard disc
t
Ra-226Std
s Time of measurement of the standard disc
m
Ra-226SS
g Mass of Ra-226 standard solution
A
Ra-226SS
Activity of Ra-226 in standard solution
R
Ra-226Std
Radium standard disc recovery
P
Ra-266
:
Type A summarised
Mean: 7516
Standard Uncertainty: 86
t
Ra-266
:
Constant
Value: 300 000 s
Addendum II: Measurement uncertainty calculation
GumWorkbench
118
V
sample
:
Type B triangular distribution
Value: 1 L
HalIwidth oI limits: 0.005 L
P
Ba-133sample
:
Type A summarised
Mean: 10 914
t
Ba-133sample
:
Constant
Value: 3000 s
m
Ba-133sample
:
Type B rectangular distribution
Value: 0.30120 g
HalIwidth oI limits: 0.001 g
t
Ba-133Std
:
Constant
Value: 3000 s
m
Ba-133Std
:
Value: 0.11280 g
P
Ba-133Std
:
Type A summarised
Mean: 5090
P
Ra-226Std
:
Type A summarised
Mean: 12 785
t
Ra-226Std
:
Constant
Value: 2000 s
119
m
Ra-226SS
:
Value: 0.01022 g
A
Ra-226SS
:
Type A summarised
Mean: 2729
Standard Uncertainty: 10.734
R
Ra-226Std
:
Type A summarised
Mean: 0.9344
Standard Uncertainty: 0.015
Uncertainty budgets:
A
Ra-266
: Activity of Ra-266 in sample
Quantity Value
Standard
Uncertainty
Distribution
Sensitivity
Coefcient
Uncertainty
Contribution
Index
P
Ra-266
7516.0 86.0 normal 17 10
-6
1.5 10
-3
Bq L
-1
3.3 %
t
Ra-266
300.0 10
3
s
V
sample
1.00000 L 2.04 10
-3
L triangular -0.13 -260 10
-6
Bq L
-1
0.1 %
P
Ba-133sample
10 914 104 normal -12 10
-6
-1.2 10
-3
Bq L
-1
2.3 %
t
Ba-133sample
3000.0 s
m
Ba-133sample
0.301200 g 577 10
-6
g rectangular 0.42 240 10
-6
Bq L
-1
0.0 %
t
Ba-133Std
3000.0 s
m
Ba-133Std
0.112800 g 577 10
-6
g rectangular -1.1 -650 10
-6
Bq L
-1
0.7 %
P
Ba-133Std
5090.0 71.0 normal 25 10
-6
1.8 10
-3
Bq L
-1
4.9 %
P
Ra-226Std
12785 113 normal -9.9 10
-6
-1.1 10
-3
Bq L
-1
2.0 %
t
Ra-226Std
2000.0 s
m
Ra-226SS
0.010220 g 577 10
-6
g rectangular 12 7.2 10
-3
Bq L
-1
79.9%
A
Ra-226SS
2729.0 10.7 normal 47 10
-6
500 10
-6
Bq L
-1
0.4 %
R
Ra-226Std
0.9344 0.0150 normal 0.14 2.0 10
-3
Bq L
-1
6.5 %
A
Ra-266
0.12719 Bq L
-1
8.04 10
-3
Bq L
-1
120
det
: Efciency of alfa detector
Quantity Value
Standard
Uncertainty
Distribution
Sensitivity
Coefcient
Uncertainty
Contribution
Index
P
Ra-226Std
12 785 113 normal 19 10
-6
2.2 10
-3
2.2 %
t
Ra-226Std
2000.0 s
m
Ra-226SS
0.010220 g 577 10
-6
g rectangular not valid! -0.014 90.1 %
A
Ra-226SS
2729.0 10.7 normal -90 10
-6
-960 10
-6
0.4 %
R
Ra-226Std
0.9344 0.0150 normal -0.26 -3.9 10
-3
7.2 %
e
det
0.2453 0.0146
R
chem
: Radiochemical yield (recovery)
Quantity Value
Standard
Uncertainty
Distribution
Sensitivity
Coefcient
Uncertainty
Contribution
Index
P
Ba-133sample
10 914 104 normal 74 10
-6
7.7 10
-3
28.8 %
t
Ba-133sample
3000.0 s
m
Ba-133sample
0.301200 g 577 10
-6
g rectangular -2.7 -1.5 10
-3
1.2 %
t
Ba-133Std
3000.0 s
m
Ba-133Std
0.112800 g 577 10
-6
g rectangular 7.1 4.1 10
-3
8.3 %
P
Ba-133Std
5090.0 71.0 normal -160 10
-6
-0.011 61.7 %
R
chem
0.8030 0.0143
Result:
Quantity Value
Expanded
Uncertainty
Coverage factor Coverage
A
Ra-266
0.127 Bq L
-1
0.016 Bq L
-1
2.00 95 %
121
Chapter 4
Determination of Polar Pesticides by Liquid
Chromatography Mass Spectrometry
Allan Knnapas, Koit Herodes, Ivo Leito
122
Measurand
Concentration of imazalil and thiabendazole in tangerines by liquid
chromatography-mass spectrometry
Authors of the example Allan Knnapas, Koit Herodes, Ivo Leito
Determination of concentration of imazalil and thiabendazole in
tangerines by liquid chromatography-mass spectrometry. The sample
preparation procedure is modifed AOAC 985.22 procedure. The
measurement procedure is an in-house developed procedure.
Customers requirement
The quality of the results should comply with the requirements given
in the EU Directives 93/58/EEC and 00/42/EEC on pesticide residues
analysis
123
File number,
type and name
Content of the fle
File is
attached
Remark
Yes No
1

-

I
Ex-04-1-I-
Pesticides-
Food-LCMS-
2006-Ver1.ppt

Given by the
leacturer
2

-

Y
e
l
l
o
w
Ex-04-2-Y-
Pesticides-
Food-LCMS-
2006-Ver1.doc

Each
participant
receives own
copy and may
keep it
PART II
The customers requirements concerning
the quality of the measurement result

PART III
measurement data
PART IV
relevant equations and measurement
data
3

-

G
r
e
e
nEx-04-3-G-
Pesticides-
Food-LCMS-
2006-Ver1.doc
PART I
chemistry

PART II

PART III



0 April 2007 -
1
II. Attached fles
Determination of Polar Pesticides by Liquid
124
Determination of Polar Pesticides by
Liquid Chromatography Mass
Spectrometry
The Customer`s Requirements
The Analytical Procedure
Sample preparation
LC-MS analysis
Model Equation
Pesticides are used for
example to prolong the
shelf life of fruit and
vegetables (they act against
e.g. molds). Besides the
useful action they are
potentially hazardous and
must be monitored.
125
The Customer`s Requirements
Post-registration control and monitoring oI pesticides based on
MRLs set by the EU Directives 93/58/EEC and 00/42/EEC Ior
imazalil and thiabendazole respectively:
LOD > 0.02 mg kg
-1
(imazalil), LOD > 0.05 mg kg
-1
(thiabendazole)
Recovery between 70110
Identity conIirmed by MS/MS experiments
LC-MS as an Analytical Tool
Unites two powerIul methods:
LC (liquid chromato-
graph) separates the
analytes from each
other
MS (mass
spectrometer)
detects, identifies
and quantifies the
analytes
126
The Analytical Procedure
Sample preparation procedure is a modiIication oI the AOAC
oIIicial method 985.22
ModiIications were made to cut down sample size and
solvent consumption
Changes were made in the solvent oI Iinal extract to suite
LC-MS system
Analysis is carried out on an LC-MS system using a selI-
developed chromatographic method
PartiaI VaIidation Required!
Sample Preparation
50 g oI homogenized sample is extracted with 100 ml oI acetone
using high speed blender
Mixture is Iiltered and the volume oI extract is measured
50 ml oI extract is extracted with 100 ml dichloromethane
petroleum ether mixture (1:1), organic layer is Iiltered through a
layer oI sodium sulphate (Ior drying purpose)
Water phase is saturated with NaCl and extracted twice with 50 ml
oI dichloromethane
Organic extracts are dried
Solvent is evaporated to almost dryness and the sample is
dissolved in 1020 ml oI methanol
Sample is Iiltered through a syringe Iilter and analysed using LC-
MS system
CompIex SampIe Preparation!
127
The LC-MS Analysis Procedure
In the LC-MS system the samples are separated chromatographically
Eluent: acetontrile (B) and buIIer solution (1mM ammonium acetate,
0.1 Iormic acid) (A) as eluent
The gradient program : B 20 ~ 100 15 min, B 100 17 min at 0.8
ml min
-1
Analysed substances were then ionized using the ESI procedure and
analysed with the ion-trap MS using Iragmentation oI quasimolecular
ions (|M H|
)
Using the 20 mg kg
-1
standard solution and other dilutions the calibration
solutions are prepared in methanol in the concentration range oI 50.003
mg kg
-1
Calibration graphs are compiled using peak areas oI certain characteristic
Iragment ions on diIIerent concentrations
Model Equation
w mass Iraction oI extractable pesticide in sample
(mg oI pesticide per kg oI sample) |mg kg
-1
|
w
c
mass Iraction oI extractable pesticide in analysed extract
|mg kg
-1
| (Iound Irom the calibration graph)
J
10
the volume oI Iinal extract in methanol |ml|
density oI methanol |g ml
-1
|
J
e
the Iull volume oI acetone extract |ml|
J
50
the volume oI acetone extract to be puriIied |ml|
M mass oI homogenised sample to be extracted |g|
m J
J J w
w

=
50
e 10 c

128
Determination of concentration of imazalil and thiabendazole
in tangerines by liquid chromatography mass spectrometry.
in the EU directives 93/58/EEC and 00/42/EEC/ on pesticide
residues analysis
PART I ............................................................................................................................... 129
PART II .............................................................................................................................. 133
The customers requirement concerning quality of the measurement result
PART III ............................................................................................................................. 135
measurement data
PART IV ............................................................................................................................ 137
data
129
The objective oI this analysis is post-registration control and monitoring oI imazalil and
thiabendazole (polar pesticides) based on Maximum Residue Limits (MRLs) set by the
EU Directives 93/58/EEC and 00/42/EEC.
Sample preparation procedure is modifed AOAC oIfcial method 985.22. Analysis was
carried out on an LC-MS system using a selI-developed chromatographic method.
1. Scope
A modifed AOAC 985.22 sample preparation procedure was used, to suite LC-MS-
MS analysis. The analysis was carried out using liquid chromatographic separation and
atmospheric pressure electrospray ionisation with tandem mass spectrometric detection
(AP-ESI-LC-MS-MS).
Sample preparation procedure is suitable Ior berries, Iruits and vegetables containing
less then 2 oI Iat and more than 70 oI water (water can be added iI its content is
insuIfcient). All in all 14 residues oI polar pesticides are analysed in this analytical
procedure. In this example, only two oI them will be discussed in detail: imazalil and
thiabendazole.
2. Principle
An aliquot oI homogenized sample is extracted with acetone and fltered. A portion
oI the extract is subjected to liquid-liquid clean-up step consisting oI one extraction
with petroleum ether (4060 C)-dichloromethane mixture and two extractions with
dichloromethane Irom saturated NaCl solution. Organic extracts are dried using anhydrous
sodium sulphate. Then the solvent is exchanged to methanol through evaporation and
dissolving. The obtained extract is analysed using LC-MS.
In the LC-MS system the samples are separated chromatographically using acetontrile
(B) and buIIer solution (1 mM ammonium acetate, 0.1 Iormic acid) (A) as eluent.
The gradient programme was as Iollows: B 20 ~ 100 15 min, B 100 17 min at
0.8 mL min
-1
. Analysed substances were then ionised through the ESI procedure and
analysed with the ion-trap MS using Iragmentation oI quasimolecular ions (|M H|
).
Calibration graphs are compiled using peak areas oI certain characteristic Iragment ions
on diIIerent concentrations.
The result is calculated in mg oI pesticide residue per kg oI sample, or ppm.
130
Laboratory sample is homogenized using appropriate equipment
50 g aliquot of homogenized sample is extracted with 100 mL of acetone using high
speed blender. Before the extraction standard solution can be added for recovery studies
Mixture is vacuum filtered through filter paper, the extraction vessel is rinsed and
filter cake is washed with approximately 30 mL of acetone
The volume of the extract is measured and a 50 mL aliquot is taken for further
purification through liquid-liquid extraction
In a separatory funnel the aliquot is extracted for 1 min with 100 mL of petroleum
ether and dichloromethane mixture (1:1)
The lower (water) phase is drained into volumetric cylinder, the upper organic layer
is filtered/dried through approximately 3 cm layer of anhydrous Na
2
SO
4
in a funnel
The water phase is saturated in the separatory funnel with NaCl and extracted twice
for 1 min with 50 mL of dichloromethane. The lower (organic) layer is also
filtered/dried through the same Na
2
SO
4
layer
The combined extract is brought down to couple of millilitres using rotary
evaporator, taking care not to evaporate to dryness
The extract is brought to almost complete dryness in slow flow of N
2
, then the
residue is reconstituted with 10 mL of methanol
If necessary the extracts are diluted in order to fit in the calibration range
Figure 5. Flow chart of the analytical procedure
131
3. Interferences
ESI procedure is dependent on ionization eIfciencies oI the species. The ionisation
eIfciencies can be aIIected by co-eluting polar matrix components. Thus sample
preparation and in most part chromatographic separation should be able to cope with
these circumstances. For this reason retention times should be reasonably large compared
to the dead volume oI the column. In addition suitable buIIer solution should be used.
The best ways to correct these eIIects are using matrix matched calibration, standard
addition or labelled internal standards. However these means will make the analysis
procedure signifcantly more complex and are not used in the current procedure.
4. Reagents
1000 mg kg
-1
individual pesticide standard solutions
Prepare pesticide standard solutions by dissolving 10 mg oI substance in 10 g oI acetone
(1000 mg kg
-1
) in 15 mL vials.
20 mg kg
-1
combined pesticide standard solution
Weigh 0.2 g oI each individual pesticide standard solution into 15 mL vial and fll it up
with methanol (9.6 g in the case oI two components)
Calibration solutions
Using the 20 mg kg
-1
standard solution and other dilutions the calibration solutions can
be prepared in methanol. Suitable number oI solutions should be prepared in the range oI
50.003 mg kg
-1
.
Solvents/eluent:
Gradient grade methanol, ultra pure water, ammonium acetate and Iormic acid (suitable
Ior LC-MS buIIer), petrol ether, dichloromethane and acetone (Ior residue analysis or
GC/HPLC grade iI suitability checked)
Other:
NaCl, Na
2
SO
4
pure Ior pesticide analysis (e.g. heated beIore use)
5. Sampling and pre-treatment
Sampling shall be carried out in accordance with European Commission Directive
2002/63/EC. While getting a laboratory/analytical sample one has to obtain homogenous
and representative sample, also a great care has to be taken in order to avoid cross-
contamination beIore or during or aIter sample preparation. Standard solutions should
be kept separate Irom samples.
132
6. Calculation
The residue content w in the sample is Iound according to the Iollowing equation.
w
c
is Iound Irom the calibration graph.
w
w V V
V m
c e 10
50
w mass Iraction oI extractable pesticide in sample (mg oI pesticide per kg oI

sample) |mg kg
-1
|
w
c
mass Iraction oI extractable pesticide in analysed extract |mg kg
-1
|
V
10
the volume oI fnal extract in methanol |mL|
density oI methanol (extract) |g mL
-1
|
V
e
the Iull volume oI acetone extract |mL|
V
50
the volume oI acetone extract to be purifed |mL|
m mass oI homogenised sample to be extracted |g|
7. Results
Calculations are perIormed using calibration graph and the model equation given
above. Obtained results are compared against MRLs set by EU Council 5 mg kg
-1

Ior both pesticides. The samples at or over MRL must be reanalysed and/or otherwise
confrmed.
133
PART II. The customers requirement concerning quality of the
measurement result
The laboratory should provide at least the Iollowing LODs Ior pesticide residues:
Imazalil 0.02 mg kg
-1
(Ior citrus) (Directive 93/58/EEC)
Thiabendazole 0.05 mg kg
-1
(Ior citrus) (Directive 00/42/EC)
Extract from the EU Quality Control Procedures for Pesticide Residues Analysis,
SANCO/10232/2006
**************************************
58. The method must be tested to assess Ior sensitivity, mean recovery (as a measure oI
trueness or bias) and precision. This eIIectively means that spiked recovery experiments
to check the accuracy oI the method should be undertaken. A minimum oI 5 replicates
is required
Mean recovery range should be within 70110 . In that case no recovery correction is
perIormed.
Exceptionally, where recovery is low but consistent (i.e. demonstrating good precision)
and the basis Ior this is well established (e.g. due to pesticide distribution in partition), a
mean recovery below 70 may be acceptable. However, a more accurate method should
be used, iI practicable.
In the case oI low recovery one has to take it into account when making decisions at or
above MRL.
78. EI-MS or MS/MS, perIormed with acquisition oI spectra, may provide good evidence
oI identity and quantity in many cases. In other cases, as with mass spectra produced
by other processes (e.g. CI, API) that can be too simple Ior absolute confrmation oI
identity, Iurther evidence may be required. II the isotope ratio oI the ion(s), or the
chromatographic profle oI isomers oI the analyte, is highly characteristic it may provide
suIfcient evidence. Otherwise, the evidence may be sought using:
(i) a diIIerent chromatographic separation system;
(ii) a diIIerent ionisation technique;
(iii) MS/MS;
(iv) medium/high resolution MS; or
(v) inducing 'in-source Iragmentation in LC-MS.
134
Table 3. Recommended maximum permitted tolerances for relative ion intensities
using a range of spectrometric techniques
Relative intensity
(% of base peak)
EI-GC-MS
(relative)
CI-GC-MS, GC-MS
n
, LC-MS, LC-MS
n
(relative)
>50 % 10 % 20 %
>2050 % 15 % 25 %
>1020 % 20 % 30 %
10 % 50 % 50 %
135
Equations
R
w
w
STDEV
n x x
n n
AVERAGE
x
n
RSD
STD

( )
exp
%
( )
theor
100
1
2
2
EEV
AVERAGE
100 %
w
exp
experimentally measured mass Iraction oI the pesticide residue in the
sample, in the recovery studies the pesticide is spiked into the sample
homogenate |mg kg
-1
|
w
theor
theoretically calculated mass Iraction oI the pesticide residues in the
spiked sample |mg kg
-1
|
n the number oI data points in the set
x Individual data points (in our case x denotes R) in the set
STDEV standard deviation ||
AVERAGE average value oI the data set ||
RSD relative standard deviation ||
136
Measurement data
Imazalil Thiabendazole Imazalil
Thiaben-
dazole
w
exp

(mg kg
-1
)
w
theor
(mg kg
-1
)
R
(%)
w
exp

(mg kg
-1
)
w
theor

(mg kg
-1
)
R
(%)
Peak area Peak area
0.06427 0.05597 0.03120 0.04244 3 996 669 300 802
0.07516 0.05871 0.03281 0.04452 3 459 066 281 164
0.04812 0.05821 0.03181 0.04413 3 838 651 230 775
0.10238 0.07342 0.04095 0.05567 3 727 188 274 366
0.04201 0.06088 0.03400 0.04616 3 414 893 296 724
0.05741 0.06241 0.03331 0.04732 3 553 740 258 916
AVERAGE recovery AVERAGE recovery AVERAGE mass fraction
STDEV of recovery STDEV of recovery STDEV of mass fraction
RSD of recovery (u
rel_rec
) RSD of recovery (u
rel_rec
) RSD of mass fraction
(u
rel_meth
)
* The recovery determinations were carried out two per day on three consecutive days.
137
Equations
u u u
u
u u
w
d w w
u
w

+

sys rnd
rnd
rel_rec rel_meth
ref
ref
2 2
2 2
100 %
+
s
n
u
d
n
u u u
1
2
2 2
dev
sys ref dev
u
w
standard uncertainty oI mass Iraction oI pesticide |mg kg
-1
|
u
sys
systematic component oI uncertainty |mg kg
-1
|
u
rnd
random component oI uncertainty |mg kg
-1
|
u
relrec
relative uncertainty oI recovery ||
u
relmeth
relative uncertainty oI analysis method ||
w pesticide mass Iraction in standard sample as obtained with the measurement
procedure |mg kg
-1
|
d diIIerence in mass Iraction between our laboratory and reIerence value
(laboratory bias) |mg kg
-1
|
w
reI
reIerence mass Iraction oI pesticide in the reIerence sample |mg kg
-1
|
s the standard deviation oI the results oI the participants oI the interlaboratory
comparison |mg kg
-1
|
n
l
the number oI laboratories who took part in interlaboratory comparison
(ILC)
n number oI completed ILCs
u
dev
uncertainty maniIested by the deviation oI the laboratory`s result Irom the
reIerence value |mg kg
-1
|
u
reI
uncertainty oI the reIerence value |mg kg
-1
|
138
Measurement data
Imazalil Thiabendazole Comments
u
rel_rec
27 % 2 %
The relative standard deviation of recovery
calculated from parallel measurement results (two
measurements per day on three consecutive days)
u
rel_meth
10 % 6 %
The relative standard deviation of results obtained
for the same solution from repeated injections of
the same solution
w 1.3350 mg kg
-1
3.5230 mg kg
-1
w
ref
1.2975 mg kg
-1
3.2863 mg kg
-1
consensus value of interlaboratory comparison
measurement
s 0.0530 mg kg
-1
0.5571 mg kg
-1
n
l
2 3
n 1 1
139
TrainMiC Exercises
Determination of concentration of imazalil and thiabendazole
in tangerines by liquid chromatography-mass spectrometry.
in pesticide residues analysis directives and guidelines
EXERCISE 1:
EXERCISE 2:
EXERCISE 3:
Addendum I. By spreadsheet approach
Addendum II. By dedicated software
140
Analyte Residues of imazalil and thiabendazole
Measurand Acetone-extractable imazalil and thiabendazole residues in tangerines
Units mg kg-1 (ppm)
Measurement
procedure
50 g of homogenized sample is extracted with 100 mL of acetone using high speed
blender. Mixture is fltered and the volume of extract is measured.
50 mL of the extract is extracted with 100 mL dichloromethane petroleum ether
mixture (1:1), the organic layer is fltered through a layer of sodium sulphate (for
drying purpose). Water phase is saturated with NaCl and extracted twice with 50
mL of dichloromethane. Organic extracts are dried as before. Solvent is evaporated
to almost dryness and the sample is dissolved in 1020 mL of methanol. Sample is
fltered through a syringe flter and analysed using LC-MS system.
Sample preparation procedure is based on the AOAC ofcial method 985.22
Organochlorine and Organophosphorus Pesticide Residues Gas Chromatographic
Method. The modifcations were made in order to cut down sample size and thus
solvent consumption. Also changes were made in the solvent of fnal extract to suite
LC-MS system. LC-MS analysis method was developed within laboratory.
Type of calibration standard curve Standard addition internal standard
Model equation
w
w V V
V m
c e 10
50
w mass Iraction oI extractable pesticide in sample (mg oI pesticide per kg oI

sample) |mg kg
-1
|
w
c
mass Iraction oI extractable pesticide in analysed extract |mg kg
-1
| (Iound Irom
the calibration curve)
V
10
density oI methanol |g mL
-1
|
V
e
V
50
141
1 Mass fraction of extractable pesticide in analysed extract (w
c
, mg kg
-1
)
2 The full volume of acetone extract (V
e
, mL)
3 The volume of fnal extract in methanol (V
10
, mL)
4 The volume of acetone extract to be purifed (V
50
, mL)
5 The density of methanol (, g mL
-1
)
6 The mass of homogenized sample (m, g)
For the analyte
1
Name/ChemicalFormula/
Producer:
Imazalil (solid substance)/C
14
H
14
Cl
2
N
2
O/Dr. Ehrenstorfer
Value including uncertainty (with units):
Imazalil: purity 97.5 % (tolerance 0.5 %) (data obtained from
corresponding Certifcate of Analysis)
2
Name/ChemicalFormula/
Producer:
Thiabendazole (solid substance)/C
10
H
7
N
3
S/Dr. Ehrenstorfer
Value including uncertainty (with units):
Thiabendazole: purity 99.0 % (tolerance 0.5 %) (data obtained from
corresponding Certifcate of Analysis)
1
None
Are all important parameters included in the model
equation?
Yes No
1 Participate in EU profciency testing programme
2 Analyse a CRM (in future, when such CRM becomes available)
142
143
SINGLE LABORATORY VALIDATION OF
MEASUREMENT PROCEDURES
The measurement
procedure
Sample preparation procedure is modifed AOAC ofcial method 985.22. Analysis
was carried out on an LC-MS system using a self-developed chromatographic
method.
Analyte Residues of imazalil and thiabendazole (polar basic pesticides)
The measurand
Acetone-extractable pesticides in tangerines.
Results are not recovery corrected, thus extractable pesticides are determined, not
total amounts.
Unit mg kg
-1
(ppm)
Matrix Tangerines
Measuring range
imazalil 0.0040.9 mg kg
-1
thiabendazole 0.0030.7 mg kg
-1
Post-registration control and monitoring of pesticides based on MRLs set by
the EU Directives 93/58/EEC and 00/42/EEC for imazalil and thiabendazole
respectively.
Mark the customers
requirements and give their
values
LOD
LOD < 0.02 mg kg
-1
(imazalil), LOD < 0.05
mg kg
-1
(thiabendazole)
LOQ
Repeatability
Within-lab
reproducibility
Trueness Recovery between 70110 %
Measurement
uncertainty
O
t
h
e
r

-

s
t
a
t
e
Identity/confrmation: retention time (compared with
standard) + MS
^2
fragmentation: imazalil (297 201),
thiabendazole (202 175) + additional qualifer ion
comparison if necessary. Guidance document refers to
sufcient confrmation when MS
^2
is used and ion ratios in
standard and sample agree within the limits specifed in
Table 3 (Yellow sheet, Part II).
144
VALIDATION
145
At approximate concentration level of 0.05 mg kg
-1
Chromatographic separation and mass-spectrometric identifcation (including MS
^2
confrmation of
identity)
The method has been proved via ILC to perform with tangerine, orange and tomato
Confrmation of identity: chromatographic retention time and MS^2 confrmation of identity
6. Measuring range
Linearity
Imazalil: 0.0040.9 mg kg
-1
; Thiabendazole: 0.0030.7 mg kg
-1
Upper limit
Imazalil: 0.9 mg kg
-1
; Thiabendazole: 0.7 mg kg
-1
LOD
Imazalil: 0.004 mg kg
-1
; Thiabendazole: 0.003 mg kg
-1
LOQ
7. Spread precision
Repeatability
Instrumental: standard deviation of the measurement method: 10 % for imazalil and 6 % for
thiabendazole (repeated injection of the same standard solution).
Reproducibility (within Lab)
Full procedure: standard deviation of recovery experiments carried out on three consecutive days
27 % for imazalil, 2 % for thiabendazole (full sample preparation included )
Reproducibility (between Lab)
in ILC the diference between results were 5.6 and 4.4 % for imazalil and thiabendazole respectively
146
8. Robustness
Variation of some of the parameters: during method development two diferent columns were used
(C182504.6 5, C18150150 2.1), mobile phase composition and velocity were changed in
increments and obtained data analysedd, fnal extract volumes of 10 and 20 mL were utilized.
9. Quality control
Control charts
R square
Other-state: Confrmation of identity: in accordance with requirements in Section 3.
147
Parameters requested
to be validated
Calculations
LOD
Since the method permits in principle to obtain signifcantly lower LOD values
than requested by the customer, LOD was estimated in a conservative way by
taking the lowest points on the respective calibration graphs as LOD estimates.
Values obtained:
Imazalil: 0.004 mg kg
-1
Thiabendazole: 0.003 mg kg
-1
LOQ
Repeatability
Within-lab
reproducibility
Trueness
Average recovery; for data and equations see frst document
Imazalil 104 %
Thiabendazole 73 %
Recovery is found according to the following equation:
R
w
w

exp
%
theor
100
R recovery of the method [%]
w
exp
experimentally measured mass fraction of the pesticide residue in
the sample, in recovery studies the pesticide sis spiked into the sample
homogenate [mg kg
-1
]
w
theor
theoretically calculated mass fraction of the pesticide residues in the
spiked sample [mg kg
-1
]
Measurement
uncertainty
Other - please
state
148
Parameter
customer
Value obtained
during validation
The requirement
is fulflled
Yes/No
LOD
Imazalil < 0.02 mg kg
-1
Thiabendazole < 0.05 mg kg
-1
0.004 mg kg
-1
0.003 mg kg
-1
Yes
Yes
LOQ
Repeatability
Within-lab
reproducibility
Trueness 70110 %
Imazalil 104 %
Thiabendazole 73 %
Yes
Yes
Measurement
Other
Confrmation based on similarity to
standard (MS
^2
spectrum)
MS
^2
spectrum in
sample is similar to
standard
Yes
Yes No
149
Measurand Extractable pesticide content in fruit/vegetable
Unit mg kg
-1
equation
50 g oI homogenised sample is extracted with 100 mL oI acetone using high speed blender.
Mixture is fltered and the volume oI extract is measured. 50 mL oI extract is extracted with
100 mL dichloromethane petroleum ether (4060 C) mixture (1:1), organic layer is
fltered through a layer oI sodium sulphate (Ior drying purpose). Water phase is saturated
with NaCl and extracted twice with 50 mL oI dichloromethane. Organic extracts are
dried as beIore. Solvent is evaporated to almost dryness and the sample is dissolved in
1020 mL oI methanol (volume might be even larger iI the concentration does not ft
in the calibration graph range). Sample is fltered through a syringe flter and analysed
using LC-MS system.
Sample preparation procedure is modifed AOAC oIfcial method 985.22. Analysis was
carried out on an LC-MS system using selI developed method.
Model equation
w
w V V
V m
c e 10
50
w mass Iraction oI pesticide in sample |mg kg

-1
|
w
c
mass Iraction oI pesticide in analysed extract |mg kg
-1
|
V
10
-1
|
V
e
V
50
150
Uncertainty estimation is carried out using the Nordtest method
1
. In principle the
Nordtest approach can be regarded as a special case oI the ISO method where very
general uncertainty sources are considered.
The main equation:
u u u
w sys rnd
+
2 2
u
w
standard uncertainty oI mass Iraction oI pesticide |mg kg
-1
|
u
sys
systematic component oI uncertainty |mg kg
-1
|
u
rnd
-1
|
The two main uncertainty sources are:
1. Uncertainty u
sys
, which is due to the systematic eIIects laboratory bias and method
bias. In the original document it is denoted also as u(bias). This component is Iound Irom
analysis oI CRM-s or Irom participating in ILC-s.
2. Uncertainty u
rnd
, which is due to the random eIIects within-laboratory between-day
reproducibility. In the original document it is denoted also as u(R
w
). This component is
Iound Irom routine between-day reproducibility monitoring oI the method (using e.g. a
control chart).
The component u
sys
in turn is Iound according to the Iollowing Iormula:
u u u
w
+
dev ref
2 2
where u
dev
denotes uncertainty maniIested by the deviation oI the laboratory`s result
Irom the reIerence value (denoted by RMS
bias
in the original document) and u
reI
denotes
the uncertainty oI the reIerence value (denoted as u(c
reI
) in the original document).
The quantifcation oI the uncertainty components is carried out according to the Iollowing
Iormulae:
1
Nordtest Report TR 537. Handbook Ior Calculation oI Measurement Uncertainty in
Environmental Laboratories. B. Magnusson, T. Nykki, H. Hovind, M. Krysell. Available
on the web at http://www.nordicinnovation.net/nordtestfler/tec537.pdI
151
u
u u
w
d w w
u
s
n
u
d
n
rnd
rel_rec rel_meth
ref
ref
1
dev
2 2
2
100 %
u
relrec
u
relmeth
w pesticide mass Iraction in a reIerence sample as determined by the
measurement procedure |mg kg
-1
|
w
reI
reIerence mass Iraction oI pesticide in the reIerence sample |mg kg
-1
|
d diIIerence in mass Iraction between our laboratory and reIerence value
|mg kg
-1
|
s the standard deviation Ior reIerence value |mg kg
-1
|
n
l
the number oI laboratories who took part in ILC
n number oI completed ILCs (n 1 in our case)
u
dev
uncertainty maniIested by the deviation oI the laboratory`s result Irom the
reIerence value |mg kg
-1
|
u
reI
-1
|
Method bias
Matrix efect: matrix efects on ionisation of pesticides (repeatability)
Other: repeatability of extraction of the pesticides
Other: stability of standard solutions, integration
Other: calibration graph linearity
152
Input
quantity
Value
Unit Remark
Imazalil Thiabendazole
w
c
2.801 7.398 mg kg
-1
Mass fraction of residue in extract, calculated based
on calibration
V
10
10 10 mL Volume of fnal methanol extract
0.791 0.791 g mL
-1
Density of methanol
V
e
150 150 mL Volume of extract after fltration
V
50
50 50 mL Volume of extract taken for further cleaning
m 49.8003 49.8003 g Sample amount taken for extraction
2
Standard uncertainty
Unit Remark
Imazalil Thiabendazole
u
ref
0.0375 0.3216 mg kg
-1
systematic uncertainty component evaluated based
on the results of ILC
u
dev
0.0375 0.2367 mg kg
-1
random component of uncertainty, calculated using
relative uncertainty (repeatability) of recovery and
method
u
rel_rec
27 2 %
relative standard deviation of recoveries calculated
using addition experiments
u
rel_meth
10 6 %
relative standard deviation of measuring method
(repeated analysis of the same solution)
w
w V V
V m
c e 10
50
w mass Iraction oI pesticide in sample |mg kg

-1
|
w
c
mass Iraction oI pesticide in analysed extract |mg kg
-1
|
V
10
-1
|
V
e
V
50
2
The Nordtest method does not require separate uncertainty evaluation Ior each input
quantity.
153
w( )
. .
.
. imazalil mg kg
-1

2 801 10 0 791 150
50 49 8003
1 335
w( )
. .
.
. thiabendazole mg kg
-

7 398 10 0 791 150
50 49 8003
3 523
11
w
Uncertainty
components
Value
Standard
uncertainty
Unit Remark
u
sys
(imazalil) - 0.0530 mg kg
-1
systematic uncertainty component
evaluated based on the results of ILC
u
rnd
(imazalil) - 0.3844 mg kg
-1
random component of uncertainty,
calculated using relative uncertainty
(repeatability) of recovery and method
u
sys
(thiabendazole) - 0.3993 mg kg
-1
systematic uncertainty component
evaluated based on the results of ILC
u
rnd
(thiabendazole) - 0.2228 mg kg
-1
random component of uncertainty,
calculated using relative uncertainty
(repeatability) of recovery and method
Uncertainty is Iound according to the the Nordtest approach using the Iollowing
set oI equations:
u u u
u
u u
w
d w w
u
w sys rnd
rnd
rel_rec rel_meth
ref
ref
+

2 2
2 2
100 %
+
s
n
u
d
n
u u u
1
dev
sys ref dev
2
2 2
u
w
standard uncertainty oI mass Iraction oI
pesticide |mg kg
-1
|
u
sys
systematic component oI uncertainty |mg
kg
-1
|
u
rnd
-1
|
u
relrec
u
relmeth
w pesticide mass Iraction in sample |mg kg
-1
|
d diIIerence in mass Iraction between our
laboratory and reIerence value |mg kg
-1
|
w
reI
reIerence mass Iraction oI pesticide in
sample |mg kg
-1
|
s the standard deviation Ior reIerence value
|mg kg
-1
|
n
l
the number oI laboratories who took part in
ILC
n number oI completed ILCs
154
u
dev
uncertainty maniIested by the deviation oI
the laboratory`s result Irom the reIerence
value |mg kg
-1
|
u
reI
-1
|
w
) and specify the coverage factor k and the units
Imazalil
u
u
w
-1
mg kg

+
0 05303 0 3844 0 388

27 10
100
1
2 2
2 2
. . .
% %
%
.
rnd
33350 0 3844
1 3350 1 2975 0 0375
0
.
. . .
mg kg
mg kg
-1
-1
d
u
ref
..
.
.
.
0530
2
0 0375
0 0375
1
0 0375
0
2
mg kg
mg kg
-1
-1
u
u
dev
sys
.. . . 0375 0 0375 0 05303
2 2
+ mg kg
-1
Thiabendazole
u
u
w
rnd
+
+

0 3993 0 2228 0 457
2 6
100
3 860 0
2 2
2 2
. . .
% %
%
. .
mg kg

-1
22228
3 5230 3 2863 0 2367
0 5571
2
0 32
mg kg
mg kg
-1
-1
d
u

. . .
.
.
ref
116
0 2367
1
0 2367
0 3216 0 2367
2
2 2
mg kg
mg kg
-1
-1
u
u
dev
sys

+
.
.
. . 00 3993 . mg kg
-1
U(imazalil) 2 u
w
(imazalil) 2 0.388 mg kg
-1
0.676 mg kg
-1
(norm, k 2)
U(thiabendazole) 2 u
w
(thiabendazole) 2 0.457 mg kg
-1
0.914 mg kg
-1
(norm, k 2)
155
contributing the most to u
w
1 u
rnd
contribution: 98.13 % (imazalil), 23.74 % (thiabendazole)
2 u
sys
contribution: 1.87 % (imazalil), 76.27 % (thiabendazole)
The Nordtest approach does not permit to obtain an uncertainty budget similar to that
obtained with the ISO GUM approach. This is because it is not a model-based approach
to uncertainty estimation: although the model equation is used Ior calculating the value
oI the result, it is not used to calculate the uncertainty.
It is however possible to separate the overall uncertainty into random and systematic
contributions. From the above table it can be deduced that the uncertainty oI determination
oI imazalil is mostly attributable to random sources and the uncertainty oI determination
oI thiabendazole is mostly attributable to systematic sources. This conclusion should
however be treated with care. The reason is that the systematic uncertainty component
oI thiabendazole is signifcantly larger than that oI imazalil. Both systematic components
were determined Irom the results oI a single interlaboratory comparison and the result
oI thiabendazole deviated more Irom the reIerence value oI the ILC than the result oI
imazalil and also the reliability oI the thiabendazole reIerence value was signifcantly
lower (see calculations in Section 7). Since all the inIormation is based on a single
ILC, Iar-reaching conclusions are not possible at this stage and the uncertainty estimate
should be regarded as the frst estimate`. As the work oI the laboratory evolves and
more data become available, better uncertainty estimates can be Iound using the same
calculation scheme.
156
Further readings
1. AOAC OIfcial Method 985.22. Organochlorine and organophosphorus pesticide
residues gas chromatographic method.
2. Document No. SANCO/10476/2003. Quality control procedures Ior pesticide residues
analysis (5 February 2004), 130 (the current revision oI this document is Document No.
SANCO/2007/3131). Method validation and quality control procedures Ior pesticide
residues analysis in Iood and Ieed (31 October 2007).
3. B. Magnusson, T. Nykki, H. Hovind and M. Krysell, Handbook for Calculation of
Measurement Uncertainty in Environmental Laboratories, 2nd ed. (2003), ISSN 0283-234
(Nordtest technical report: http://www.nordicinnovation.net/nordtestfler/tec537.pdI).
157
Chapter 5
Determination of Ammonium in Water by Continuous
Flow Analysis (CFA) and Spectrometric Detection
Bertil Magnusson
The summary form (blue page)
158
The TrainMiC example summary form
Measurand Mass concentration of ammonium in drinking water in mg L-1
Author(s) of the example Bertil Magnusson
Determination of concentration of ammonium in drinking water by
continuous fow analysis (CFA) and spectrometric detection (ISO 11732:
2005)
Customer requirement
Directive 98/83/EC on the quality of water intended for human
consumption
159
Determination of Ammonium in Water by Continuous Flow Analysis (CFA) and Spectrometric Detection
II. Attached fles
File number, type
and name
Content of the fle
File is
attached Remark
Yes No
1

-

I
Ex-07-1-I-
NH4-water-
Photometry-
2006-Ver1.ppt
About the analytical procedure: short
introduction

Given by the
lecturer
2

-

Y
e
l
l
o
w
Ex-07-2-Y-
NH4-water-
Photometry-
2006-Ver1.doc
PART I
Description of the analytical
procedure

Each participant
receives own
copy and may
keep it
PART II
measurement result
PART III
measurement data
PART IV
Measurement uncertainty of the
result relevant equations and
measurement data
3

-

G
r
e
e
nEx-07-3-G-
NH4-water-
Photometry-
2006-Ver1.doc
PART I
chemistry

PART II

PART III

Addendum 1: By spreadsheet
approach
-

0 April 2007 -
1
Determination of Ammonium in Water by Continuous Flow Analysis (CFA) and
Spectrometric Detection
160
Photometric determination of
ammonium in drinking water
Model equation
Water quality -- Determination of ammonium nitrogen
-- Method by flow analysis (CFA and FIA) and
spectrometric detection
ISO 11732 (2005)
1he quality of the results should comply with the
requirement in the directive 98/83/EC on the quality of
water intended for human consumption (in preparation)
161
Ammonium present in the sample reacts in alkaline
solution with hypochlorite to Iorm chloramines.
The chloramines Iormed reacts under catalysis oI
nitroprusside with salicylate at a temperature oI 3750 C
to Iorm a bluegreen indophenol dye.
The absorbance oI the nitroprusside is quantitatively
measured in a Ilow photometer at 640660 nm by
comparing with a calibration curve.
Instrumental setup
Sample
Reagent
Reagent
Reagent
Reaction coil
Uv
detector
162
C |(A
sample
- b
0
) / b
1
| ( f
dil
/ R)
Model Equation
Recovery Iactor oI the analysis unitless R
Dilution Iactor unitless f
dil
Slope oI calibration line AU L mg
-1
N b
1
Intercept oI calibration line AU b
0
Absorbance oI the sample solution AU A
sample
Concentration oI NH
4
in the sample solution mg L

-1
N C
Definition Unit Quantity
163
Model equation and equation for
measurement uncertainty calculation
( )
4
NH
c
u k U =
C |(A
sample
- b
0
) / b
1
| ( f
dil
/ R)
164
Determination of concentration of ammonium in drinking water
by fow analysis (CFA and FIA) and spectrometric detection.
in the Directive 98/83/EC on the quality intended for human
consumption
PART I ................................................................................................................................ 165
PART II .............................................................................................................................. 168
The customers requirement concerning quality of the measurement result
PART III ............................................................................................................................. 169
measurement data
PART IV ................................................................................................................. 171
data
165
1. Scope
This International Standard specifes methods suitable Ior the determination oI ammonium
nitrogen in various types oI waters (ground, drinking, surIace and waste waters) in mass
concentration ranging Irom 0.1 to 1 mg L
-1
(in the undiluted sample), applying either FIA
(Clause 3) or CFA (Clause 4). Here the CFA is presented.
2. Principle CFA Continuous Flow Analysis
In a continuously fowing, gas-segmented carrier stream, ammonium present in the
sample reacts in alkaline solution with hypochlorite, which has previously been liberated
Irom dichloroisocyuanurate.
The chloramines Iormed reacts under catalysis oI nitroprusside with salicylate at a
temperature oI 3750 C to Iorm a blue-green indophenol dye which is quantitatively
measured in a fow photometer at 640660 nm.
3. Interferences CFA method
Low-molecular amines react similarly to ammonia and their presence will consequently
lead to erroneously high results. InterIerences may also occur with strong acidic or
buIIered samples as well as samples with particles or high concentration oI metals
Iorming hydroxide precipitates.
166
4. Reagents here only calibrant is described
Ammonium stock solution, C
N
= 1000 mg L
-1
Dissolve in a 1000 mL volumetric fask 3.819 g oI ammonium chloride (p.a. min 99 ,
dried at 105 C + 2 C to constant mass) in approximately 900 mL oI water, acidiIy to pH
2 by drop wise addition oI dilute sulphuric acid and make up to a volume with water.
Standard solutions, 10 mg L
-1
Pipette 1 mL oI the ammonium stock solution into a 100 mL graduated fask, add
approximately 80 mL oI water, acidiIy by drop wise addition oI dilute sulphuric acid and
make up to a volume with water.
Calibration solutions
Prepare calibration solutions by diluting the ammonium standard solutions. At least
fve calibration standards per working range are recommended. As an example, iI six
standard are applied proceed Ior the working ranges 0.11.0 mg L
-1
N, as Iollows.
Working solutions 0.11.0 mg L
-1
Pipette into a series oI 100 mL graduated fasks 1, 2, 4, 6 and 10 mL respectively oI the
ammonium standard solution 10 mg L
-1
and make up to volume with water.
Prepare all calibration solutions Ireshly beIore use.
Other chemicals needed see ISO standard 11732:2005
5. Sampling and pre-treatment
Sampling shall be carried out in accordance with ISO 5667-1, ISO 5667-2, ISO 5667-3.
Containers oI glass, PE, PP and PTFE are suitable Ior sample collection. Clean all containers
with HCl (1 M, 0.1 and 0.01 M) and rinse with water. Analyse the samples immediately aIter
collection. For preservation up to 24 h, add dilute sulphuric acid to pH about 2 and store at
25 C in the dark.
167
6. Procedure
Instrument set-up
Prior to measurement, continuously run the reagent solutions Ior approximately 10 min
through the system, and record the baseline. The system is operational when the baseline
does not show any driIt. A satisIactory signal-to-noise relation should be obtained.
PerIorm the calibration with the blank solutions and 4 to 5 equidistant calibration
solutions Ior an appropriate concentration range. It should be stressed that the linearity
oI the calibration curve is oIten limited. Correct the absorbance values oI the calibration
solutions by subtracting the absorbance value oI the blank calibration solution. For
plotting oI a calibration curve or Ior calculation oI the calibration Iunction, use the
resulting values together with the analyte concentrations oI the calibration solutions.
Analyse the test samples in the same way as the calibration solutions with the continuous
fow system.
7. Method of calculation
Read the values oI the analyte concentrations oI the test sample solutions, the reagent blank
solution and the blank test solution Irom the calibration graph or calculate them Irom the
calibration Iunction. Correct the analyte concentrations oI the test sample solutions by
subtracting the analyte concentrations oI the reagent blank solutions or the blank test solution.
Correct Ior dilution steps, iI appropriate. Report results ammonia expressed as nitrogen
in mg L
-1
.
168
Extract from the Directive 98/83/EC (Draft annex 2005/04/20), on the quality of water

The parametric value (max limit) Ior ammonium in drinking water is 0.5 mg L
-1
.
The requirements Ior the analyses are the Iollowing:
Parameter Trueness of parametric value
(Note 1)
Precision of
parametric value
(Note 2)
Limit of detection of
parametric value
(Note 3)
Ammonium 10 % 10 % 10 %
Note 1
(*):
Trueness is the systematic error and is the diIIerence between the mean
value oI the large number oI repeated measurements and the true value
Note 2
(*):
Precision is the random error and is usually expressed as the standard
deviation (within and between batch) oI the spread oI results about the
mean. Acceptable precision is twice the relative standard deviation.
(*) These terms are Iurther defned in ISO 5725.
Note 3: Limit oI detection is either:
three times the relative within batch standard deviation oI a natural
sample containing a low concentration oI the parameter, or
fve times the relative within batch standard deviation oI a blank
sample.
PART II. The customers requirement concerning quality of the
measurement result
169
Limit of Detection
Equation
Calculate detection limit as 5 standard deviations see Directive 98/83/EC.
Measurement data
A synthetic control sample at a level oI 0.020 mg L
-1
has been run Ior over a period oI 7
months. The results in mg L
-1
are given in the table below.
0.021 0.032 0.023
0.023 0.023 0.024
0.023 0.025 0.022
0.024 0.026 0.024
0.026 0.022 0.025
0.015 0.021 0.022
0.015 0.019 0.021
0.017 0.025 0.020
0.016 0.026 0.021
0.014
Internal quality control
The standard deviations obtained on these control samples are estimates on the within-
laboratory reproducibility.
Measurement data
Results Irom two control samples are given in the table below
Unit QC1 QC2
Mean value mg L
-1
0.114 0.605
s mg L
-1
0.005 0.021
n - 27 28
Time period months 7 7
Nominal value mg L
-1
0.100 0.600
170
External quality control participating in PT studies
Year/Exercise
Nominal value
x
ref
[Mg l
-1
]
laboratory result x
i
[mg L
-1
]
Bias
[%]
s
R
[%]
Number of
labs
1999/1 81 83 2.4 10 31
1999/2 73 75 2.7 7 36
2000/1 264 269 1.9 8 32
2000/2 210 213 1.4 10 35
2001/1 110 112 1.8 7 36
2001/2 140 144 2.9 11 34
171
The relevant equations
C = (A
sample
b
0
) / b
1
f
dil
/ R
172
M
e
a
s
u
r
e
m
e
n
t

d
a
t
a
Q
u
a
n
t
i
t
y
U
n
i
t
V
a
l
u
e
S
t
a
n
d
a
r
d

u
n
c
e
r
t
a
i
n
t
y

R
e
l
a
t
i
v
e

s
t
a
n
d
a
r
d

u
n
c
e
r
t
a
i
n
t
y

T
y
p
e

o
f

u
n
c
e
r
t
a
i
n
t
y
T
y
p
e

o
f

d
i
s
t
r
i
b
u
t
i
o
n
(
u
)
(
%
)
n
o
r
m
a
l
r
e
c
t
a
n
g
u
l
a
r
t
r
i
a
n
g
u
l
a
r
C
C
o
n
c
e
n
t
r
a
t
i
o
n

o
f

N
H
4
+

i
n

t
h
e

s
a
m
p
l
e

s
o
l
u
t
i
o
n
m
g

L
-
1
0
.
2
4
6
5
0
.
0
0
3
1
1
.
3
x
A
s
a
m
p
l
e
A
b
s
o
r
b
a
n
c
e

o
f

t
h
e

s
a
m
p
l
e

s
o
l
u
t
i
o
n
A
U
0
.
2
5
6
0
0
.
0
0
1
5
0
.
5
8
x
b
0
I
n
t
e
r
c
e
p
t

o
f

c
a
l
i
b
r
a
t
i
o
n

l
i
n
e
A
U
0
.
0
1
4
3
0
.
0
0
2
1
1
4
x
b
1
S
l
o
p
e

o
f

c
a
l
i
b
r
a
t
i
o
n

l
i
n
e

u
n
i
t

A
U

d
i
v
i
d
e
d

b
y

m
g

N

L
-
1
A
U

L

m
g
-
1
0
.
9
9
0
2
0
.
0
0
7
0
0
.
7
1
x
f
d
i
l
D
i
l
u
t
i
o
n

f
a
c
t
o
r
u
n
i
t
l
e
s
s
1
0
.
0
0
0
.
0
0
R
R
e
c
o
v
e
r
y

f
a
c
t
o
r

o
f

t
h
e

a
n
a
l
y
s
i
s
u
n
i
t
l
e
s
s
0
.
9
9
0
0
0
.
0
0
5
8
0
.
5
9
x
173
TrainMiC Exercises
Determination of concentration of ammonium in drinking
water by continuous fow analysis (CFA) and spectrometric
detection
in the Directive 98/83/EC on the quality of water intended for
human consumption
EXERCISE 1:
EXERCISE 2:
EXERCISE 3:
Addendum I: By spreadsheet solution
174
Analyte Ammonium
Measurand Dissolved ammonium in water sample arriving in the laboratory
Units mg L
-1
Measurement
procedure
ISO 11732:2005 using the continuous fow analysis and photometric detection
Model equation
C (A
sample
b
0
) / b
1
f
dil
/R
C Concentration oI NH
4
in the sample solution |mg L

-1
|
A
sample
Absorbance oI the sample solution |AU|
b
0
Intercept oI calibration line |AU|
b
1
Slope oI calibration line |AU divided by mg L
-1
|
f
dil
Dilution Iactor
R Recovery Iactor oI the analysis
The calibration line evaluated by linear regression is based on fve standards in the range
0.041 mg L
-1
.
The dilute standard solution oI 10 mg L-1 is prepared Irom a stock solution oI 1000 mg
L
-1
. This stock solution is prepared Irom ammonium chloride.
1 Recovery factor contributing 30 % to the expanded uncertainty
2 Absorbance of the sample - here the main source is the drift contributing about 20%
3 Calibration standard solution purity of ammonium chloride
4 Calibration volumetric fasks and pipettes
175
For the analyte
1 Name/ChemicalFormula/Producer: Ammonium chloride, NH4Cl, Merck pa min 99 %
2 Name/ChemicalFormula/Producer:
1
Absorbance relative measurement. Not direct part of
the traceability chain
2 Quantity/Equipment/Calibration: Volumetric fasks Class A quality
Volumetric pipettes calibrated by producer and
regularly checked by the laboratory
model equation?
Yes No
Other important parameters are: Within-lab reproducibility, contamination
1 Participating in PT rounds
2
3
176
The measurement procedure Measurement procedure is based on EN/ISO11732
Analyte Ammonium
The measurand Dissolved ammonium in water sample arriving in the laboratory
Unit mg L
-1
Matrix Drinking water
Measuring range up to 1 mg L
-1
for undiluted samples
Intended use of the results To analyse drinking water according to the EU requirements in the EU directive
Mark the customers
their values
LOD
LOD 0.05 mg L
-1
: - 3s on a natural sample,
5s on a blank: s is repeatability
LOQ
Repeatability
Within-lab
reproducibility
at 0.5 mg L
-1
, s = 0.025 mg L
-1
:
at 0.2 mg L
-1
, s the demand estimated to be
s = 0.010 mg L
-1
or 5 %
Trueness
at 0.5 mg L
-1
less than 0.05 mg L
-1
or less than
10 % relative
Measurement
uncertainty
Other-state
VALIDATION
177
6. Measuring range
Linearity
Upper limit
LOD
LOQ
7. Spread precision
Repeatability
8. Robustness
178
9. Quality control
Control charts
R square
179
3
Parameters requested
to be validated
Calculations
LOD
s = 0.004 mg L
-1
LOD = 5s = 0.02 mg L
-1
LOQ
Repeatability
At a level of 0.1 mg L
-1
s
Rw
is 4.4 % and at a level of
0.6 mg L
-1
s
Rw
is 3.5 %.
Trueness
From PT results the trueneness is estimated to be less than
3 %. The trueness is probably around 2 % - then mean value of
the PT results for levels over 0.08 mg L
-1
.
The measurement uncertainty at a level of 0.2 mg L
-1
is estimated
to be 2.5 %. According to EA guideline this value should be
rounded of to 3 %.
3
3
Comment: This value oI 2.5 obtained using the model approach is probably an
underestimate since some Iactors are not taken into account e.g. contamination, within-
lab reproducibility. A routine laboratory would typically have an uncertainty oI 15 at
this level due to contamination and an expert laboratory may typically have an uncertainty
oI 5 to 10 .
180
Parameter
customer
Value obtained
during validation
The requirement
is fulflled
Yes/No
LOD 0.05 mg L
-1
0.02 mg L
-1
Yes
LOQ
Repeatability
Within-lab
reproducibility
5 % at a level of 0.2 mg L
-1
4 % Yes
Trueness 10 % 2-3 % Yes
Measurement
uncertainty
Other
The analytical procedure is t for the intended use:
Yes No
181
Measurand Dissolved ammonium in water sample arriving in the laboratory
Unit mg L
-1
equation
Ammonium present in the sample reacts in alkaline solution with hypochlorite. The
chloramines Iormed reacts under catalysis oI nitroprusside with salicylate at a temperature
oI 3750 C to Iorm a blue-green indophenol dye which is quantitatively measured in a
fow photometer at 640660 nm.
Model equation
C = (A
sample
b
0
) / b
1
f
dil
/ R
C concentration oI NH
4
in the sample solution |mg L

-1
|
A
sample
absorbance oI the sample solution |AU|
b
0
intercept oI calibration line |AU|
b
1
slope oI calibration line |AU divided by mg L
-1
|
f
dil
dilution Iactor
R recovery Iactor oI the analysis
Method bias
Matrix efect
Other: measurement of sample
Other: Preparation, measurement of calibration solutions and constructing the calibration graph
Other:
182
A
sample
0.256 AU
b
0
0.01734 AU
b
1
986.3 AU L mg
-1
f
dil
1 unitless
R 0.99 unitless
Input
quantity
Standard
uncertainty
Unit Remark
A
sample
1.49 10
-3
AU Takes into account repeatability, drift and rounding
b
0
0.00207 AU
b
1
0.0070 AU L mg
-1
Takes into account reference solution (0.3 % relative
uncertainty, preparation and measurement of calibration
standards and constructing the calibration graph
f
dil
0 unitless
Dilution of sample in this case the sample was not
diluted
R 0.0058 unitless A rough estimate of recovery of 99 1 %
6. Calculate the value of the measurand, using the model equation.
C = (A
sample
b
0
) / b
1
f
dil
/ R;
C

0 256 0 01734
986 3
1 0 99 0 247
. .
.
. . mg L
-1
183
7. Calculate the combined standard uncertainty (uc) of the result and specify units
Using: Mathematical solution; Spreadsheet Approach; Commercial SoItware
Input
quantity
Value
Standard
uncertainty
Unit Remark
A
sample
0.256 1.49 10
-3
AU
b
0
0.01435 0.00207 AU
From calibration graph note regression without
weights and a slight curvature. A too high
estimate but here we are interested in higher
concentrations.
b
1
0.9902 0.0070 AU L mg
-1
f
dil
1 0 Unitless Sample was not diluted
R 0.99 0.01 Unitless
C = (A
sample
b
0
) / b
1
f
dil
/ R
The combined standard uncertainty is 0.0031 mg L
-1
.
8. Calculate expanded uncertainty (Uc) and specify the coverage factor k and the
units
U k u 2 0 0031 0 006 . . mg L
-1
The expanded uncertainty using a coverage Iactor oI 2 is 0.006
2
mg L
-1
N or 2.5
relative.
c
1 Recovery factor contributing 20 % to the expanded uncertainty
2
Absorbance of the sample - here the main source is the drift contributing about 20 % to the expanded
uncertainty
3
Preparation of standard solution 10 mg L
-1
0.13 mg L
-1
(k = 2) - main components dilution using a
1mL pipette and purity contribution about 25 %
184
Further readings
1. EN standard ISO 11732.
2. EC 1998, Council Directive 98/83/EC oI 3 November. The quality oI water intended
Ior human consumption, Ofc. J. Eur. Commun. L330 (1998).

3. B. Magnusson, T. Nykki, H. Hovind and M. Krysell Handbook for Calculation of
Measurement Uncertainty in Environmental Laboratories, 2nd ed. (2003), ISSN 0283-234
(Nordtest technical report: http://www.nordicinnovation.net/nordtestfler/tec537.pdI).
185
Addendum: Measurement uncertainty calculation -
GumWorkbench
The values oI uncertainty components oI volumetric ware and photometric equipment
are taken according to experience, experiments carried out in the lab and data Irom
equipment manuIacturers. The calculations are here based on a manual method Ior a
clearer view oI the calculations but an automated method will give similar or lower
uncertainty.
Model equation:
{ The main equation }
C = (A
sample
- b
0
) / b
1
f
dil
/ R;
{ Nitrogen- Ammonium ion stock solution - 1000 mg N L
-1
. Prepared from ammonium
chloride.}
C
st_0
= m
NH4Cl
/ V
1000
P
NH4Cl
f
NH4Clconv
1000;
{ Ammonium standard solution - 10 mg N/L. Prepared from ammonium stock solution.
The standard solution is further used for preparation of the calibration standard
solutions. }
C
st
= C
st_0
V
1
/ V
100
;
{ Concentrations of calibration standard solutions 0.1 to 1 mg N L
-1
.
1 to 10 mL of the standard solution is transferred to 100 mL volumetric asks.
The reagents are added and the solution is made up to the mark. The solution is left to
stand for 60 min and then the absorbance at 655 nm is measured. }
C
1
= C
st
(V
1_st
/ V
1_100
);
C
2
= C
st
(V
2_st
/ V
2_100
);
C
3
= C
st
(V
3_st
/ V
3_100
);
C
4
= C
st
(V
4_st
/ V
4_100
);
C
5
= C
st
(V
5_st
/ V
5_100
);
f
dil
= 1;
{in this case the sample was not diluted}
{ Photometric measurements
It is assumed that the uncertainty of all photometric measurements consists of three
components (on the example A
sample
):
Repeatability uncertainty (included in A
sample_rep
);
Uncertainty due to drift (A
sample_drift
)
Uncertainty due to rounding of the reading (A
sample_round
) (The photometer use din this
example has three decimal places)
The absorbance of blank is not subtracted but all the measurements are made against
blank}
{ Absorbance of sample solution }
A
sample
= A
sample_rep
+A
sample_drift
+A
sample_round
;
186
{ The regression equations for nding the slope (b
1
) and intercept (b
0
) of the calibration
line }
AC = C
1
A
1
+ C
2
A
2
+ C
3
A
3
+C
4
A
4
+ C
5
A
5
;
AvgC=(C
1
+C
2
+C
3
+C
4
+C
5
)/n;
AvgA=(A
1
+A
2
+A
3
+A
4
+A
5
)/n;
CC=C
1
C
1
+C
2
C
2
+C
3
C
3
+C
4
C
4
+C
5
C
5
;
b
1
=(AC-n

AvgC

AvgA)/(CC-n

AvgC

AvgC);
b
0
=AvgA-b
1
AvgC
List of quantities:
C mg N L
-1
Concentration of NH
4
+
in the sample solution
A
sample
AU Absorbance of the sample solution
b
0
AU Intercept of calibration line
b
1
AU L mg
-1
Slope of calibration line
f
dil
unitless Dilution factor
R unitless Recovery factor of the analysis
C
st_0
mg N mL
-1
Concentration of NH
4
+
in calibration stock solution
m
NH4Cl
g Weight of NH
4
Cl
V
1000
mL Volume of 1 L volumetric fask
P
NH4Cl
unitless Purity of NH
4
Cl
f
NH4Clconv
unitless
Conversion factor for converting the amount of ammonium chloride (NH
4
Cl)
to the amount of nitrogen
C
st
mg N L
-1
Concentration of NH
4
+
in the ammonium standard solution
V
1
mL Volume of 1 mL pipette
V
100
mL Volume of 100 mL volumetric fask
C
1
mg N L
-1
Concentration of the 1st ammonium calibration standard solution
V
1_st
mL
Volume of ammonium standard solution taken for preparing the 1st
ammonium calibration standard solution
V
1_100
mL Volume of the 1st ammonium calibration standard solution
C
2
mg N L
-1
Concentration of the 2nd

V
2_st
mL
Volume of ammonium standard solution taken for preparing the 2nd
V
2_100
mL Volume of the 2nd ammonium calibration standard solution
C
3
mg N L
-1
Concentration of the 3rd ammonium calibration standard solution
187
V
3_st
mL
Volume of ammonium standard solution taken for preparing the 3rd
V
3_100
mL Volume of the 3rd ammonium calibration standard solution
C
4
mg N L
-1
Concentration of the 4th ammonium calibration standard solution
V
4_st
mL
Volume of ammonium standard solution taken for preparing the 4th
V
4_100
mL Volume of the 4th ammonium calibration standard solution
C
5
mg N L
-1
Concentration of the 5th ammonium calibration standard solution
V
5_st
mL
Volume of ammonium standard solution taken for preparing the 5th
V
5_100
mL Volume of the 5th ammonium calibration standard solution
A
sample_rep
A
sample_drift
A
sample_round
AC - Interim quantity for regression statistics calculation
A
1
AU Absorbance of the 1th ammonium calibration standard solution
A
2
AU Absorbance of the 2nd ammonium calibration standard solution
A
3
AU Absorbance of the 3rd ammonium calibration standard solution
A
4
A
5
AvgC mg N L
-1
Interim quantity for regression statistics calculation
n unitless Number of points on the calibration line
AvgA AU Interim quantity for regression statistics calculation
CC - Interim quantity for regression statistics calculation
R:
Value: 0.99 unitless
HalIwidth oI limits: 0.01 unitless
m
NH4Cl
:
Value: 3.819 g
188
V
1000
:
Value: 1 mL
HalIwidth oI limits: 0.001 mL
P
NH4Cl
:
Value: 0.995 unitless
HalIwidth oI limits: 0.005 unitless
f
NH4Clconv
:
Constant
Value: 14.0067/(14.0067 4 1.0079 35.4527)
V
1
:
Value: 1 mL
V
100
:
Value: 100 mL
V
1_st
:
Value: 1 mL
V
1_100
:
Value: 100 mL
V
2_st
:
Value: 2 mL
V
2_100
:
Value: 100 mL
189
V
3_st
:
Value: 4 mL
V
3_100
:
Value: 100 mL
V
4_st
:
Value: 6 mL
V
4_100
:
Value: 100 mL
V
5_st
:
Value: 10 mL
V
5_100
:
Value: 100 mL
A
sample_rep
:
Type A summarized
Mean: 0.256
Standard Uncertainty: 654 10
-6
Degrees oI Freedom: 50
A
sample_drift
:
Type A summarized
Mean: 0
Standard Uncertainty: 1.3 10
-3
Degrees oI Freedom: 50
190
A
sample_round
:
Value: 0
HalIwidth oI limits: 0.0005
A
1
:
Value: 0.108 AU
HalIwidth oI limits: 0.001 AU
A
2
:
Value: 0.214 AU
A
3
:
Value: 0.412 AU
A
4
:
Value: 0.606 AU
A
5
:
Value: 0.9979 AU
n:
Constant
Value: 5 unitless
191
Uncertainty budgets:
C: Concentration oI NH
4
in the sample solution

Quantity Value
Standard
uncertainty
Distribution
Sensitivity
coefcient
Uncertainty
contribution
Index
A
sample
0.25600 AU 1.48 10
-3
AU
b
0
0.01435 AU 2.07 10
-3
AU
b
1
0.99023
AU L mg
-1
7.01 10
-3
AU L mg
-1
f
dil
1.0 unitless 0.0 unitless
R 0.99000 unitless
5.77 10
-3

unitless
rectangular -0.25 -1.4 10
-3
mg L
-1
21.3 %
C
st_0
995.01 mg mL
-1
2.96 mg mL
-1
m
NH4Cl
3.81900 g 1.15 10
-3
g rectangular 0.065 75 10
-6
mg L
-1
0.0 %
V
1000
1.000000 mL 577 10
-6
mL rectangular -0.25 -140 10
-6
mgL
-1
0.2 %
P
NH4Cl
0.99500 unitless
2.89 10
-3

unitless
rectangular 0.25 720 10
-6
mg L
-1
5.3 %
f
NH4Clconv
0.26185152642501
unitless
C
st
9.9501 mg L
-1
0.0649 mg L
-1
V
1
1.00000 mL 5.77 10
-3
mL rectangular 0.25 1.4 10
-3
mg L
-1
20.9 %
V
100
100.0000 mL 0.0577 mL rectangular -2.5 10
-3
-140 10
-6
mgL
-1
0.2 %
C
1
0.099501 mg L
-1
868 10
-6
mg L
-1
V
1_st
1.00000 mL 5.77 10
-3
mL rectangular 0.035 200 10
-6
mg L
-1
0.4 %
V
1_100
100.0000 mL 0.0577 mL rectangular -350 10
-6
-20 10
-6
mg L
-1
0.0 %
C
2
0.19900 mg L
-1
3.15 10
-3
mg L
-1
V
2_st
2.0000 mL 0.0289 mL rectangular 0.031 900 10
-6
mg L
-1
8.4 %
V
2_100
-6
-36 10
-6
mg L
-1
0.0 %
C
3
0.39800 mg L
-1
6.31 10
-3
mg L
-1
V
3_st
4.0000 mL 0.0577 mL rectangular 0.023 1.3 10
-3
mg L
-1
17.9 %
V
3_100
-6
-53 10
-6
mg L
-1
0.0 %
192
Quantity Value
Standard
uncertainty
Distribution
Sensitivity
coefcient
Uncertainty
contribution
Index
C
4
0.59701 mg L
-1
4.00 10
-3
mg L
-1
V
4_st
6.00000 mL 8.66 10
-3
mL rectangular 0.014 120 10
-6
mg L
-1
0.2 %
V
4_100
-6
-49 10
-6
mg L
-1
0.0 %
C
5
0.99501 mg L
-1
6.67 10
-3
mg L
-1
V
5_st
10.0000 mL 0.0144 mL rectangular -2.7 10
-3
-39 10
-6
mg L
-1
0.0 %
V
5_100
100.0000 mL 0.0577 mL rectangular 270 10
-6
16 10
-6
mg L
-1
0.0 %
A
sample_rep
0.256000 654 10
-6
normal 1.0 670 10
-6
mg L
-1
4.6 %
A
sample_drift
0.0 1.30 10
-3
normal 1.0 1.3 10
-3
mg L
-1
18.2 %
A
sample_round
0.0 289 10
-6
rectangular 1.0 290 10
-6
mg L
-1
0.9 %
AC 1.5720 - 0.0108 -
A
1
0.108000 AU 577 10
-6
AU rectangular -0.36 -210 10
-6
mgL
-1
0.4 %
A
2
0.214000 AU 577 10
-6
-6
mg L
-1
0.3 %
A
3
0.412000 AU 866 10
-6
-6
mg L
-1
0.4 %
A
4
0.60600 AU 1.15 10
-3
-6
mg L
-1
0.3 %
A
5
0.99790 AU 1.15 10
-3
AU rectangular 0.027 31 10
-6
mg L
-1
0.0 %
AvgC 0.45771 mg L
-1
3.27 10
-3
mg L
-1
n 5.0 unitless
AvgA 0.467580 AU 404 10
-6
AU
CC 1.5544 - 0.0211 -
C 0.24650 mg L
-1
3.11 10
-3
mg L
-1
Results:
Quantity Value
Expanded
uncertainty
Coverage factor Coverage
C 0.2465 mg L
-1
2.5 % (relative) 2.00 manual
193
Appendix 1
195

TrainMiC Exercises
Analytical procedure:
EXERCISE 1:
EXERCISE 2:
EXERCISE 3:
196
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.
197
Analyte
Measurand
Units
Measurement
procedure
Type of calibration standard curve Standard addition internal standard
Model equation
198
judgement should be based on your previous experience only
1
2
3
4
5
For the analyte
1
(k = 2) see also data yellow sheet
Are all important parameters included in the model
equation?
Yes No
1
2
3
199
200
The measurement procedure
Analyte
The measurand
Unit
Matrix
Measuring range
Intended use of the results:
Mark the customers
their values
LOD
LOQ
Repeatability
Trueness
Other-state
VALIDATION
201
6. Measuring range
Linearity
Upper limit
LOD
LOQ
7. Spread precision
Repeatability
8. Robustness
202
9. Quality control
Control charts
Participation in profciency testing schemes
R squared
203
validated
Calculations
LOD
LOQ
Repeatability
Trueness
Parameter
customer
(the same as stated in question3)
Value obtained
during validation
The requirement
is fulflled
Yes/No
LOD
LOQ
Repeatability
Within-lab
reproducibility
Trueness
Measurement
Other
Yes No
204
EXERCISE
Measurand
Unit
equation
Model equation:
Method bias
Matrix efect
Other:
Other:
Other:
205
Input quantity
Standard
uncertainty
Unit Remark
583.5
c
Input
quantity
Value
Standard
uncertainty
Unit Remark
c
units
206
c
1
2
3
207
Addendum I: Measurement uncertainty calculation:
Addendum II: Measurement uncertainty calculation
GumWorkbench
209
Appendix 2
210
Introduction to the TrainMiC
example session
TrainMiC example session
Introduction
example session
How does the TrainMiC
example session look like?
1. About a TrainMiC example: how is it designed?
2. How are we going to organise ourselves Ior the example
session?
3. Introduction to the analytical procedure we will be
working on
211
example session
A TrainMiC example includes:
All the input information needed to do the three exercises
i.e.
About the analytical procedure
About the customer`s requirement(s) on the quality oI the
measurement results
Measurement data.
This is known as vellow pages.
example session
A TrainMiC example also includes:
A list oI questions Ior which you should Iind the answers
in the yellow` pages (sometimes some calculations are be
needed too)
Questions are grouped as Iollows:
Exercise 1. Establishing traceability in analytical chemistry
Exercise 2. Single laboratory validation oI measurement
procedures
Exercise 3. Building an uncertainty budget
Exercises are known as white pages.
212
example session
Exercise 1: Establishing traceability
in analytical chemistry
1. SpeciIying the analyte and the measurand
2. Choosing a suitable measurement procedure with associated model
equation
3. List the input quantities according to their inIluence on the
uncertainty oI the measurement (Iirst the most important ones). At
this point, your judgement should be based on your previous
experience only.
4. List the reIerence standards needed and state the inIormation
regarding traceability oI the reIerence value
6. How would you prove traceability oI your result
7. Any other comments, questions, ..
example session
How to organise ourselves for
the TrainMiC example session?
Forming the groups, each consisting oI maximum 5
participants
Each group nominates a reporter
Each oI you will get
the yellow page` (input inIormation) and
the white page` (Exercises on traceability, validation
and measurement uncertainty)
213
example session
How to organise ourselves for the
TrainMiC example session time wise?
30(15) minutes are available Ior the group work per exercise
AIterwards, all the reporters will be asked to report the answers and to
explain to the others about the questions and comments his/her group
was discussing during the exercise (30 minutes are available Ior a
discussion)
IMPORTANT:
the reporting & discussion panel is not about correct/wrong answers,
but about sharing your questions, experiences, ideas, suggestions and
comments you would be having during the exercise session with all the
attendees, including the trainers.
example session
Exercise 2: Single laboratory validation
of measurement procedures
1. SpeciIy the measurement procedure, analyte, measurand and units
2. SpeciIy the scope
4. Origin oI the measurement procedure
1. Selectivity/InterIerence/Recovery
2. Measuring range
3. Spread Precision
4. Robustness
5. Quality Control
1. Calculation oI parameters requested by the customer
2. Does the analytical procedure IulIil the requirement(s) Ior the intended use?
214
example session
Exercise 3: Building an uncertainty budget
1. SpeciIy the measurand and units
2. Describe the measurement procedure and provide the associated
model equation
3. IdentiIy (all possible) sources oI uncertainty
4. Evaluate values oI each input quantity
5. Evaluate the standard uncertainty oI each input quantity
6. Calculate the value oI the measurand, using the model equation
c
) oI the result and
speciIy units
8. Calculate the expanded uncertainty (U
c
) and speciIy the coverage
Iactor k and the units
9. Analyse the uncertainty contribution and speciIy the main three
input quantities contributing the most to U
c
example session
Measurements oI xxxx in xxxx by xxxx
Introduction to the analytical procedure
we will be working on today
215
Notes
216
Notes
European Commission Joint Research Centre Institute for Reference Materials
and Measurements
EUR 22791/2 EN Practical examples on traceability, measurement uncertainty
and validation in chemistry Vol. 1
Editors: Nineta Majcen, Philip Taylor
Luxembourg: Publications OIfce oI the European Union
2010 217 pp. 18.2 x 25.7 cm
EUR - Scientifc and Technical Research series ISSN 1018-5593
ISBN 978-92-79-12021-3
JRC 59026
Abstract
Case studies on traceability, measurement uncertainty and validation Ior measurements
oI gold in gold alloys, calcium in serum, radium in water, polar pesticides in Iood and
ammonium in water are presented in this report. Additionally, the idea and structure oI
the TrainMiC
examples, which complement the TrainMiC
theoretical presentations,
are described in detail to give a complete overview oI the TrainMiC
teaching material.
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 Ofhcial 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 Offce of the European
Union (http://publications.europa.eu/others/agents/index_en.htm).

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L

programme |www.trainmic.org|. This programme

to create harmonised training material as well as to disseminate knowledge

trainers. AIterwards, the

courses in various European countries,

example session has been developed to

example has been developed and some guidelines

example session have been draIted. As this is

example includes a part on

example session they are given

national team leader, who is organising a TrainMiC

example session, taking into account the knowledge

example session at a certain TrainMiC

programme and is chairing the TrainMiC

team leader, member oI the TrainMiC

Editorial Board. She is currently working at the

program in 2002 as a member oI Bulgarian team oI

coordinator, member oI the TrainMiC

Editorial and Management

team leader Ior Bulgaria.

Management Board and National TrainMiC

Editorial Board and a team leader oI the Nord/

Calculation of signal standard uncertainty

ESTABLISHING TRACEABILITY IN ANALYTICAL CHEMISTRY

6. Combined model equation for calculation of Au mass fraction ()

7. Combined model equation for calculation of Au mass fraction ()

w mass Iraction oI extractable pesticide in sample (mg oI pesticide per kg oI

w mass Iraction oI extractable pesticide in sample (mg oI pesticide per kg oI

w mass Iraction oI pesticide in sample |mg kg

w mass Iraction oI pesticide in sample |mg kg

0 05303 0 3844 0 388

in the sample solution mg L

in the sample solution |mg L

in the sample solution |mg L

in the sample solution

examples, which complement the TrainMiC

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