H. Albus
Basic Course Experiments to Demonstrate Validation
Basic Course Experiments to
Demonstrate Validation
(1) Fundamental concepts
(2) Experimental arrangements
(3) Results
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
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H. Albus
Basic Course Experiments to Demonstrate Validation
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Objective of the Lesson
Elaboration of the fundamental meaning
of validation:
Validation is the process of making sure
that an analytical method is fit for the
intended purpose.
or in other words:
Make sure that the obtained results are
as good as you need them!
(But first define properly what is „good enough“!)
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
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Fit for Purpose
Question:
How can one achieve good results?
Answer:
by using a method with the appropriate
performance characteristics

Task of method validation
Investigation and evaluation of the
performance characteristics of an analytical method
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
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Basic Performance Characteristics
of Analytical Methods
Precision
or: how good is the closeness of agreement
between the obtained measurement results?
measured values
true value
good precision
measured values
true value
poor precision
Note:
Precision is only influenced by random errors. This type
of error shows no systematics and occurs randomly
with statistical variability.
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
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Basic Performance Characteristics
of Analytical Methods
Trueness
or: how good is the closeness of agreement
between the average value calculated
from a series of test results and the
(assumed) true value of the analyte
(= substance under investigation)?
Note:
Trueness is only influenced by systematic errors.
This type of error modifies the result only in one direction
(too low or too high results).
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
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Basic Performance Characteristics
of Analytical Methods
Accuracy
or: how good is the closeness of agreement
between the result of a single measurement
and the true value of the analyte?
Note:
Accuracy is a measure which combines precision and
trueness (i.e. the effects of random and systematic
errors respectively). If the obtained results are not
affected by systematic errors, their accuracy becomes
equivalent to their precision.
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
Key Question of Analytical
Chemistry
How can one achieve accurate results?
Answer:
(1) by performing measurements using
appropriate samples and a well
described analytical method
( estimation of precision)
(2) by performing measurements using an
independent, validated comparison
method ( estimation of trueness)
Note:
“Independent“ in this connection means that two
methods are based on different physical and/or
chemical principles.
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
7
H. Albus
Basic Course Experiments to Demonstrate Validation
Strategy
Sample
(homogeneous)
Subsamples
Measurement
Method A
(under investigation)
Result
Method B
(validated)
independent
Result
agreement

YES ?
NO


Method A is suitable / not suitable
for the intended purpose
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
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H. Albus
Basic Course Experiments to Demonstrate Validation
Example 1
Validation of the gravimetric determination of
Fe3+ , Al3+ , PO43- and SO42by application of a one-channel FIA-system.
Background:
Gravimetric determinations
are difficult for beginners!

The flexible constructed One-channel FIA-system
allows reliable control measurements which can
be performed by the students very fast
and without great expenditure.
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
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H. Albus
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Basic Course Experiments to Demonstrate Validation
One-Channel FIA-System
sample route
Pos. A
recorder
ion-exchange column
sample route
Pos. B
detector
reagent
(carrier-) stream
peristaltic
pump
Injection valve
chemical
reactor
waste
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
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Working parameters
Detector:
Flow rate:
Injection volume:
Wavelength:
photometer with flow cell
1,8 mL/min
40 µL
470 nm (Fe3+ ); 532 nm (Al3+);
390 nm (PO43-); 232 nm (SO42-)
Reagent solutions
Fe3+ :
Sulfosalicylic acid solution (1.0 % (m/m)); pH = 1.8
Al3+ :
Xylenol orange (0.1 % (m/m)) in buffer solution; pH = 4.4
PO43- :
Ammonium heptamolybdate (c = 0.005 mol/L)
in nitric acid (c = 0.4 mol/L)
SO42- :
dist. water (carrier)
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
12
Working Procedure
Determination of Fe3+, Al3+ and PO43Sample route Position A
 direct injection of sample into reagent stream
via injection valve
Linear measurement range
Fe3+ 100 - 700 mg / L
Al3+ 10 - 70 mg / L
PO43- 100 - 700 mg / L
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
Working Procedure
Determination of SO42Sample route Position B
 pumping of sample solution through
ion-exchange column (nitrate-form);
quantitative replacement of sulphate:
R(NR3+)n(NO3-)n + n/2 SO42-  R(NR3+)n(SO42- )n/2 + n NO3-

Direct UV-detection of NO3- at 232 nm
Calculation factor: 0.775
1.29 mg NO3-  1 mg SO42-
Linear measurement range
100 - 800 mg / L
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
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H. Albus
Basic Course Experiments to Demonstrate Validation
Results
Typical analyte concentrations
 500 mg /L ( Fe3+ , PO43- and SO42- )
 50 mg /L (Al3+ )
Typical performance parameters
Analyte
average deviation
average rel. standard
mean value nominal value
deviation (n = 3)
Gravimetry
FIA
Gravimetry
FIA
Fe3+
1.2 %
0.7 %
1.2 %
1,0 %
3+
1.0 %
0.8 %
0.9 %
1.1 %
3-
0.7 %
0.9 %
1.0 %
1.1 %
2-
1.0 %
0.7 %
1.1 %
0.9 %
Al
PO
4
SO4
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
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H. Albus
Basic Course Experiments to Demonstrate Validation
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Conclusion
Gravimetry, performed with typical
practical course equipment, and FIA
show
comparable accuracy!

One-channel FIA is an appropriate
independent comparison method for the
validation of gravimetric determination
of Fe3+, Al3+, PO43- and SO42-.
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
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Example 2
Validation of the electrogravimetric
determination of Cu2+ by application of
a didactically designed photometer.
Background:
Modern analytical instruments are
„black box“ for beginners!

The use of an „open“ instrument enhances
the learning effect to a great extent.
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
17
Basic Concept of Photometer
•
•
•
•
cheap (unit price 500 DM)
no complex optical system
robust and easy to handle
modular construction; one electronic
and one measurement case, connected
with cables
• All important parts (lamp; slit; filter;
detector) can directly be seen in full
view.
• Display shows transmission;
corresponding absorbance has to be
calculated by the students.
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
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Basic Course Experiments to Demonstrate Validation
Diagram of Course of Beam
within the Measurement Case
tungsten lamp
slit
colour
filter
cuvette
photodiode
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
Basic Course Experiments to Demonstrate Validation
Electronic Case
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
19
H. Albus
Basic Course Experiments to Demonstrate Validation
Measurement Case
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
20
H. Albus
Basic Course Experiments to Demonstrate Validation
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Procedure
Cu2+- solution +
conc. ammonia (25 % (m/m))
 formation of [Cu(NH3)4(H2O)2]2+
(max = 580 nm)

Transfer in 1cm cuvette and threefold
measurement (every time with new
solution!)

calculation of concentration
Linear measurement range:
100-700 mg / L
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000
H. Albus
22
Basic Course Experiments to Demonstrate Validation
Results
Typical analyte concentration:
 500 mg Cu2+/ L
Typical performance parameters:
average deviation
average rel. standard
mean value nominal value
deviation (n = 3)
El.-Gravimetry
Photometry
El.-Gravimetry
Photometry
1.2 %
0.8 %
1.5 %
1.0 %

Photometry is easy to implement into first
practical courses und of great usefulness
for validation purposes in this context!
B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000