Validation of t he Calibration Procedure in

Atomic Absorption Spectrometric

Methods
J ournal of
Analytical
Atomic
Spectrometry
W. PENNI NCKX, C. HARTMANN, D. L. MASSART AND J . SMEY ERS-VERBEKE
ChemoAC, Pharmaceutical Institute, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
A general strategy for the validation of the calibration
procedure in AAS was developed. In order to accomplish this,
the suitability of different experimental designs and statistical
tests, to trace outliers, to examine the behaviour of the
variance and to detect a lack-of-fit, was evaluated. Parametric
as well as randomization tests were considered. For these
investigations, simulated data were used, which are based on
real measurements. The results obtained indicate that to
validate a straight-line model, the measurement points should
preferably be distributed over three or four concentration
levels. In order to check the goodness-of-fit, the significance of
the quadratic term should be investigated. A lack-of-fit to a
second degree model is better detected when the measurement
points are distributed over more than four concentration levels.
For an unweighted second degree model, an analysis of
variance (ANOVA) lack-of-fit can be used, while a
randomization test is proposed for a weighted model. A one-
tailed Res t or an alternative randomization test should be
used to trace a non-constant variance.
Keywords: Method validation; calibration; randomization test
Method validation is the process of demonstrating the ability
of a newly developed method to produce reliable results.'
Generally, one starts this process by validating the applied
calibration procedure. The calibration model is used to describe
the relationship between the analytical signal ( y ) and the
concentration (x). One can assume, for example, that the
Lambert-Beer law is valid within the applied concentration
range, so that a straight-line model (y=b,+b,x) can be used.
However, if the Lambert-Beer law is not valid and the straight-
line model is fitted to the data, the calibration procedure
introduces a systematic error in the analysis results. The
calibration method has to be evaluated prior to its routine
use, since the limited number of data points used routinely
does not permit such an evaluation.
Different approaches can be followed to validate the
calibration procedure. A number of guidelines to validate
the calibration function are, for example, published by the
International Organisation for Standardisation ( ISO).273
However, some important problems, such as the investigation
of the lack-of-fit of a weighted and a second degree calibration
line, are not discussed by ISO. Therefore, in this paper a more
general strategy for the validation of AAS calibration pro-
cedures is given.
This work investigates the validation of straight-line models
( y =b, +blx) and second degree models ( y =bo +blx +b,x2),
which are the most applied in practice and are included in the
I S0 guideline^.^,^Other calibration models are reported in the
literature. Some workers4 use, for example, a cubic model ( y =
b, +blx +b,x2 +b3x3). However, such a model has little physi-
cal meaning and requires a large number of calibration stan-
dards during routine analysis. Barnett' has described the
calibration model that is included in the Perkin-Elmer atomic
absorption spectrometers. Other models have been reported
by Phillips and Eyring6 and Ko~cielniak.~ Since these models
are not generally applied their validation is not discussed
further here.
The validation of the calibration procedure involves an
examination of the behaviour of the variance and of the
goodness-of-fit of the selected model. In order to accomplish
this, aqueous standards are measured at different concentration
levels, covering the complete calibration range. IS0,233 for
example, recommends to distribute ten standards uniformly
over the calibration range and to perform ten replicate analyses
of each of the lowest and highest concentrations. The exper-
imental design is important since it influences the probability
that a problem, such as a lack-of-fit or a non-constant variance,
is detected. Therefore, this work evaluates the applicability of
different experimental designs.
After the performance of the experiments, one should first
evaluate the data graphically. Often this permits an easy
detection of important problems. A good way to do this is by
examining the residuals.' Since this is not included in the
I S0293 recommendations, it is discussed briefly in this paper.
For a statistical evaluation of the results, one should check
whether the data are free of outliers. Outlying points may
disturb the normality of the data, which is required by most
of the tests used to examine the behaviour of the variance and
the goodness-of-fit. Moreover, the occurrence of multiple out-
liers would indicate a fundamental problem with the method.
In this paper, two tests to trace single outliers, namely the
Dixon' and the Grubbs" tests, and a test to trace paired
outliers," are studied. Next, the behaviour of the variance is
investigated. This work evaluates the suitability of tests which
compare variances at different concentration levels, such as
the F, 2, 3 Cochran,12 Hartley12 and Bartlettl3 tests, as well as
alternatives for these tests, where the standard deviations at
the different levels are estimated by the range.I4 Finally, in
order to trace a lack-of-fit, the applicability of an analysis of
variance (ANOVA) procedure15 and the significance of the
quadratic terrnI6 are evaluated. An alternative test is considered
for weighted m0de1s.l~This work also investigates the suit-
ability of a number of randomization tests." In that case, the
computed test statistic is not compared with a critical value,
but with a distribution which is obtained by random assign-
ment of the experimental data. By deriving the distribution
from the data themselves, these tests should be less sensitive
to deviations from normality.18
The different tests and experimental designs are evaluated
in a systematic way by means of simulations which are based
on a number of real data sets. The results of this evaluation
are used to construct a general validation strategy for the
calibration procedure in AAS. This could form the basis for a
more general strategy applicable to other measurement
techniques.
EXPERIMENTAL
The symbols that are used throughout this paper are summar-
ized in Table 1.
Journal of Analytical Atomic Spectrometry, April 1996, Vol. 11 (237-246) 237
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Table 1 Symbols that are used
~
Total number of measurements: N
Concentration levels: 1; ...; i; ...; n
Number of replicates: m,; ...; mi; ...; m,
xi=concentration at level i (i =l , ..., n)
yij=jth absorbance measured at level i ( j = 1, . . ., mi)
Yi =mean absorbance at level i
si =the standard deviation of the absorbances of level i
wi =the range of the absorbances of level i
b, ; bl ; bz =the estimated calibration parameters
j i =estimated absorbance at level i
=b, +b , Xj for a straight-line model
=b, +b,xi +b,x? for a second degree model
eij =yij - ji =jth residual at level i
Ci=mean of the residuals at level i
Z =mean of all residuals
ewij =,,h%K [ y z j - ji] =jth weighted residual at level i
q = ,=the weight at level i
-
1
3,
Instrumental
All programming was performed on a Compaq ProLinea 4/25s
personal computer. Visual Basic 3.0 (Microsoft) was used as
the programming environment. A Perkin-Elmer (Norwalk, CT,
USA) Zeeman 3030 atomic absorption spectrometer equipped
with an HGA-600 graphite furnace, an AS-60 autosampler and
a PR-100 printer were used for ETAAS determinations. For
flame AAS determinations, a Perkin-Elmer 373 spectrometer
with a PRS-10 printer sequencer were used.
Planning of Simulations
Description of experimental data
In order to simulate the data as realistically as possible, some
real experimental results were obtained first. The investigated
data sets contain absorbances measured in aqueous solutions,
at different levels and over a large concentration range. Zn, Fe
and Cu measurements were obtained with flame AAS, while
ETAAS was used for Pb, Cd and Mn measurements.
For the different data sets, the standard deviation is constant
at low concentration levels, but from a certain level it increases
with the absorbance. This is illustrated in Fig. 1 for Fe measure-
ments obtained with flame AAS. For the given example it is
clear that, from a certain concentration level, the standard
deviation increases linearly with the absorbance, but this could
T
0.008 } ,-
I
T
0.006 \ w
v) t
I
0.004 t
A
----.-- rn
i- -+-+ t--+
Fe concentration/mg I-
I .
0.002
0 2 4 6 8 10 12 14 16 18 20
Fig. 1
the Fe concentration (data obtained with flame AAS)
Standard deviation of the absorbance (n =6 ) as a function of
1.2
1 .o
0.8
ct 0.6
0.4
0.2
0
1 2 4 6 8 10 12 14 16
-0.2
Zn concentration/mg I-
Fig. 2 Absorbances as a function of the Zn concentration (data
obtained with flame AAS). The line represents the weighted second
degree function that is computed from these data
not be shown for all examined cases. The concentration level
where the behaviour of the variance changes can be situated
above, below or within the selected calibration range. This
must be investigated during the validation, because the first
situation (constant variance) permits the use of an unweighted
model, while for the other two (non-constant variance) the
most precise results are obtained with a weighted model.
Generally, up to a certain concentration, a straight-line
model can be used to describe the relationship between concen-
tration and absorbance. For higher concentrations a second
degree calibration model is needed. This is also the case for
the inspected data sets. Moreover, it is difficult to build a good
calibration model in a concentration range where the cali-
bration line is partially straight (lower part of the range) and
partially curved (upper part of the range). Fig. 2 shows, for
example, the absorbance values measured with flame AAS as
a function of the Zn concentration. It can be seen that the
weighted second degree model that is computed from the data
does not describe the measurement results accurately. In such
a situation the calibration range should be split into two parts.
For the given example, an unweighted straight-line model can
be used for concentrations up to 2mgl-, while a weighted
second degree model has to be used in the range between 2
and 15 mg 1- (results not shown). Consequently, in the selected
calibration range, three situations can occur. The calibration
line can be straight or curved over the complete range, or it
can be partially straight and partially curved. Moreover, it is
found that in the region where a straight-line model can be
used, the variance remains constant, while it increases when
the calibration line starts to bend.
Simulations
In order to evaluate the different statistical tests and experimen-
tal designs for each situation considered, 100 data sets were
simulated. Randomized normal distributed numbers were gen-
lerated in Visual Basic, using the method proposed by Box and
Mu1ler.l The suitability of the random generator was con-
firmed by simulating a large number of data of which the
normality of the distribution was checked graphically (with a
]histogram) as well as statistically (with a x2 test). Moreover,
in0 correlation was observed between data that were success-
ively simulated.
The simulations are based on the Pb data that were obtained
by ETAAS. The applied parameters are given in Table 2. The
calibration range (0-100 pg I-) is divided into two equal parts
in which different conditions can be valid. For example, a data
set can be simulated with a constant standard deviation in the
lower part, but an increasing standard deviation in the upper
part of the calibration range. Similarly, a first degree model
can be valid in the lower part but not in the upper part. For
238 Journal of Analytical Atomic Spectrometry, April 1996, Vol. 11
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Table 2 Parameters applied for the simulation of the data
I .
I
.
.
.
0 - - : ~
0.05 0.10 a.15 0.50 0.25 0.30. 0.35
Experimental conditions-
Concentration range:
Equation calibration line:
b1 =0.003
0-100 pg I -' (divided into two equal
parts)
y =Po +plxi +& x i 2
with Po =0.02
pz= -4.0 x -3.8 x ..., -0.2 x 0.0 x
Standard deviation:
homoscedastic: oi =0.002
heteroscedastic: oi =0.002 +yi 0.02
y i j ' =y i j +ka with k=4, 6, 8, 10 or 12
Introduction outlier:
In case of a problem at the lowest concentration level (see
Section 4):
Yl j ' = ~ 1 j + c ,
0.010
0.005
Experimental design-
Number of
concentration
Design levels (n)
( d )
-~
c11
c21
c31
c41
1151
0 .
3
4
6
9
12
.
m
.. . 0.05 0.10 0.15 0.20 ,0.25 0.30
rn
Number of
replicates at
each level (mi)
12
9
6
4
3
each investigation (evaluation of outlier tests, tests to trace
heteroscedasticity and goodness-of-fit tests) different simu-
lations were performed, which are specified further.
Five different ways to distribute 36 measurement points
symmetrically over the calibration range are considered (see
Table 2). Design 1, for example, positions 12 measurements at
three concentration levels, namely 0, 50 and 100 pg 1-'. The
choice of 36 points is arbitrary. The main reason why this
number was selected is that it permits the residual variance to
be estimated with a large number of degrees of freedom (>30)
and the measurement points can be distributed evenly over
three or four concentration levels. These are the minimum
number of levels to investigate a lack-of-fit to a straight line
and second degree model, respectively (see below).
0.006
0.004
0.002
0
-0.002
-0.004
. m
.
. . . m .
1
.
I
: ! :
.
0.05 ' 0.10 0.15 O. ; O 0.2: 0.30 ; 0.35
. '
m .
I
- v) -0.006
a
' 0.008 1
3
I
0.004
I
!
I :
1
. .
-0.008
I
:
The design that is proposed by IS0,293 namely ten replicates
at both extremes and a single measurement point at the eight
other concentration levels, is not considered, mainly for practi-
cal reasons. In the first place, this design cannot be applied for
the validation of a weighted model. In order to determine the
weight factors the variance must be estimated at the different
concentration levels which requires the performance of repli-
cate measurements. The lack of replicates also hampers the
application of outlier tests at the different levels, as well as the
evaluation of tests which compare variance estimates at differ-
ent levels, such as the Cochran test. Moreover, if one does not
take into account the replicates at the extremes of the cali-
bration range for the investigation of the goodness-of-fit, as is
proposed by KO, several tests that are evaluated here (e.g.,
ANOVA lack-of-fit) cannot be performed.
RESULTS AND DISCUSSION
1. Examination of the Residuals
The residuals ( ei j ) as given in Table 1 are the differences
between the responses actually measured (yij) and those pre-
dicted by the calibration model ( j i ) . In order to examine the
residuals they are plotted against the predicted value. When a
weighted model is used the weighted residuals (see Table 1)
are plotted against the predicted value. Fig. 3 illustrates four
situations that can occur. In the first situation [Fig. 3(a)] the
residuals form a horizontal band which indicates no abnor-
mality. In the second situation [Fig. 3(b)] the spread of the
residuals increases with the size of the predicted value (and
thus also with the concentration), which indicates that the
variance is not constant. In such a situation the use of a
weighted calibration model should be considered. Fig. 3 (c)
shows a trend in the residuals, which indicates that the model
is inadequate. Fig. 3( d) illustrates the residual plot in a situation
where the calibration set contains an outlier.
Draper and Smith' have shown that the estimated residuals
are correlated but they indicate that this correlation does not
invalidate the residual plot when N is large compared with
the number of regression parameters estimated.
0.008 i ( b )
0.004 1
.
.
= I
m
I
-0.004
-0.008
. . rn '
I
.
.
I
! 8
rn
I I .
. ' 0.05 '0.10 :0.15 : 0.25~ 0.301 0.35
= 0.20
-0.005 1
-0.01 0 I
Predicted value
Fig. 3 The residuals (Le., the difference between the measured and estimated absorbances) are plotted against the estimated absorbance to detect
a calibration problem. The following plots can be obtained, which indicate: (a), no abnormality; (b), a variance that increases with the estimated
absorbance (i.e., heteroscedasticity); (c), a lack-of-fit; and ( d) , an outlier
Journal of Analytical Atomic Spectrometry, April 1996, Vol. 1 1 239
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Table 3 Test criteria to trace an outlier at concentration level i. The
applied symbols are explained in Table 1
Test criteria f or Dixons test, as presented by ISO:5
When 2<m<8:
When 7<m<13:
Q=
When 12<rn<41:
Yi 2- Yi 1 and Q= Yi m- Yi ( m- 1)
Y i m- Y i 2 Yi ( m - 1) - YI
Yi 3- Yi 1 and Q= Yi m- Yi ( m- 2)
Y i m- Y i 3
Q=
Yi ( m- 2) - Y i l
Test criteria for Grubbs single outlier test?
and z= -
Test criteria for Grubbs paired outlier test:7
z2= -
with si the standard deviation of leaving out a pair of values
Y i m - j i Y i l - P i
z= __.
Si Si
1 - s i t
Si
2. Detection of Outliers
2.1. Description of tests
After the performance of the experiments, one inspects each of
the concentration levels for outliers. This work considers the
Dixon and the Grubbs tests which are the most generally
applied tests for the detection of a single outlier. The Dixon
test is applied as originally presented in the I S 0 guidelines.
The test criteria are given in Table 3. In contrast to the Dixon
test, which is based on a range, the Grubbs or maximum
normalized deviation (MND) test makes use of the standard
deviation to detect outliers. The Grubbs test criterion is also
given in Table 3. The use of the Grubbs test is recommended
by the Association of Official Analytical Chemists (AOAC)
and is also preferred in the latest draft I S0 document.21
Multiple outliers can be detected by repeated application of a
test for single outliers. However, paired outliers may mask
each other so that neither of them is detected with a single
outlier test. The Grubbs pair statistic for the detection of two
outliers, given in Table 3, investigates the decrease in the
standard deviation when removing a pair of values.
2.2. Evaluation
In order to evaluate the performance of the described tests,
samples containing 3- 18 normally distributed measurement
points were simulated. Single outliers were introduced at 4, 6,
8, 10 or 12 standard deviations (0) of the population mean. In
order to introduce two paired outliers, the first was positioned
at 4, 6, 8, 10 or 12 standard deviations, while the second was
put at two times the position of the first. For each situation
100 samples were simulated and the tests were performed at
the 5% significance level.
For all investigated situations with sample sizes larger than
three, similar results are obtained with both single outlier tests.
Fig. 4 shows, for example, the percentage of positive test results,
when an outlier is positioned at 60 or when no outlier is
present. The probability of correctly detecting an outlier
decreases with a decreasing sample size. One can see that for
sample sizes below six, the probability of not detecting an
outlier when it is present ( p error) becomes very high. The
probability of falsely detecting an outlier (a error) varies around
5%, which is in agreement with the specified significance level.
However, for a sample size equal to three the number of false
positive results (a error) obtained with the Grubbs test is
-a
- ---_ _ _
i t
- q----_ ----$-
2 4 6 8 10 12 14 16 18
Sample size
r 1 - 4
Fig.4 Percentage of positive test results obtained with the Grubbs
(squares) and the Dixon (triangles) test when an outlier is positioned
at 6s (solid line) and when no outlier is present (broken line)
unexpectedly high. There is clearly a problem with the Grubbs
test for very small sample sizes.
For a data set that contains 2 outliers, the Grubbs test
performs better than the Dixon test. The best results are
obtained with the test for the detection of paired outliers. For
example, with nine measurements, of which one is at 40 and
another at 80, a problem is detected in 21, 53 and 81% of the
cases, with the Dixon test, Grubbs test and the Grubbs test
for paired outliers, respectively. The probability of falsely
detecting a problem (a error) with the paired outlier test varies
around 5% (significance level) for samples containing 18, 12
and 9 measurements. However, for samples that contain only
six measurements the a error is about 11 YO.
These results indicate that, to avoid difficulties with false
outliers, the number of replicate measurements at each concen-
tration level should be sufficiently high (at least >4). When
no significant outlier is detected with the single outlier test,
one should check the data set for two paired outliers that may
mask each other. This test, however, requires even larger
sample sizes (>6).
The Behaviour of the Variance
3.1. Description of the evaluated tests
Parametric tests. I S0 proposes to use a one-tailed F-test to
check (a) whether the variance at the highest concentration
level is significantly larger than at the lowest concentration
level and (b) whether the variance at the lowest concen-
tration level is significantly larger than at the highest con-
centration l e~el . ~?~ For AAS applications, only the first test is
meaningful, because one knows that the variance, when not
constant, increases with the concentration. Other tests, which
can be applied to trace a non-constant variance, use estimated
variances at different levels. In this work the suitability of the
Cochran, Hartley12 and BartlettI3 tests is investigated. Table 4
shows how the test statistics are computed. Cochran compares
the ratio between the highest variance and the sum of the
variances with a critical value. Hartley, on the other hand,
uses the ratio between the highest and the lowest variance.
Theoretically, both these tests, for which specific tables exist,
require an equal number of measurement points at each
concentration level. However, I S0 indicates that for the
Cochran test small differences in the number of points can be
ignored, and applies the Cochran criterion for the number of
measurements that occur at most concentration levels. The
more complex Bartlett test does not assume an equal number
of points at the different levels. A quantity M/ C is computed
I(seeTable 4), which is distributed as x2 when the variances are
not significantly different. All these tests (Cochran, Hartley,
Bartlett and F-test) are based on the comparison of estimated
variances (s2). Some workers14 propose to test the sample
range (w). Correction terms are published14 by which the range
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Table 4 Evaluated test parameters to detect a non-constant variance.
The symbols are explained in Table 1
Smm2
r = ~
Smin'
Hartley
F-test
Bartlett M/ C, with:
where vi =mi - 1
must be divided to obtain an estimation of s. These estimations
can then be applied in parametric statistical tests, such as the
F-test. Alternatives for the Cochran and Hartley tests, based
on the ranges, have also been described.14 The test statistics
for these tests are also given in Table 4.
Randomization test. Apart from the parametric tests, which
are generally applied, this work also investigates the applica-
bility of a randomization test" to trace heteroscedasticity. The
randomization F-test that wepropose is illustrated in Table 5.
First, at each concentration level, the squared differences
between the individual and the mean measurement results are
computed (di;). Since the size of the d2 values depends on the
variance at that level, the test statistic for the experimental
data ( Re) is computed as the sum of the d2 values at the upper
concentration level divided by the sum of the values at the
lowest level. In a next step, the d2 values are randomly
permuted between the concentration levels, and for each
permutation an R, value is computed. If the d2 value (and thus
the variances) at the upper and the lower concentration levels
are similar, R, values will be found that are distributed around
Re. However, if the d2 values at the upper concentration level
are significantly larger, most of the R, values will be smaller
than Re. Consequently, the significance level can be computed
as the ratio between the number of permutations with R,>R,
to the total number of permutations. In this work the test
results are based on 1000 permutations obtained by means of
a random data permutation program.18
3.2. Performance under normal conditions
As already mentioned, five different ways to distribute the 36
measurement points symmetrically over the calibration range
were considered. Fig. 5 shows the number of positive test
results for these designs, in a situation where the variance is
1
h
s?
v
Q)
v)
0
.- .- .I-
n
\
0 ' I
I I I
Design 1 Design 2 Design 3 Design 4 Design 5
Fig. 5 Percentage of positive test results obtained with Cochran (A),
Hartley (A), Bartlett ( 0) and F (H) tests in a heteroscedastic situation
constant in the lower part of the calibration range, but increases
in the upper part. It can be seen that for all tests the best
results are obtained with the design that positions all meas-
urement points at three concentration levels (design 1).
Distributing the measurements over more levels, and thus
reducing the number of replicates at each level, decreases the
probability of detecting the heteroscedasticity. When the
different tests are compared, one can conclude that the best
results are obtained with the Bartlett and the F-tests. The F-
test has its simplicity as an additional advantage. Moreover,
this test is recommended by IS0.273 For homoscedastic
measurements the evaluated tests produce between 2 and 10%
of false positive results which is in agreement with the specified
significance level of 5%.
For the tests that estimate the standard deviation by the
range, a similar performance is observed. The test that uses
the ratio of the smallest to largest absorbance range, for
example, performs similarly to the classical Hartley test.
Moreover, determining the significance level of the F-test by a
randomization procedure gives similar results as using a criti-
cal value.
3.3. Performance in the presence of outliers
Since there is always a probability that an outlier is not
detected it is important to examine the effect of such a
measurement point on the applied tests. Here, a number of
situations are considered, where an outlier at one of the
concentration levels results in an overestimation of the variance
at that level.
With homoscedasticity, this overestimated variance can lead
to an increased number of false positive conclusions. Fig. 6
illustrates this, in a situation where the lower or the upper
concentration level contains an outlier at 60. With the Bartlett
test, for example, between 50 and 80% of false positive results
are obtained, depending on the applied design. Similar results
are obtained with the Cochran and Hartley tests (not shown).
The F-test is only affected by the outliers at the extreme levels,
because it does not use the data of the other levels. Moreover,
since this test is one-sided, an outlier at the lowest concen-
tration level does not increase the number of false positive
results (see Fig. 6). When the upper concentration level con-
tains an outlier, the F-test gives a comparable number of false
positive results as the other tests. However, for the most
suitable designs (designs 1, 2 and 3), this number largely
decreases when the significance level of the test is determined
by a randomization procedure (see Fig. 6). A similar conclusion
is obtained for data sets where one of the concentration levels
is contaminated with two outliers. The number of false positive
results obtained with the F-test is only increased by outliers
: : L*+ , ,
0
Design 1 Design 2 Design 3 Design 4 Design 5
Fig. 6 Percentage of positive test results obtained with the Bartlett
test (squares), the F-test (triangles) and the randomization F-test
(diamonds) in a homoscedastic situation, but with an outlier (60) at
the lower (solid line) or upper (broken line) concentration level
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at the upper concentration levels, while the other tests are also
affected by an overestimation of the variance at the other levels.
When, in a heteroscedastic situation, the variance at the
lowest concentration level is overestimated owing to an outlier,
an increased number of false negative conclusions can be
obtained. In that case, the real difference between the variance
at the highest and lowest concentration levels is underesti-
mated, so that an existing heteroscedasticity is masked. As an
example, a situation is considered where design 1 is applied
and the real standard deviations at the lowest, the middle and
the upper level of the concentration range equal 0.002, 0.002
and 0.008, respectively. The lowest level is contaminated with
an outlier, which leads to an overestimation of the variance at
this level. With an outlier positioned at 40 or at 60, the
probability to detect the heteroscedasticity with the F-test
decreases from about 100 to 65 and 30%, respectively.
Determining the significance levels of the I;-test by a randomiz-
ation procedlure does not improve the results. With the Bartlett
test, this decrease is also observed, but the number of positive
test results stabilizes and even slightly increases for important
outliers. This is because, in this situation, the variance at the
lowest level is overestimated in such a way, that it becomes
significantly larger than the variance at the other levels. This
effect is not experienced with the one-tailed F-test which checks
whether the variance at the upper level is significantly higher
than at the lower level.
4. Goodness-of-fit for Homoscedastic Data
4.1. Descriplion of the tests
Parametric tlests. I S02,3 recommends, as a goodness-of-fit test,
to check whether the data are better fitted by a second degree
model ( y =bo +blx +b, x2) than by a straight-line model ( y =
b, +blx) using an F-test. In this work we preferred to check
whether b2 iis significantly different from zero, using a t-test
(see Table 6). When it is shown that the data are better fitted
by a second degree than by a straight-line model, one concludes
that the straight-line model does not fit the data accurately.
This does, however, not prove that the second degree model
fits the data correctly, as is illustrated in Fig. 2. ANOVA lack-
of-fit is a test which can be applied to check the goodness-
of-fit of a model such as a straight-line or second degree model.
The test, which is described in Table 6, compares the error due
to lack-of-fit with the pure experimental error.
Randomization tests. Van der VoetZ2 has proposed a randomiz-
ation test to compare the predictive accuracy of two calibration
models. In this work we applied this test to check whether
better predictions are made by a second degree model than by
a straight-line model. Consequently, it can be considered as a
Table 5 Procedure of the randomization F-test
1. Computation of d2 values:
2. Test param,eter for experimental data:
3. Permutations:
The following steps are repeatedly performed:
3.1. The d2 values are randomly permuted between the
3.2. The test parameter is computed for each permutation: R,
concentration levels
4. Determination of signijicance level:
= =umber of permutations
number of permutations with R, >Re
Table 6 Parameters applied to detect a lack-of-fit of an unweighted
calibration model. The symbols are explained in Table 1
ANOVA lack-of-@ test:
Degrees of Mean squares
Sum of squares (SS) freedom (df) ( MS)
N- n
ss,,
df pe
Lack of fit: n- k SSlOf
i =l dhof
mi c ~i - j i 12
with k =2 for a straight-line model
k =3 for a second degree model
MSlof
MSF
--
F(iV - n) , ( n- k) , a -
Signijicance of second degree term:
randomization alternative of the parametric test proposed by
IS0.293 The test compares the squared residuals for the straight-
line model (ei?l) and the squared residuals for the second
degree model (eij22). If both models have the same predictive
ability, ei:l and e i t 2 have equal distributions (HO =null
hypothesis). However, if better predictions are obtained with
the second degree model, the eij21 values are generally larger
than the eij22 values. In order to test this, the difference between
the squared residuals, dsi j = eij21 - eij22, is computed for each
measurement point. The mean of these values, which is called
T, is then used as the test parameter. This value is first
computed for the experimental data (T,). If both models have
the same predictive ability, the dsij values are small and
distributed around zero so that T, will be almost zero. However,
if the second degree model provides better estimations, the dsij
values will generally be positive so that a T, value larger than
zero is obtained. In the randomization test at each iteration
random signs are then attached to the dsij values, and the T
value is computed (T,). If the original dsij values are distributed
around zero (HO true), this operation has little effect on T so
that T, values are obtained which are sometimes larger and
sometimes smaller than T,. However, if random signs are
attached to ds, values that are predominantly positive (HO not
true), most T, values will be smaller than T,. Therefore, the
significance level is then computed as the ratio between the
number of iterations with T,> T, and the total number of
iterations.
In this paper we propose another randomization test to
trace a lack-of-fit. This test investigates whether the prediction
error, estimated by the residuals, is independent of the concen-
tration level. Asshown in Fig. 3(c), a lack-of-fit can be detected
by demonstrating that the size of the residuals depends on the
level where they are measured. Table 7 explains how this can
beused in a randomization test. The numerator of the applied
test parameter is an estimate of the variation of the mean
residuals between the different concentration levels, while the
denominator estimates the variation within the levels. First,
the parameter value is computed for the experimental data
(LJ. Then, data permutations are performed. At each step the
residuals are re-assigned to the different concentration levels,
and the test parameter is re-calculated (I+). Re-assigning the
data has little effect on the test parameter when the residuals
are randomly distributed over the concentration levels [see
Fig. 3 (a)]. Consequently, for the different permutations one
will find test values which are sometimes larger and sometimes
smaller than that obtained for the experimental data. However,
when the size of the residuals depends on the concentration
level [see Fig. 3(c)] re-assigning the data will affect the test
parameter. In fact, for most permutations a test value will be
242 Journal of Analytical Atomic Spectrometry, April 1996, Vol. 11
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Table7 Procedure of the randomization test to investigate the
goodness-of-fit
1. The test parameter:
m i [ ~ i - ~ z
2. Computation of the test parameter for the experimental data:
100
80
60
40
20
0
W
.I -20 -1 5 -1 0 -5 0
c. The residuals [el I ; . . .; ei j ; . . .; enm] are used to compute L,
.-
3. Permutations:
The following steps are repeatedly performed:
3.1. The residuals are randomly assigned to the n concentration
3.2. The test parameter is computed for the ith permutation: L,
levels
4. Determination of signi,ficance level:
= total number of permutations
number of permutations with L, >L,
found which is lower than that obtained for the experimental
data. The significance level is then calculated as the ratio
between the number of permutations with a test parameter
higher than that computed with the experimental data (L,. >L,)
and the total number of permutations. In this work the test
results are again based on 1000 random permutations.
Strictly speaking, the proposed test, as with most statistical
tests, is applicable only when the data are independent. For
the estimated residuals this is not the case owing to the
correlation that exists between them. However, when the
number of measurement points is large compared with the
number of regression parameters estimated, the effect is small,
so that it can beignored.
4.2. Performance under normal conditions
First, the validation of a straight-line model is investigated.
For the test that is based on the significance of the quadratic
term the best result is theoretically found with design 1. It
follows from the D-optimality principle23 that the volume of
the confidence region for the calibration parameters of a
second degree polynomial is minimized when the measure-
ments are equally distributed over the two extremes and the
middle of the concentration range. It is clear that the more
precisely the calibration parameters are estimated, the easier
it is to demonstrate that the quadratic term is significantly
different from zero. For the ANOVA lack-of-fit it is more
difficult to explain theoretically which is the optimum design.
The simulations show, however, that the design that distributes
all measurement points over three concentration levels
(design 1 ) is the best to trace a lack-of-fit of the straight-line
model with both tests. The sensitivity of the tests decreases
when the number of concentration levels over which the
measurements are distributed increases and the number of
replicates at each level decreases. This is illustrated in Fig. 7(u)
for the ANOVA lack-of-fit. One can see, for example, that for
a calibration line with a quadratic term equal to - 12 x l op7,
the lack-of-fit to the straight-line model is almost certainly
detected when design 1 is used. With design 5, on the other
hand, the probability of correctly detecting the lack-of-fit is
about 30%. The test based on the significance of the quadratic
term is less affected by the applied design [see Fig. 7(b)].
Compared with the ANOVA lack-of-fit, this test gives similar
results when the most suitable design is applied, but better
results when the other designs are applied. The randomization
tests give less good results. For example, ANOVA lack-of-fit
detects a problem in 97, 88 and 34% of the cases when the
100
80
60
40
20
0
-20 -15 -1 0 -5 0
Quadratic term (x lo-)
Fig. 7 Percentage of cases for which (a) a lack-of-fit is detected with
an ANOVA, and (b) a significance of the quadratic term is detected,
for a curved calibration line and for different designs. The lines
correspond to: H, design 1; 0, design 2; +, design 3; 0, design 4;
and A, design 5
calibration lines contain a quadratic term equal to - 12 x
-8 x and -4 x respectively (design 1 applied).
With the randomization test described in Table 7, this is in 93,
72 and 18% of the cases. The randomization test proposed by
van der Voet22 detects a lack-of-fit in 82, 56 and 13% of the
cases. The number of false positive conclusions obtained with
the randomization tests is situated between 2 and 6%, which
is in agreement with the specified significance level of 5%.
Similar conclusions are obtained for situations where the
lack-of-fit to the straight-line model is the result of problems
other than a curvature to the x-axis. For example, a situation
is considered where data following a straight-line model are
simulated, and a constant value cb is added to all measurement
points of the lowest concentration level. Also, in that situation,
the test based on the significance of the quadratic term and
the ANOVA lack-of-fit give the best results, and they should
preferably be combined with design 1.
In order to validate a second degree model, an ANOVA
lack-of-fit is applied. Design 1, which uses only three concen-
tration levels, cannot be applied here. Table 6 illustrates that
with three parameters to estimate ( k =3 ) and three concen-
tration levels (n=3), the degrees of freedom of the error due
to lack-of-fit would be zero. In order to simulate a lack-of-fit
to a second degree calibration model, curved calibration lines
were simulated and a constant value (cb) was added to the
measurement results of the lowest concentration level. Fig. 8
gives the number of positive test results, for the different cb
values added, and for different designs. For the given situations,
the most suitable designs to detect a lack-of-fit are designs 3
and 4. For example, when cb=0.005, with design 2 a lack-of-
fit is detected in 30% of the cases, while designs 3 and 4 detect
it in about 50% of the cases. Thus, in contrast to the straight-
line model, the distribution of the measurement points over
the minimum number of concentration levels (four in this
situation) does not guarantee the best results. The reason for
this is explained further for the weighted second degree model,
where this problem is even more important. The randomization
test gives less good results than the ANOVA lack-of-fit. For
example, for cb values of 0.002,0.004 and 0.006, a lack-of-fit is
detected in 16, 33 and 62% of the cases, respectively, when
Journal of Analytical Atomic Spectrometry, April 1996, VoL 11 243
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'OiI 60 A
501- 4-==
' Design 2 Deskn 3 Des'ign 4 Deiign 5 '
1
Fig. 8 Percentage of positive test results obtained with an ANOVA
lack-of-fit for a second degree model, in a situation where the lowest
concentration level is contaminated. To simulate this problem, constant
values (cb) of 0.00 (H), 0.02 (0), 0.03 (+), 0.04 (0 ), 0.05 (A) and 0.06
(A) were added to the absorbances of the lowest concentration level
ANOVA lack-of-fit and design 3 are applied. With the same
design, the randomization test detects a lack-of-fit in 4, 16 and
42% of the cases, respectively.
4.3. Performance in the presence of outliers
The effect of outliers is only evaluated for the validation of a
straight-line model. Two outlier problems are examined,
namely a curvature that is masked by one or two too high
measurement results at the upper concentration level and a
curvature that is falsely detected owing to one or two too high
measurements at the middle concentration level. For the most
suitable design (design 1) the effect of an outlier at the upper
level is the same for the ANOVA lack-of-fit and the test based
on the significance of the quadratic term. When the curvature
is strong and the outlier is not very large, the probability of
detecting a lack-of-fit to the straight-line model is little affected.
However, when the curvature is weak and the outlier is
important, the probability of detecting the curvature decreases.
For example, when the quadratic term equals - 12 x
-8 x l op7 and -4 x a lack-of-fit is detected in 97, 95
and 34% of the cases, respectively. When an outlier (60) is
introduced at the upper concentration level, the probabilities
decrease to 89, 28 and 3%. With two outliers at the upper
concentration level (40 and 80), the probabilities to detect the
lack-of-fit are 25, 3 and 1%. In a similar way to what was
described above, also in the presence of an outlier the signifi-
cance of the quadratic term is less affected by the applied design.
The probability of falsely detecting a curvature increases if
the middle concentration level contains one or two outliers.
With design 1, for example, and a single outlier (60) at the
middle concentration level, one falsely detects a lack-of-fit to
the straight-line model in 10% of the cases, with the ANOVA
lack-of-fit as well as with the test based on the significance of
the quadratic term. When the middle concentration level is
contaminated with two outliers (40 and 80), a lack-of-fit is
detected in 22% of the cases. The tests are performed at a
significance level of 5%, so that a number of false positive
results around this level is expected.
In a similar way to what was described above, also in the
presence of outliers the randomization tests are less sensitive
than the parametric tests. This means that the number of false
positive results, owing to an outlier in the middle of the
concentration range, is lower with the randomization tests.
However, the probability of correctly detecting a curvature
when the upper concentration level contains an outlier is also
lower with the randomization than with the parametric test.
Therefore, one cannot say that the randomization tests are
more robust to outliers.
5. Goodness-of-fit for Heteroscedastic Data
5.1. Description of the tests
In order to examine the goodness-of-fit of a weighted cali-
bration model, two parametric and two randomization tests
are evaluated. The first testI7 computes the sum of squares
S = c K( Ji - 9i ) 2, where the weight factor is the inverse of
the variance of yi. If the calibration model describes the data
accurately, the value of S has a x2 distribution, with n- 2 or
n - 3 degrees of freedom for a straight-line and a second degree
model, respectively. As a second test, to validate a straight-
line model, one can also check the significance of the quadratic
term for a weighted model.
The evaluated randomization tests are similar to those
described in Section 4.1. In order to apply the test proposed
by van der Voet22 for weighted models, the weighted residuals
pi ( yi j -j i ) are used. Thus, one computes the mean difference
between the squared weighted residual for the straight-line
model and the squared weighted residual for the second degree
model and applies the test on these values. For the test that is
proposed by us (see Table 7), one states that the weighted
residuals must be randomly distributed over the concentration
levels. Thus, the randomization test which is explained in
Table 7 can also be applied on the weighted residuals. Here it
is also assumed that the correlation that exists between the
weighted residuals does not affect the test results.
5.2. Performance
Fig. 9 compares the performance of the evaluated tests for the
validation of a weighted straight-line model. A situation is
considered where all measurement points are distributed over
three concentration levels (design 1) and where the relative
standard deviation equals 2%. It can be seen that the best
results are obtained with the test that determines the signifi-
cance of the quadratic term. Less good results are obtained
with the randomization test that is proposed by us. The other
tests seem less suitable. Regarding the selection of the design,
the same conclusions can be made as for the unweighted
straight-line model, namely that the best results are obtained
with design 1. It should also be remarked that when the
heteroscedasticity is not detected, and the goodness-of-fit tests
for an unweighted model are performed, satisfactory results
are still obtained when the data set is free of outliers. However,
when the middle concentration level is contaminated with two
outliers (40 and 84, the probability of obtaining a false positive
result with the unweighted test is very high (between 20 and
30%). The weighted tests, on the other hand, are little affected
by these outliers. This is probably because the outliers increase
80
h
$?
- 60
a)
.- .- U
a 40
20
0
B
I I
-30 -25 -20 -15 -10 -5 0
Quadratic term (x
Fig. 9 Percentage of cases where a lack-of-fit to a weighted straight-
line model is detected with the x2 test (O), the randomization test
proposed in Table 7 (A), the randomization test proposed by van der
Voet (+) and by determining the significance of the quadratic term (H)
244 Journal of Analytical Atomic Spectrometry, April 1996, Vol. 11
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.
.
0.5
0 -
-1
-2
. .
.
0
-0.5
I
10 20 30. 40 50 60 '70 80 90 100
I
~ : .I'
10 20 -30 40 50 60 70, 80. 99 :
rn
~. -
100
--
.
I
m
.
.
. .
I .
.
. .
.
-41
Concentration
Fig. 10 Weighted residuals for a second degree calibration model in
a situation where a contamination of the lowest concentration level
occurs. Design 2 (a) and design 5 (b) are applied.
the variance at the middle concentration level so that a smaller
weight is given to this level.
In order to evaluate the goodness-of-fit of a weighted second
degree model, the randomization test described in Table 7,
performed on the weighted residuals, is found to be the most
suitable test. The probability to detect a lack-of-fit increases
when the measurement points are spread over an increasing
number of concentration levels, so that the best results are
obtained with design 5. This is because, for a small number of
concentration levels, an alternative model can be found which
fits the actual data accurately, but does not give a correct
description of the relation between concentration and
absorbance. This is illustrated in Fig. 10. The weighted
residuals for a calibration line obtained with design 2 and
design 5 are given in a situation where a curved line is
simulated, but a value cb is added to all data points of the
lowest level. Design 5 clearly indicates a problem, because the
weighted residuals are not randomly distributed over the
concentration levels. With design 2, on the other hand, no
problem is detected. However, the calibration model that is
found with this latter design cannot be used to make correct
estimations. The x2 test performs less well than the randomiz-
ation test. With design 4, for example, and Cb=0.002, 0.004
and 0.006, the randomization test detects a lack-of-fit in 24, 58
and 90% of the cases, respectively. With the x2 test, the lack-
of-fit is only detected in 1,16 and 38% of the cases, respectively.
In contrast to what is concluded for the straight-line model,
the goodness-of-fit tests that assume homoscedasticity fail
when they are applied on a second degree model in a heterosc-
edastic situation.
Recommended Strategy
The described results were used to build a strategy for the
validation of atomic absorption calibration models. The pro-
posed strategy is based on the assumption that the analyst has
an idea of the linear range of the calibration line before he
starts the validation. Consequently, he will preferably try to
demonstrate the suitability of a straight-line model within this
range. However, sometimes the linear range is so small that
one is obliged to work in the curved concentration range. The
analyst will then try to demonstrate the suitability of a second
degree model within the specified calibration range.
A number of experienced analysts, whose opinion was asked,
stated that to ensure the general acceptance of the validation
strategy it should be combined with information on how to
continue when a problem, such as a lack-of-fit, is detected.
Although this is not part of the validation, the results of the
validation experiments can be used to give these recommen-
dations. In order to avoid confusion, in this section a distinction
is made between the real validation of the calibration line and
a number of additional tests that are performed to advise the
analyst on how to continue.
Validation strategy
In order to validate a straight-line model, the analyst is advised
to apply experimental design 2 (ie., nine replicates at four
concentration levels). Design 1 (three concentration levels) is
more sensitive but does not permit a further investigation
when a lack-of-fit is detected. Designs 4 and 5, on the other
hand, do not permit an accurate outlier detection at the
different concentration levels and are less suitable for the
validation of a straight-line model. Moreover, owing to the
small number of replicates at each concentration level, these
designs are not really suitable to evaluate a possible het-
eroscedasticity.
For the validation of a second degree model, an alternative
design is proposed because none of the evaluated designs seems
optimum. The designs that position all measurement points at
a small number of concentration levels (designs 1, 2 and 3) are
the most appropriate to detect a heteroscedasticity but are the
least suitable to detect a lack-of-fit, especially for a weighted
second degree model. The designs that spread the measurement
points over a large number of levels (designs 4 and 5) , on the
other hand, are the most suited to detect a lack-of-fit, but the
small number of replicates makes them scarcely appropriate
to investigate the behaviour of the variance. Therefore, it is
proposed to position nine replicates at both extremes and six
replicates at five other concentration levels, equally spread
over the concentration range. This design requires more
measurement points than for that proposed for the validation
of a straight-line model (48 instead of 36), but additional effort
can be required for the validation of a more complex model.
After the performance of the experiments, the results should
first be evaluated visually. The most appropriate way to do
this is by plotting the residuals uersus the predicted value. The
statistical evaluation of the experimental results for a straight-
line model is summarized in Fig. 11. First, single outliers are
traced at the different concentration levels, applying a Grubbs
test. When no single outliers are found, the presence of possible
paired outliers is investigated. When, in the complete data set,
no more than two outliers are detected, the analyst can remove
them and continue the evaluation of the data. More outliers
indicate a fundamental problem with the analysis method,
which must be investigated. When the problem that is respon-
sible for the outliers is solved, a new data set should be
prepared. The homoscedasticity of the data is then investigated.
One investigates whether the variance at the highest concen-
tration level is significantly larger than at the lowest concen-
tration level. In order to accomplish this one can use a one-
tailed F-test or the alternative randomization test which is
even more suitable. Depending on the result, an unweighted
or a weighted model must be used.
In order to investigate the goodness-of-fit of the straight-
line model, one checks whether the data are better fitted by a
second degree model. If this is not the case, one confirms the
suitability of a straight-line model with an ANOVA lack-of-fit
(for an unweighted model) or with a randomization test (for
Journal of Analytical Atomic Spectrometry, April 1996, Vol. 1 I 245
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method does
not perfow
as expected
Straight
line model
is not
suitable
outliers
from data
F test
n l y
171 Use unweighted 'rl U s e weighted
n l
Unueighted
straight
line model
Weighted
straight
line model
Fig. 11 Strategy for the evaluation of the experimental results
a weighted model). These tests are also those used to demon-
strate the suitability of a second degree model.
Recommendations after the detection of a lack-of--t
When the validation shows that calibration data cannot be
described by a straight-line model, two actions can be taken.
First, one can check whether a straight-line model can be valid
over a smaller concentration range. Therefore, the upper
concentration level of the design is eliminated and the tests for
the validation of a straight-line model are applied on the
reduced calibration set. Possibly, a decrease of the calibration
range also allows the use of an unweighted instead of a
weighted model. When no suitable straight-line model can be
built, one can investigate the suitability of a second degree
model. It must beclear that those tests on a reduced calibration
set are only applied to give an indication of how the analysis
method can be adapted. They cannot be used as validation
results. If these tests indicate, for example, that a straight-line
calibration model seems suitable over a smaller concentration
range than specified at the start of the validation, the analyst
can adapt his method and start the validation of this new
method.
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Paper 5/07400B
Received November 10, 1995
Accepted December 13, 1995
246 Journal of Analytical Atomic Spectrometry, April 1996, VGl. 11
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