Talanta 52 (2000) 329 336

www.elsevier.com/locate/talanta

Standardization of a multivariate calibration model applied

to the determination of chromium in tanning sewage
Florenci Sales a,*, Antoni Rius b, M. Pilar Callao a, F. Xavier Rius a
a
Departament de Qumica Analtica i Qumica Orga`nica, Uni6ersitat Ro6ira i Virgili, Pl. Imperial Ta`rraco 1, 43005 Tarragona,
Catalonia, Spain
b
Escola Superior dAdoberia, EUETII Uni6ersitat Polite`cnica de Catalunya, Pl. Rei 15, 08700 Igualada, Catalonia, Spain

Received 6 January 2000; received in revised form 1 March 2000; accepted 3 March 2000

Abstract

A multivariate standardization procedure was used to extend the lifetime of a multivariate partial least squares
(PLS) calibration model for determining chromium in tanning sewage. The Kennard/Stone algorithm was used to
select the transfer samples and the F-test was used to decide whether slope/bias correction (SBC) or piecewise direct
standardization (PDS) had to be applied. Special attention was paid to the transfer samples since the process can be
invalidated if samples are selected which behave anomalously. The results of the F-test were extremely sensitive to
heterogeneity in the transfer set. In these cases, it should be taken as an interpretation tool. 2000 Elsevier Science
B.V. All rights reserved.

Keywords: Multivariate standardization; Multivariate calibration; Chromium analysis

1. Introduction governing the environment.

Chromium salts are frequently used in the tan-
ning industry to stabilize the structure of the
collagen. Tanning floats and sewage are conse-
quently generated during the process. These con-
tain certain quantities of chromium. It is
interesting, therefore, to determine the chromium
contents at the various stages of the tanning pro-
cesses and monitor them, both for economic rea-
sons and so that they comply with regulations
* Corresponding author. Tel.: +34-77-558122; fax: + 34-
77-559563.
E-mail address: sales@quimica.urv.es (F. Sales)
The reference method for determining chromic
oxide [1] usually takes up to several hours mainly
due to a process of sample oxidation. Other pro-
posed alternatives [2] also have the drawback of
the sample oxidation step. New methods based on
spectroscopy and multivariate calibration [3,4] are
promising alternatives to the reference method.
These provide several advantages: they are quick,
they consume less reagent and there is no need for
sample pre-treatment.
Multivariate models should be made to last a
long time because much time and effort is neces-
sary to develop them, e.g. a sufficient number of
samples have to be collected and analysed by

0039-9140/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 3 9 - 9 1 4 0 ( 0 0 ) 0 0 3 6 6 - 0
330 F. Sales et al. / Talanta 52 (2000) 329336
reference methods. One way of extending the use
of multivariate models is to apply standardization
procedures, which reduce the experimental work
needed to update the model if there is a drift in
the predictions [5]. Several standardization strate-
gies have been successful in solving these analyti-
cal problems [6 8].
One of these strategies is to transfer the newly
measured data, where the experimental conditions
in which the calibration model is developed are
considered as the reference, and the samples
analysed after the change (second experimental
conditions) are corrected in the direction of the
model conditions [9]. For this, a set of representa-
tive samples in the initial experimental conditions
is selected by the algorithms e.g. Kennard/Stone
[10] or hat matrix calculation [11]. These samples
have to be analysed under the new experimental
conditions and a transfer function must be estab-
lished to correct the data from the new samples to
be predicted with the initial model. Under the new
experimental conditions, the samples are expected
to behave like the transfer samples [12]. It is,
therefore, important to detect possible outliers in
the transfer samples, since standardizations could
be wrong if they are included.
The transfer technique used depends on how
great the change among the experimental condi-
tions is. Tests done before the standardization
have been used to decide the most suitable tech-
nique for solving the problem [13].
This paper studies the validity over time of a
multivariate calibration model for quantifying
chromium in residual samples from tanning treat-
ments. The model was not used continuously and
this means that its validation was only tested
when the system was used. The model was built
with UVVis spectra. The Kennard/Stone al-
gorithm was used to select the samples which, in a
first step, were used to evaluate whether there had
been changes in experimental conditions. First, a
diagnostic F-test was applied before the transfer
to determine the best correction technique to use.
MSEP (4)
The transfer step was then applied. Possible irreg-
ular tendencies of the selected samples under the
second experimental conditions were also consid-
ered. Slope/bias correction (SBC) [14] and piece-
wise direct standardization (PDS) [15] were used
to correct the deviated predictions and evaluate
the information obtained from the diagnostic F-
test. The number of transfer samples was also
varied.
2. Theoretical background
2.1. Multi6ariate calibration
The prediction step can be formulated in in-
verse multivariate calibration models [16] by Eq.
(1):
cunk = rTunkb (1)
where the concentration of an analyte in an un-
known sample cunk is predicted by multiplying the
transposed response of the sample rTunk (one sam-
ple by m variables) by the matrix of the coeffi-
cients of the model b (m variables by one analyte).
The model is constructed with both the matrix
of responses R (n samples by m variables) and the
matrix of concentrations c (n samples by one
analyte) of the calibration samples as follows:
b =R+c (2)
+
and R is the pseudoinverse of the centered ma-
trix R. The calculation of R+ characterizes the
multivariate calibration method. For instance,
PLS tries to extract the most useful information
from R while at the same time considering the
concentration [17].
Accuracy in quantification is assessed by the
error of prediction [18], for example, as the mean
squared error of prediction (MSEP):
n
% (ci ci )2
i=1
MSEP= (3)
n
or, in percentage terms, as the relative root mean
square error of prediction (RRMSEP):
100
RRMSEP=
c
In these expressions, ci is the predicted concen-
tration, ci is the real concentration, c is the mean
of the real concentrations and n is the number of
samples predicted.
 F. Sales et al. / Talanta 52 (2000) 329336
Calibration models can be validated with an
internal validation, e.g. cross-validation and,
therefore, prediction errors are called MSEPCV
and RRMSEPCV, respectively [17]. Calibration
samples can be used again as an external set to
validate calibration models after a period of time.
Although they are not completely independent,
their prediction shows whether models cope with
the possible changes in the experimental condi-
tions [15]. Obviously, it is essential to have sam-
ples that are stable over time, otherwise this
methodology could lead to incorrect results.
Sometimes, the samples used are not stable, so
other strategies must be applied [9,12].
Finally, the joint confidence interval test for the
slope and the intercept (JCIT) can detect potential
bias in the predictions, i.e. systematic error in the
predictions [19].
2.2. Standardization
2.2.1. Selecting transfer samples
The first step is to select the transfer samples, in
this case, by the Kennard/Stone sequential al-
gorithm [20]. The selection tries to spread the
samples uniformly along the multivariate space
defined by the whole set of samples. The Ken-
nard/Stone algorithm selects one sample at a time
depending on its Euclidean distance from the
other of samples. If ri is the response of a sample
i, the Euclidean distance,d12, between two samples
'
is:
m
d12 = % (r1j r2j )2
j=1
The algorithm begins by selecting the two sam-
ples with the maximum Euclidean distance, i.e.
the furthest from each other.
The third sample is the furthest from these two.
To find this sample, the distance between each
sample and the two selected ones is calculated. In
each case, the smallest distance is kept. Of this
group of smallest distances, the sample which has
the greatest distance is selected.
The fourth sample is the furthest from the three
selected ones, and so on. Each new sample is
chosen by means of the procedure explained
above. This is repeated until the number of re-
331
quired samples is complete, but the algorithm
does not indicate what the best number is. This
depends on the data studied, although subsets of
samples which form stable structures i.e. groups
of points regularly distributed, can be a criterion
to use [10].
2.2.2. Standardization algorithms
2.2.2.1. SBC [21]. This is a simple univariate
standardization technique which establishes a lin-
ear regression between cT1 and cT2. These are the
concentrations predicted by the initial model (Eq.
(1)) with the responses of the transfer samples
(small T) in the first and the second experimental
conditions, respectively:
cT1 = bias+ slope cT2 (6)
Both the slope and the intercept (so-called bias
here) of the regression are applied to obtain the
standardized concentration (c2,unk)std as follows:
(c2,unk)std = bias+ slope c2,unk (7)
2.2.2.2. PDS [22]. This is a multivariate standard-
ization technique in which the response of each
variable in the first experimental conditions r1i is
related to the response of a group of variables
[r2i j, r2i + j ] in the second experimental condi-
tions. By extending this to all the transfer sam-
ples, the relation can be written as:
r1i = R2i bi + b0i (8)
(5)
where a multivariate regression, e.g. PCR can
calculate the regression coefficients bi and the
offset term b0i. The number of columns in matrix
R2i is called the window size, i.e. the number of
variables from second experimental conditions in-
volved in the relation.
When a local model is applied for each vari-
able, vectors bi are grouped in a diagonal matrix F
and values b0i are grouped in vector bT0 . This
matrix and this vector are used to correct rT2,unk, a
sample measured in the new experimental condi-
tions, to the standardized (rT2,unk)std:
(r2,unk)std = rT2,unkF+ bT0 (9)
332 F. Sales et al. / Talanta 52 (2000) 329336
which can be predicted with the initial model by
Eq. (1).
2.2.3. F-test prior to standardization
This F-test shows us the best standardization
technique [23]. The results of this test indicates
whether the system only needs a simple correction
like SBC or, alternatively, whether a stronger
correction, like PDS, is needed.
Hence, cT1, cT2 and their real concentrations cT
are related by linear regressions, that provide the
intercept b01 and the slope b11 in the first experi-
mental conditions and the intercept b02 and the
slope b12 in the second conditions:
cT1 =b01 + b11 cT and cT2 =b02 +b12 cT
(10)
Both regressions have their own residual vari-
ances s 21 and s 22 which are compared with an
F-test in which F is calculated as:
t weeks, the samples were stored in conditions that
% (cT2i [b02 +b12 cTi ])2
s 22 i = 1
F= = t (11)
s 21
% (cT1i [b01 +b11 cTi ])2
i=1
where t is the number of transfer samples. The
calculated F-values are compared with the critical
values of one-sided F(a,t 2,t 2). If the calculated
F-values are smaller than the critical values, there
is no great difference between the predictions in
both experimental conditions. Simple correction
algorithms are therefore recommended.
3. Experimental
3.1. Instrumentation
A Hitachi spectrophotometer, model U-2000,
controlled by a personal computer equipped with
an RS-232 interface for communications, regis-
tered and stored the spectra in ASCII code.
3.2. Software
U2000 Manager software controlled the spec-
trophotometer and recorded and stored the
spectra.
Matlab [24] home-made functions and Wises
Toolbox [25] were used for all the computations.
3.3. Samples
Twenty-six samples from a real tanning process
were used as a calibration set to develop the
model. These samples were analysed by the refer-
ence method, and the concentration of chromic
oxide found ranged from 0.7 to 7.0 g l 1.
3.4. Procedure
The sample was filtered (if necessary) and then
diluted to 1:4. The spectra of the solution were
registered between 360 and 700 nm and stored in
ASCII code. Deionized water was sufficient to
dilute the samples.
The set of 26 samples was measured on two
different days, 3 weeks apart. During these 3
kept them stable and the reference method
checked that the chromium concentration did not
change.
The first measurement produced the initial data
set, or calibration set, that was used to develop
the calibration model. We called this Day1.
The second measurement produced the second
experimental conditions data set, which was used
to evaluate the stability of the model and to apply
the standardization procedure. We called this
Day2.
4. Results and discussion
The concentrations and spectra of the 26 sam-
ples in the calibration set were used to build the
PLS model. The cross-validation leave-one-out
procedure was applied to validate the model. The
evolution of the prediction error with the number
of PLS factors is shown in Fig. 1a. A model with
four factors was selected as optimum, according
to the criterion of the minimum error of predic-
tion and the plot of the model coefficients against
the response variables for each factor (Fig. 1b).
The models error of prediction is slightly smaller
with five or even six factors but with four factors
 F. Sales et al. / Talanta 52 (2000) 329336 333

the coefficients of the model do not contain so
much noise.
The scores for the samples of the calibration
set were plotted and used to check for outliers.
Neither outliers nor sub-models were found. The
accuracy of the models, evaluated by a cross-val-
idated relative error of prediction (RRM-
SEPCV), is less than 5%, which is a very good
value for this type of determination. Trueness
was assessed by linear regression. The joint test
for the slope and intercept showed that there
were no systematic errors at a 95% confidence
level.
Table 1 (S1 selection) shows the samples se-
lected when the Kennard/Stone method was used
on the 26 initial samples. The whole set of initial
samples, the Kennard/Stone selected samples and
the Kennard/Stone selected samples after their
analysis in the second experimental conditions
are plotted together in the plane of the two first
principal components on Fig. 2 (left), where over
99% of the total variance of the data is retained.
There is a displacement in all 10 samples. This
can also be seen by the unusual differences in the
spectra, which are larger than the normal ran-
dom noise. The change in the scores suggests a
deviation in the predictions, as detected when the
subset of Day2 samples is predicted with the
initial model. The prediction error rises to 16%,
which suggests the need for the standardization
procedure.
However, displacement is not regular for sam-
ple 18, whose new score, due to an experimental
error, is located much further from the others.
As this transfer sample is an outlier, it may con-
siderably influence the results. If sample 18 is

Fig. 1. (a) Prediction errors versus number of factors for the calibration model. (b) Coefficients of the model for four, five and six
factors.

Table 1
Transfer samples obtained in each selection

Selection Arrangement of transfer samples

1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th

S1 16 18 6 23 20 22 19 7 1 21
S2 16 13 6 23 20 22 19 7 1 21
S3 13 16 26 23 14 17 22 7 1 25
334 F. Sales et al. / Talanta 52 (2000) 329336

Fig. 2. Scores of the set of samples analysed using the initial experimental conditions (crosses) and the first subset of 10 samples
measured using the second experimental conditions (circles) according to procedure S1 (left) and S3 (right). Only the sample subsets
are labelled.

Table 2
Results of the diagnostic F-test for each selection of samplesa

Standardization samples

3 4 5 6 7 8 9 10

Calculated F-value for S1 387.6 21.8 25.7 26.9 23.9 19.5 38.0 37.5
Calculated F-value for S2 17.79 5.83 5.46 7.16 7.27 6.79 26.28 25.70
Calculated F-value for S3 4.28 3.89 3.43 3.14 4.26 4.23 18.14 18.76
Critical F (0.99, t2, t2) 4052.2 99.0 29.4 16.0 11.0 8.5 7.0 6.0

a
Figures in bold indicate that the calculated F-values are higher than the critical F-values.

removed, the error of prediction for the other 4.1. Standardization

nine samples is 14%, so the transfer was still
needed. To study the influence of a possible
anomalous transfer sample the standardization
process was carried out with and without this
sample.
There were two possibilities when sample 18
was removed. First, it was replaced by sample 13
(the nearest one) so that only one more sample
needed to be analysed, and second, a new selec-
tion on the remaining 25 samples was initiated.
Selections were called S2 and S3, respectively.
They are summarized in Table 1 and plotted on
Fig. 2. The displacement shown by the scores of
the S3 samples is similar to that of the S1 sam-
ples.
Table 2 shows the results of the F-test and
indicate that the change in experimental condi-
tions is not important with a small number of
samples. SBC is therefore considered the best
technique, although this can be due to the high
value of the tabulated-F when there are very few
degrees of freedom. For more than five samples,
PDS should be used to correct the measurements
if S1 is used. Also, when the ninth sample is
added in S2 and S3, i.e. sample 1 (Table 1) the
F-test varies its diagnosis. This may be due to this
sample, which is also influential since its change in
experimental conditions is greater than those of
the other samples (Fig. 2).
 F. Sales et al. / Talanta 52 (2000) 329336
Both SBC and PDS were used for the standard-
ization of the Day2 data set. The transfer samples
varied from 3 to 10 and selections S1, S2 and S3
were used (Fig. 3).
S1 does not standardize Day2 data effectively.
Neither SBC nor PDS removed the bias in the
predictions at the usual 95% level of significance
(1 a =0.95) due to the presence of sample
18 which behaves as an outlier. However, if SBC
is applied, when the number of transfer samples
increases the relative influence of sample 18 de-
creases and results are better, although PDS was
diagnosed by the F-test. The influence of sample
18 is maintained with the PDS technique even
when the number of transfer samples increases.
As expected, both S2 and S3 provide very ac-
ceptable standardization results, regardless of the
selection used since there are no noticeable differ-
Fig. 3. Predictions of data in initial experimental conditions
(Day1), second experimental conditions (Day2) and after the
standardization of the second conditions by SBC and PDS
techniques. The standardizations were applied on the three
selections of samples and the number of transfer samples was
varied from three to 10.
335
ences when either standardization technique is
applied. Taking into account the simplicity of the
technique, and the level of predictions, the best
standardizations with S2 and S3 could be found
with SBC for five transfer samples. Also, based on
the number of new transfer samples to remeasure,
S2 is better than S3. A subset of five samples for
S2 needs to collect only one sample more than the
initial S1, i.e. sample 13, while for S3 selection 3
new samples should be measured samples 13,
26 and 14 (Table 1).
The SBC standardization notably improves the
prediction of the calibration samples in the second
experimental conditions. It reduces the MSEP and
removes the bias in the predictions of the Day2
data without standardization. The number of
transfer samples is not critical for this technique
since any variation in the results is minimal.
Moreover, the best PDS outputs do not signifi-
cantly improve the SBC results. SBC is therefore
better because it is simpler and requires fewer
transfer samples to reach similar results. Also,
PDS results depend more on the number of sam-
ples than SBC does, particularly when there are
very few transfer samples. Therefore, the F-test
results, which recommended PDS when nine and
10 transfer samples were used with S2 and S3
(Table 2), should not be interpreted as a conclu-
sion but as an indication. If there is any doubt, it
is better to apply the simplest algorithm.
Fig. 4 compares the residuals for the best case
of five transfer samples for S2 with those from the
initial and the uncorrected samples. Correction is
particularly effective for the more deviated predic-
tions of the Day2 second experimental conditions,
while the other samples are slightly corrected al-
though in some cases prediction is not improved.
5. Conclusions
Multivariate standardization can cope with
changes in experimental conditions which degen-
erate UVVis multivariate models for determin-
ing chromium in tanning waste samples.
Studying the transfer samples before their use
for standardization is shown to be very important
for analysing any possible heterogeneity, which
336 F. Sales et al. / Talanta 52 (2000) 329336

Acknowledgements

We would like to thank the DGICyT, the

Spanish Ministry for Education and Culture, pro-
ject number BP 96-1008 and grant reference FP95
40930075, for their financial contribution.

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