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Received 7 June 2004; received in revised form 28 December 2004; accepted 28 December 2004
Available online 16 February 2005
Abstract
Copper(II) and mercury(II) were analyzed simultaneously employing a spectrophotometric method based on application of murexide
solution as a chromogenic reagent. A full factorial six level design was used for the construction of calibration and prediction data sets
consisting of absorption spectra recorded in 350–700 nm range from solution mixtures. A control data set, from a random design, was
applied for validation of the calibration models. The presence of non-linearities was confirmed by a recently discussed methodology based on
augmented partial residual plots (APaRPs). Combinations of principal component analysis (PCA) or linear discriminant analysis (LDA) with
radial basis function networks (RBFNs) or feed-forward neural networks (FFNNs) were built and investigated, as four calibration models.
Number of inputs and hidden nodes for each of the networks were optimized. Performances of methods were tested with relative standard
error of prediction (RSEP%), using synthetic solutions of two metal ions as prediction set. Linear discriminant analysis assisted networks
(LDRBNN) resulted in preferred models, using only one latent variable for each of the analytes. All of the methods were applied for the
analysis of a number of synthetic samples and a dental alloy sample and satisfactory results were obtained.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Artificial neural networks; PCA; Linear discriminant analysis; Metal ions; Spectrophotometry
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2004.12.079
332 Y. Akhlaghi, M. Kompany-Zareh / Analytica Chimica Acta 537 (2005) 331–338
sion procedure. Similar to PCA, LDA summarizes almost all The input value to each output node is the weighted sum
variances in the X-matrix on only a few axis, which are mu- of all the outputs of the hidden nodes. Finally, the response
tually orthogonal [20]. The main difference between the two of each output node is calculated by a linear function of its
compression methods is that LDA analysis focuses on the input (including the bias wk0 ), that is, the output of hidden
dissimilarity between classes [21–22]. This work is among layer (outk ). The relation between the value outk and the input
the first studies [23] on the application of LDA as a data com- variable xi can be represented by:
pression method, before performing calibrations using ANN.
Due to its direct and continuous contact to saliva, com- outk = wk0 + wkj Φj (xi − cj ) (2)
position of dental amalgams is of medical significance. Par- j
ticularly, heavy metals contents of these kinds of alloys are
important factor. Copper and mercury are among the impor- The weights wkj are adjusted to minimize the mean square
tant constituents of these alloys. In a number of recent stud- error of the net output. Two sets of parameters (the centers
ies, simultaneous determination of copper and mercury based and the widths) in the hidden layer and a set of weights in the
on atomic absorption spectrometry [24] and electrochemical output layer are adjusted. Therefore, the adjustment of the
methods [25,26] are reported. The aim of this study is to output layer is simple and RBFN has a guaranteed learning
profit by the advantages of spectrophotometry, as a low cost procedure for convergence. However, in back-propagation
and simple analysis method, in coincident determination of FFNN, the parameters of transfer functions both in hidden
the two metals. There are also some reports on recent spec- and output layers should be adjusted by using the Sigmoid
trophotometric quantification of Cu and Hg, but they are not transfer functions and generally it is time-consuming.
simultaneous determinations [27,28]. A considerable point is that many of authors call the
In this paper, murexide was added to mixtures of mercury RBF system of linear equations the “neural” networks, which
and copper cations solutions to form colored complexes for seems not correct. The RBF system lacks all the attributes that
spectrophotometric measurements. The reason for selecting go with the natural-inspired methods, such as ANN, genetic
murexide as coloring agent is the not complete complex algorithm (GA), and simulated annealing (SA). All natural-
formation of Hg(II) with this ligand which results to a non- inspired methods have in common (a) any form of random-
linear relation between the spectral data and concentration ness influencing the outcome of the procedure (sample order,
of analytes. The main goal in this article is the comparison initialization of weights, etc.), (b) similarity to some natural
of four artificial neural networks based methods (PCFFNN, phenomenon (functioning of biological neuron, survival of
PCRBFN, LDFFNN, and LDRBFN) for performing the fittest, movement of atoms during cooling, etc.), and (c)
non-linear calibration. The metal ions were estimated and an iterative learning procedure, with the number of iteration
methods were validated using synthetic and a dental amalgam steps influencing the results. RBFN has none of these features
samples. and can be solved straight by the multiple linear regression
(MLR) method.
2. RBFN theory
3. Experimental
The structure of radial basis function network (RBFN) is
3.1. Apparatus
comprised of three node layers of input, hidden and output.
The input layer serves only to distribute input to the hidden
A Pharmacia Ultraspec 4000 spectrophotometer with
layer. Each neuron of the hidden layer represents a kernel
Swift II (Amersham Pharmacia Biotech, Piscataway, NJ,
or a basis function, with equal dimensions to the input data.
USA), as instrument control and data acquisition soft-
RB networks generally use a Gaussian function to account
ware, was employed for spectrophotometric measurements.
for the non-linearity of the hidden layer processing elements.
A model 713 Metrohm pH meter (Metrohm Ltd., Herisau,
The Gaussian function responds only to a small region of the
Switzerland) was used for the measurement of pH of the
input space where the Gaussian is centered. The successful
solutions. A Pentium III computer and programs written in
implementation of these networks is to find suitable centers
MATLAB 6.0 (Mathworks Inc., Natick, MA, USA) were uti-
for such a Gaussian functions, which is characterized by two
lized for processing of data.
parameters, i.e. center (cj ), and peak width (σ j ). The output
for the jth Gaussian neuron for an input object xi can be
calculated by the following equation: 3.2. Reagents
outj = Φj (xi − cj ) = exp(xi − cj /σj )2 (1) All solutions were prepared with analytical reagent
grade reagents. Stock solutions of copper (1.600 mmol L−1 ),
where xi − cj is the calculated Euclidean distance between and mercury (1.600 mmol L−1 ) were prepared from
xi and cj , and σ j determines the portion of the input space Cu(NO3 )2 ·3H2 O, and Hg(NO3 )2 ·6H2 O, respectively. A bo-
where the jth RBF will have a non-significant zero response. rate buffer pH = 12 solution was prepared using 0.05 mol L−1
Y. Akhlaghi, M. Kompany-Zareh / Analytica Chimica Acta 537 (2005) 331–338 333
3.3. Procedure
Fig. 3. Augmented partial residual plots (APaRPs) for Cu (considering PC1) (a), Hg (considering PC2) (b), Hg (considering PC3) (c), and Cu (considering
PC4) (d) when all selected PCs were included in the model. The selected PCs for plots (a–c) were PC1 to PC3, and for plot (d) were PC1 to PC4.
4.3. Detection of non-linearities result of the test. The fourth PC seems to contain no consid-
erable information. Totally, the test illustrates the significant
Application of ANNs in multivariate calibrations was pro- non-linearity in the considered data, and need to non-linear
posed when a significant non-linearity is observed in the data. calibration models like ANNs.
Apparent partial residual plot (APaRP) [32], as the most uni-
versal diagnostic tool, was the applied statistical test for dis- 4.4. Reducing the number of data
tinguishing the non-linearity of the relation between some
of the first factors and the concentrations in this study. This Decreasing the data volume before using ANNs for non-
procedure was implemented in the following way: individual linear multivariate calibration was suggested as a preprocess-
analyte concentrations contained in the vector cm were first ing step in many of the previous studies [12,33]. Two common
regressed against the first A PCs of the data matrix and the methods for reducing data are linear discriminant analysis
square of the first PC [32]: (LDA) and principal component analysis (PCA). In this work,
before being reduced, the absorbance data were autoscaled
cm = b0 + b1 PC1 + · · · + bA PCA + baa (PCa )2 + eAPaRP , (using the mean and standard deviation of each column). For
a = 1, . . . , A (3) estimation of first significant score vectors using PCA, con-
centrations of calibration mixtures were not considered and
where eAPaRP was a vector collecting the APaRP fitting resid- one set of principal components for both metals was esti-
uals. The relevant plot was obtained by plotting the sum mated. The plot of first two score vectors versus the sample
[eAPaRP + ba PCa + baa (PCa )2 ] as a function of PC1 [32]. codes, which are in correlation with concentrations, is illus-
Fig. 3 shows the APaRPs for the calibration of the two trated in Fig. 4a. No correlation between the values of scores
analytes, using three or four PCs for copper and mercury. As and the concentrations can be seen, for any of the PCs. Es-
can be found from the plots, for the first PC a linear relation timation of first significant discrimination factor (DF) using
with the variation of mixtures components was obtained. For LDA included a classification step, based on concentration
the second and third PCs a significant non-linearity was the matrix. The members (samples) for each class were differ-
Y. Akhlaghi, M. Kompany-Zareh / Analytica Chimica Acta 537 (2005) 331–338 335
4.5.1. PCFFNN
Utilizing the Levenberg–Marquardt algorithm, feed-
forward neural networks (FFNNs) including one to six nodes
in the hidden layer, and one to seven PCs as input were trained.
To observe the extent of reproducibility of the results, and
the robustness of the network, the training process in each
of the conditions was repeated 10 times. From the 10 ob-
tained results, the one with minimum RSE% was considered
for investigations. As shown in Fig. 5(a) and (b), the low-
est RSE% values were obtained with five input factors and
four hidden nodes for Cu(II) and with five input factors and
two hidden nodes for Hg(II), which were included in the fi-
nal PCFFNN models. It can be seen that in the most of the
cases resulting RSE%s for 10 times repetition of trainings
in each condition are not similar. This shows the degree of
robustness of FFNNs when using PCA for data compression.
The predicted concentrations of two analyte ions using the
final obtained PC-FFNN models for seven test samples are
in Table 3.
Fig. 4. (a) Estimated first discrimination factors (DF1s) from LDA on spec-
tral calibration data, once based on copper concentrations (white) and then
based on mercury contents (gray). (b) Calculated scores for the first two
4.5.2. LDFFNN
PCs from PCA on spectral calibration data (without considering the analyte Discrimination factors (DFs) were the obtained com-
contents of solutions). pressed data from LDA for this part of work. For each of the
analytes, the first DF included the majority of the variations in
ent when the classification was on Hg contents of solutions, the spectral data (Table 2). Anyway, in addition to the effect of
compared to when it was based on Cu contents. The first DFs number of hidden nodes on the values of RSE%s for the con-
from applying LDA on spectral data for Hg and Cu are shown trol samples, the influence of utilizing more than one DF as
in Fig. 4b. Considerable correlation can be observed between the input for the calibration model was investigated. Applica-
the values of DFs and the sample codes (concentrations). The tion of more than one node in the hidden layer made consider-
eigenvalues from PCA and LDA calculations on the calibra- able improvements in the obtained RSE%s. But, application
tion data are in Table 2 and well illustrates that more than of more than one DF decreased the repeatability of the results,
99.9% of variations are in the first DFs. in addition to increasing the error values. This is due to the
classification ability of LDA. The results from application of
4.5. Optimizing the number of factors and the network 1–10 hidden nodes and one DF for each of two analytes are
variables illustrated in Fig. 5(c) and (d). One DF (one node in the input
layer) and nine hidden nodes were selected for both Cu and
One layer of hidden nodes and one node in the output layer Hg in the final LDFFNN models. The estimated concentra-
are common topological suggestions, when using an artificial tions for the two cations using the final obtained LDFFNN
neural network as a model for the non-linear calibration [12]. models in seven test samples are in Table 3. Compared to
In this way, separate networks were trained for copper and the condition of using PCA prior to FFNN, application of
Table 2
First five eigenvalues estimated from applying LDA and PCA on calibration set spectra
λ1 λ2 λ3 λ4 λ5
LDA
Cu 23362 6.42 × 10−11 4.92 × 10−11 4.77 × 10−11 4.59 × 10−11
Hg 12835 3.33 × 10−11 2.61 × 10−11 2.22 × 10−11 1.78 × 10−11
PCA 106.4147 57.4301 23.8994 15.6024 10.6913
336 Y. Akhlaghi, M. Kompany-Zareh / Analytica Chimica Acta 537 (2005) 331–338
Fig. 5. The relation of RSE% and the number of input PCs in PCFFNN using four nodes for Cu (a), and two nodes for Hg in the hidden layer (b). RSE% values
vs. the number of hidden nodes in LDFFNN, using one DF for both copper (c) and mercury (d).
LDA resulted in a considerable improvement in the obtained input variables and SPREAD. The latter parameter was in re-
RSEs. The second point is that the input variable of the net- lation with the spread of radial basis functions in the network.
work for LDFFNN was only one DF, whereas in the case of Input variables for this part were the PCs from application of
PCFFNN the number of input PCs were >4 for both analyte PCA on spectral data. RSE%s for the prediction of copper
ions. and mercury ions (in the control samples) at the number of
input PCs from 3 to 6, and SPREAD values of 1–100 were
4.5.3. PCRBFN investigated. Error values for number of PCs less than 3 and
The exact type of radial basis function networks (RBFNs), more than 6, and SPREAD values less than 1 were very high.
from ANN Toolbox of MATLAB version 6.0, was the choice Introduction of four to six PCs resulted in significantly im-
for this part. In the exact RBFNs the number of hidden nodes proved RSEs. In this way, four first PCs as input variables
are equal to the number of nodes in the input layer. In this and SPREAD = 21 were chosen for copper, and same four
way, the adjustable parameters in this part were the number of PCs and SPREAD = 45 were the choice for mercury in the
Table 3
Results for metal ions analysis in synthetic samples
Calculated conc. (mol L−1 ) Actual conc. (mol L−1 )
Cu Hg Cu Hg Cu Hg Cu Hg
0.00 23.64 0.25 19.49 0.00 23.62 0.00 20.83 0.00 23.64 1
3.99 0.00 3.86 0.08 3.98 0.02 3.75 0.07 3.99 0.00 2
3.70 111.34 3.72 112.34 3.69 111.32 3.61 112.46 3.71 111.36 3
7.51 90.14 7.79 88.85 7.51 90.31 7.38 93.52 7.51 90.14 4
11.40 68.40 11.71 72.15 11.40 68.29 11.97 69.60 11.40 68.41 5
15.38 46.15 15.50 44.24 15.40 46.14 15.52 50.76 15.38 46.15 6
19.46 23.57 19.34 21.65 19.45 23.87 19.26 21.39 19.46 23.36 7
0.03 0.12 1.85 3.69 0.11 0.35 2.38 3.99 RSE%
Y. Akhlaghi, M. Kompany-Zareh / Analytica Chimica Acta 537 (2005) 331–338 337
4.5.4. LDRBFN
This part included LDA as a compression procedure,
before modeling the data with RBNN. The adjustable pa-
rameters for the utilized exact RB network were SPREAD
and the number of input DFs, calculated using LDA. RSEs
for prediction of the concentrations of copper and mercury
(in the control set) at the number of DFs from 1 to 3,
and log(SPREAD) values of −1.3 to 2 were investigated.
The results showed that the minimum RSE% values for Cu
and Hg were at log(SPREAD) values of −1.0 and −1.2
(or SPREAD values of 0.10 and 0.07), respectively, and
only one DF as input (Fig. 6b), which were the choice for
training the final LDRBFNs. The specific RSE% values of
0.03 and 0.12% in the analysis of test samples (Table 3)
illustrates the effect of gathering the abilities of LDA as
a classification method and RBFN as a flexible and pre-
cise calibration model. In this part also, due to high repro-
ducibility of RBNN, 10 times repetition of training was not
necessary.
Table 4
Accuracy and precision for the metal ions analysis in dental alloy samples
Sample PCFFNNa LDFFNNa PCRBFNa LDRBFNa AAS
Hg (mol L−1 )
1 16.84 ± 0.15 16.38 ± 0.19 16.75 ± 0.20 16.36 ± 0.15 16.46
2 23.93 ± 0.34 24.28 ± 0.26 24.71 ± 0.29 24.38 ± 0.24 24.33
3 19.90 ± 0.17 20.21 ± 0.21 20.42 ± 0.17 20.14 ± 0.24 20.21
4 13.86 ± 0.15 14.06 ± 0.11 13.99 ± 0.13 14.10 ± 0.14 14.09
5 23.72 ± 0.25 23.55 ± 0.23 23.90 ± 0.20 23.52 ± 0.26 23.52
Cu, mol L−1
1 14.78 ± 0.13 14.77 ±0.09 14.83 ± 0.06 14.76 ± 0.10 14.75
2 15.92 ± 0.21 15.79 ±0.12 15.74 ± 0.17 15.87 ± 0.15 15.84
3 18.28 ± 0.15 18.30 ±0.17 18.21 ± 0.17 18.30 ± 0.12 18.24
4 12.51 ± 0.11 12.50 ±0.10 12.57 ± 0.08 12.55 ± 0.10 12.54
5 17.69 ± 0.16 17.60 ±0.18 17.59 ± 0.17 17.65 ± 0.13 17.63
a With three times replication of the experiments.
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