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1 Introduction
Anthocyanins are an important group of plant pigments. Due to their biological activities and their potential
benefits for human health, there is considerable interest in the development of food colorants from these
natural sources to replace synthetic food colorants. Their color is easily affected by some factors, such as
temperature, pH, solvent, light exposure, the structure of the pigment itself and the presence of other
molecules, which can be generally described as copigments[1,2]. A particular problem is the pH influence on
their colour. Depending on the acidity or alkalinity, anthocyanins adopt different chemical structures.
According to McClelland[3] and Brouillard[4], in aqueous solution, several species can be involved in this
complex set of equilibria. Flavylium cation (AH+) is the predominant species at low pH (<2), and it easily
deprotonates to form the quinoidal base (A) or the colorless pseudobase or carbinol (B) The carbinol form
does not absorb light and is in tautomeric equilibrium with cis-chalcone (Cc) that also isomerizes trans-
chalcone (Ct) via a cis-trans isomerization reaction. Color intensity increases at basic media but the color and
maximum wavelength (λmax) are different when compared to solution at pH<2. According to Maestri et
al[5,6], this interconvertion can also be observed through light excitation.
With the growth of chemometrics in recent years, a general approach involving the identification of a model
from numerical and statistical analysis of data has been proposed to solve the mixture analysis problem,
without any previous assumption about the nature or composition of the system under investigation. Mixture
analysis implies the estimation of the number of chemical species simultaneously present in the mixture, the
identification of these species, and the determination of their concentration. Among the computational and
statistical methods used to solve mixture analysis problems, factor analysis (FA), principal component
analysis (PCA), and singular value decomposition (SVD) techniques play a key role, especially in the
estimation of the number of species contributing significantly to the experimental data variance[7]. These
pure mathematical solutions obtained by means of FA-derived methods can be transformed to physically
meaningful solutions by means of multivariate curve resolution (MCR) methods[8].
The objective of this work is to investigate the effect of pH variation and light incidence on anthocyanin
degradation by using chemometric methods. The analysis were carried out using anthocyanins extracted
from 3 different plants, measuring UV-Vis spectra with and withoud exposure of UV radiation (in order to
check the influence of radiation) at different pH values over 2 hours. The results obtained from this work
may help on the development of an analytical methodology for determining the number of species involved
and to elucidate the mechanism of the anthocyanin kinetics degradation.
2 Theory
Multi-Way PCA
MPCA is equivalent to performing PCA on a large two-dimensional matrix (augmented data matrix) Daugment,
formed by unfolding a three-way array or concatenating individual data matrices as it is shown in the scheme
below. In this work, the individual data matrices obtained at each pH were organized one on top of the other
in the new augmented matrix Daugment, giving a new matrix with of dimensions (IxK, J)[10], where I is the
number of measured time spectra at each pH, J is the number of measured wavelengths in the spectra and K
is the number of different pH matrices included in the simultaneous analysis. This way of augmenting
matrices allows analyzing the variability among pH batches or samples in Daugment and summarizes the
information in the data with respect to variables and their spectra variation (the vectors defining the column
space of Dk matrices or of PT are the same for all the simultaneously analyzed matrices).
Figure 1 - Matrix augmentation and bilinear decomposition using either MPCA or MCR-ALS
In PCA and MPCA, this bilinear data decomposition is performed under orthogonality constraints (for
vectors in both T and PT), normalization of vectors in PT and in the directions of maximum explained
variances for the successively extracted components. Under these conditions, obtained solutions are unique,
but they lack physical meaning.
(a) (b)
Figure 3 – Kinetic and spectral profile recovered by MCR from the data matrix of Hibiscus acetosella at (a) pH 2,50 -
not exposed to the UV and (b) pH 2,42 - exposed to the UV radiation.
(a) (b)
Figure 4 – Kinetic and spectral profile recovered by MCR from the data matrix of Hibiscus acetosella at (a) pH 7,22 -
not exposed to the UV and (b) pH 7,12 - exposed to the UV radiation.
(a) (b)
Figure 5 – Kinetic and spectral profile recovered by MCR from the data matrix of Hibiscus acetosella at (a) pH 12,42 -
not exposed to the UV and (b) pH 12,72 - exposed to the UV radiation.
By analyzing these results for the proposed equilibra for the exposed and not exposed to radiation samples
and based on previous studies, it is suggested that in aqueous medium the equilibra between anthocyanin
forms could be represented for the scheme 1.
R R R
OH OH O-
HO O - O O - O O
OH OH
R' R' R'
+ +
H H
5 References
[1] C. E. Lewis, J. R. L. Walker and J. E. Lancaster: Effect of polysaccharides on the colour of anthocyanins. Food
Chemistry, 54:315-3I9, 1995
[2] P. Markakis. Anthocyanins as food colors. New York: Academic Press, 1982.
[3] R. A. McClelland, S. Gedge: Hydration of the Flavylium Ion. Journal of American Chemical Society, 102: 5838-
5848, 1980.
[4] R. Brouillard, G. A. Iacobucci, J. G. Sweeny: Chemistry of Anthocyanin Pigments. 9.1 UV-Visible
Spectrophotometric Determination of the Acidity Constants of Apigeninidin and Three Related 3-Deoxyflavylium
Salts. Journal of American Chemical Society, 104: 7585-7590, 1982.
[5] M. Maestri, F. Pina, A. Roque, P. Passaniti: Light and pH switching between the various forms of the 4´-
methylflavylium cation. Journal of Photochemistry and Photobiology A: Chemistry, 137: 21-28, 2000.
[6] M. J. Melo, S. Moura, M. Maestri, F. Pina, Micelle effects on multistate/multifunctional systems based on
photochromic flavylium compounds. The case of luteolinidin. Journal of Molecular Structure, 612 (2-3): 245-253,
2002.
[7] A. Izquierdo-Ridorsa, J. Saurina, S. Hernández-Cassou, R. Tauler: Second-order multivariate curve resolution
applied to rank-deficient data obtained from acid-base spectrophotometric titrations of mixtures of nucleic bases.
Chemometrics and Intelligent Laboratory Systems, 38 (2): 183-196, 1997.
[8] R. Tauler: Multivariate curve resolution applied to second order data. Chemometrics and Intelligent Laboratory
Systems, 30(1): 133-146, 1995.
[9] B. M. WISE, N. B. GALLAGHER, R. BRO, J. M. SHAVER, W. WINDIG, R. KOCH: PLS_Toolbox Version 4.0
for use with MATLAB, Eigenvector Research, Inc., Manson, WA 98831.
[10] A. de Juan, R. Tauler: Chemometrics applied to unravel multicomponent processes and mixtures: Revisiting latest
trends in multivariate resolution. Analytica Chimica Acta, 500 (1-2):195-210, 2003.
[11] http://www.oceanoptics.com/products/px2.asp: accessed at 08/01/2008.
[12] Tauler webpage: http://www.ub.es/gesq/mcr/mcr.htm: accessed at 08/01/2008.