Multway PCA and MCR investigation of the effect of pH and UV

radiation on kinetic degradation of anthocyanin mixtures extracted

from flower dyes.

Paulo Henrique Março1, Ronei Jesus Poppi1 and Romà Tauler2

1
Universidade Estadual de Campinas, Instituto de Química, C.P. 6154, CEP 13084-971, Campinas SP, Brazil.
2
Department of Environmental Chemistry, Institute of Chemistry and Environmental Research – CSIC Jordi Girona,16,
08034, Barcelona, Spain.

Keywords: Anthocyanin, Dyes, MPCA, MCR-ALS, Augmented Data Matrix.

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.

Multivariate Curve Resolution

MCR is a chemometric method included in the Factors Analysis (FA) family of techniques. The main goals
are the isolation, resolution, and quantification of the sources of variation in a particular data set. The
outstanding feature of this technique is that no a priori assumption about the contribution of the different
factors in the global response is necessary[7]. All resolution methods decompose mathematically a global
mixed instrumental response (following a bilinear model) into the pure contributions due to each of the
components in the system. Like for PCA, this global response may be organized in augmented data matrix
Daugment containing raw information about all the components present in the data set. MCR methods allow for
the bilinear decomposition of the initial mixture data matrix D into the product of two data matrices, called
now C and ST, each of them including the pure response profiles of the n mixture or process components
associated with the row and the column vector spaces of the initial data matrix, respectively. In matrix
notation, the expression valid for all resolution methods is:
D = CS T + E (1)
T
where D (r × c) is the original data matrix, C (r × n) and S (n×c) are the matrices containing the pure
response profiles related to the data variation in the row direction and in the column direction, respectively,
and E (r × c) is the error matrix, i.e. the residual variation of the data set that is not related to any chemical
contribution. r and c are the number of rows and the number of columns of the original data matrix,
respectively, and n is the number of chemical components in the mixture or process. C and ST often refer to
concentration profiles and spectra (hence their names), although resolution methods are proven to work in
many other diverse problems[11]. The bilinear decomposition is now performed under a completely different
set of constraints, searching for solutions with physical meaning, for instance instead of using orthogonality
constraints like in PCA, non-negativity constraints are applied. MCR may be also easily extended to the
study of augmented data matrices and to multiway data analysis as it is done in the present work. In
references 7, 8, 10 and 12 more details are given about the application of MCR methods.
3 Material and methods
Plant material was collected in Londrina city, Parana state, Brazil. The vouchers were deposited at the
herbarium of the State University of Londrina – UEL, with access number FUEL 33816 (Hibiscus
Acetosella), FUEL 37275 (Hibiscus Sabdariffa) and FUEL 33818 (Malvaviscus Penduliflorus). The calices
of the flowers were crushed and the anthocyanins extracted with acidified ethanol (HCl 0,01 mol/L) then
concentrated up with a rotary evaporator connected to a high-vacuum pump at 35°C during 1 hour. After
completely evaporation of ethanol the concentrated were dissolved with hexane followed by
dichloromethane, three times each under agitation at room temperature. The latter was evaporated and
cleaned up through a XAD-7 column (50 x 4 cm) by passing ultra pure water followed by methanol, with the
anthocyanins being eluted by methanol, which was evaporated. The concentrated was dissolved with HCl
0,01 mol/L and then stored in refrigerator at 0° C. The buffer solutions presented pH values from 1.9 to 12.7.
UV-Vis analyses were carried out using a diode array Agilent 8453 spectrophotometer with quartz cuvette (d
= 1cm). Temperature was hold on 25 (±0.1) ºC at constant agitation. Ultraviolet-visible absorption spectra at
different pH values were obtained from 190 to 1100 nm. The samples were diluted in each of the buffer
solutions at the moment of analysis.
Concentration was placed in a value suitable for an absorbance around 0.8 at the λmax on the visible region.
To the experiments using UV light the same procedure described above was taken, but with an ocean
optics® Xenon lamp “PX-2”[12] UV light of 9.9 watts emitting from 220 to 750 nm, working on 220Hz,
lamp supported on the cuvette with the radiation incidence being directly in the sample. The spectra were
monitored during 2 hour, with and without UV radiation exposure, and then the data were rearranged.
PCA was applied in order to estimate the pseudo rank at each pH value, then, a MPCA was used to separate
the most similar matrices. The MCR was applied to obtain the relative concentration values and the pure
spectra from the sample components.
MCR was used on the MatLab 6.5 ® program implemented with tools available from prof. Dr. Romà Tauler
Ferré and profª Drª Anna de Juan webpage[13].

4 Results and discussion

MPCA was applied to find out the similarity between the matrices, and results of the analysis of the 3
different types of flowers are presented in the figure 2 - (a) to Hibiscus Acetosella, (b) Hibiscus Sabdariffa
and (c) to Malvaviscus Penduliflorus. Table 1 show pH values and the colors indicate whether the samples
were exposed to UV light or not. It is observed from the results that MPCA separated the data matrices from
samples exposed to light from those not exposed to UV radiation. In the case of Hibiscus Sabdariffa and
Malvaviscus Penduliflorus it was possible to separate even more according to the pH value.
PCA analysis of the different data matrices showed different number of components in each case depending
on the number of coexisting species in equilibrium at the considered pH value, between 2 species at low pH
to 4 species at basic pH.
The number of Principal Components (PCs) observed were related with the probable number of species that
could be present in equilibrium to a determined pH value and compared to the suggested one in the literature.
To investigate the presence of the suggested species, MCR-ALS was used to resolve the species spectra, and
the kinetic profiles which were used to compare the degradation between exposed and not exposed samples.
As example, only the results of application of MCR-ALS to Hibiscus acetosella flower are given in this
summary. A more complete description of the three cases and of their simultaneous analysis using matrix
augmentation strategies will be shown in the presentation. Figures 3, 4 and 5 presents the kinetic and spectral
profiles resolved by MCR for the Hibiscus acetosella data at acidic, neutral and alkaline medium,
respectively.
 (a) (b) (c)
Figure 2 – MPCA results to the samples of (a) Hibiscus Acetosella, (b) Hibiscus Sabdariffa and (c) Malvaviscus
penduliflorus not exposed (x) to the UV radiation and exposed (x).

Table 1 – pH value to the samples

Not exposed to UV light (x) pH Exposed to UV light (x) pH
0 1.90 - -
1 2.50 1 2.42
2 3.98 2 4.08
3 5.12 3 5.12
4 6.13 4 6.12
5 7.22 5 7.12
6 8.12 6 8.16
7 8.67 7 8.47
8 9.32 8 9.32
9 9.88 9 9.75
10 10.80 10 10.93
11 11.83 11 11.70
12 12.42 12 12.72

(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

O-Açúcar O-Açúcar O-Açúcar

OH OH OH
Cátion Flavílico (AH+) Anidrobase Quinoidal (A) Ionização das Anidrobases
- H 2O
+ H 2O
-
H+ OH
R OH
-
R H+ OH- R
- H 2O H+
+ H2O
OH O- O-
OH HO OH O
HO O - OH
- O O
OH R'
R' R'
H+ H+

O-Açúcar O-Açúcar O-Açúcar

OH OH O-
OH
- Quinoidal Ionizada (A-)
Carbinol (B) Cis-Chalcona ( CC )
H+
OH
- R
H+
O-
R R
-O
- O- O
HO O- O- O
O -Açucar
OH
- O -Açucar R'
H+
R' R' O-Açúcar
-O O
OH O -O

Trans-Chalcona ( Ct ) Trans-Chalcona Ionizada ( C t- ) Cis-Chalcona Ionizada ( CC- )

Scheme 1 - Possible structural transformation in aqueous medium as a pH function.

5 References
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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.