Chimiometrie 2009

Effect of pH and UV radiation on anthocyanin mixtures extracted

from Malvaviscus penduliforus investigated with MCR-ALS

Paulo Henrique Março1, Ronei Jesus Poppi1, Romà Tauler2

1
Universidade Estadual de Campinas, Instituto de Química, CP 6154, CEP 13084-971, Campinas-SP, Brazil.
2
Department of Environmental Chemistry, Institute of Environmental Diagnostics and Water Studies
IDAEA-CSIC Jordi Girona, 16, 08034, Barcelona, Spain.

Keywords: MCR-ALS, MPCA, Anthocyanin.

1 Introduction
Anthocyanins are an important group of plant pigments, playing the major role in the appeal of many flowers
and fruits, being considered as indicators of fruit ripening. Due to their biological activities and their
potential benefits for human health [1]. A particular aspect of these products is the pH influence on their
color. Depending on the acidity or alkalinity, anthocyanins can form different chemical structures. According
to McClelland [2] and Brouillard [3], in aqueous solution, several species can be involved in complex
equilibria. Flavylium cation (AH+) is the predominant species at low pH (< 2), and it is easily deprotonated
to form the quinoidal base (A) or the colorless pseudobase carbinol (B). The carbinol form does not absorb at
the visible range and it is in tautomeric equilibrium with cis-chalcone (Cc). The equilibrium between
carbinol and trans-chalcone is always described as being a very fast equilibrium and sometimes the forms
can not be distinguished, being described as B + CC or showed as only one form [4]. Color intensity
increases at basic medium but the color and maximum wavelength (max) are different when compared to
solutions at pH < 2 [5]. Cis-chalcone also isomerizes in trans-chalcone (Ct) via a cis-trans isomerization
reaction. According to Maestri et al [6], this transformation can also be observed through light excitation,
indicating that light exposure can cause photodegradation of anthocyanins. Photodegradation processes can
be monitored in the laboratory by UV spectroscopy, where a series of spectra are collected as a function of
time. Changes in absorption bands as the photodegradation takes place can provide important information
about the evolution and mechanism of the kinetic process under study. However, experimental spectra of
anthocyanin raw materials provided by UV absorption are strongly overlapped and, most often, no selective
wavelengths are expected to be present. Using traditional means is obviously very difficult to estimate
simultaneously the number, concentration and pure spectra profile of so many species present in such a
complex chemical mixture system without spectral resolution. Therefore, to get information about this
system, the use of powerful spectra resolution chemometric mehods is proposed [7]. A general approach
involving the identification of a model from numerical and statistical analysis of spectrometric data is
proposed to solve the chemical mixture analysis problem, which implies the estimation of the number of
chemical species simultaneously present in the mixture, the spectral identification of these species, and the
determination of their concentration without any previous assumption about the nature or composition of the
system under investigation [8].

2 Theory
In this work, all the data were concatenated to generate a augmented data matrix (Daug). The Daug is
decomposed in the product of two matrices, one augmented matrix describing respectively the different row
spaces Caug of the original individual data matrices, and one matrix describing the common column spectra
space ST of them. Eaug gives the spectral variation not explained by the bilinear model. Another form to write
it in a more compact way is using equation 1:

Daug = CaugST + Eaug (1)

Multiway Principal Component Analysis, MPCA, is equivalent to performing Principal Components,
Analysis (PCA) on the augmented data matrix, formed concatenating individual data matrices.
The main objectives of MCR are the isolation, resolution, and relative quantification of the main sources of
variation in a particular data set. The outstanding feature of this technique is that no a priori chemical
assumption about the contribution of the different components is necessary. Multivariate Curve Resolution
methods decompose mathematically the measured instrumental response (assumed to follow a bilinear model
like in PCA) into a mixture of pure contributions of each of the components of the system. The bilinear
decomposition is now performed under a completely different set of constraints than PCA, searching for
solutions with physical meaning, for instance instead of using orthogonality constraints like in PCA, non-
negativity constraints are applied [9].
3 Material and methods
In this work the individual k data matrices of dimensions Dk(I, J) (I = 239 spectra and J = 531 wavelengths)
obtained at each pH, (k =1,..,K, K = 12, varying from pH = 1,90 to pH = 12,72, were concatenated one on
top of the other in the new augmented matrix Daug, giving a new matrix with dimensions of (239x12,531).
This method of matrix augmentation allows analyzing the variability among pH batches or samples in Daug
and summarizes the information in the data with respect their spectra variation (in Figure 2, the vectors
defining the column vector space of all Dk matrices (or of ST) are the same for all the simultaneously
analyzed matrices).

4 Results and discussion

In order to have an initial estimation of the number of species, the chemical or pseudo rank
(mathematical rank in absence of experimental noise) of the data matrix obtained at each pH value
was estimated using Principal Components Analysis (PCA) and Singular Value Decomposition
(SVD). MPCA was used to check and match the number of components present simultaneously in
the different individual data matrices obtained at different pH values and also to perform an
exploratory cluster analysis of the whole set of flower samples and to find out which of them were
more similar. The result shows that MPCA was efficient to separate samples exposed to not
exposed to the UV radiation, as well as separete the acidic medium group from neutral and basic
groups, as shows Figure 1.

Figure 1 – MPCA result to Malvaviscus penduliforus sample exposed and non-exposed to the UV-radiation.
Sample number is referred to the correspondent pH as in the side-table.
MCR-ALS was then applied to resolve the pure spectra and concentration profiles of all the different species
in equilibrium at the different pH and experimental conditions (with and without UV exposure). The
cosntraints applied during the ALS optimization were spectra and concentration profiles non-negativity and
concentration profiles closure, besides species correspondence among different samples at different pH
values. This information about species correspondence was preliminary acquired from the singular value
decomposition (SVD) of the individual and augmented data matrices and it was optimized afterwards during
the MCR-ALS resolution process. The species AH+, A, CC, some A- structures and CC- could be detected.
When the analysis was carried out under UV radiation was possible to detect one species not detected when
the analyses were carried out in the dark.

Figure 2 – Spectra (at the top) and Kinetic evolving with pH (at the botton) and time resolved by MCR-ALS:
(1) Flaviliun Cation (AH+); (2) Quinoidal (A); (3) Chalcone (CC); (4 & 5) Ionized Quinoidal forms(A-); 6) Ionized
Chalcone (CC-). The dashed line (….) shows the behavior of the species when exposed to the UV radiation, while the
continuous line (___) shows the behavior at the dark. The species ilustrated in (7) was detected only under UV radiation.

5 Conclusion
Multivariate Curve Resolution allowed the investigation of the kinetic (photo)degradation study of
antocyanins at different pH values, with and without radiation exposure. Up to 7 different species were
resolved, identified by their pure spectra and kinetic profiles which would be very hard to obtain by using
tradicional methods.
6 References

[1] - Netzel, M., Netzel, G., Kammerer, D. R., Schieber, A., Carle, R., Simons, L., Bitsch, I., Bitsch, R., & Konczak, I.
(2007). Cancer cell antiproliferation activity and metabolism of black carrot anthocyanins. Innovative Food
Science and Emerging Technologies, 8, 365–372
[2] - McClelland, R. A., & Gedge, S.(1980). Hydration of the Flavylium Íon. Journal of American Chemical Society,
102, 5838-5848.
[3] - Brouillard, R.; Iacobucci, & G. A.; Sweeny, J. G.(1982). Chemistry of Anthocyanin Pigments. 9. UV-Visible
Spectrophotometric Determination of the Acidity Constants of Apigeninidin and Three Related 3-Deoxyflavylium
Salts, Journal of American Chemical Society, 104, 7585.
[4] - Asenstorfer, R. E., & Jones, G. P.(2007). Charge equilibria and pK values of 5-carboxypyranomalvidin-3-
glucoside (vitisin A) by electrophoresis andabsorption spectroscopy, Tetrahedron, 63, 4788–4792
[5] - Brouillard, R., & Dubois, J. E. (1977). Mechanism of the Structural Transformations of Anthocyanins in Acidic
Media. Journal of American Chemical Society, 99, 1359.
[6] - Melo, M. J., Moura, S., Maestri, M., & Pina, F.(2002), Journal of Molecular Structure, 612, 245-253.
[7] - Schiozer, A. L., Março, P. H., Barata, L. E. S., Poppi, R. J. (2008). Exploratory Analysis of Arrabidaea chica
Deoxyanthocyanidins using Chemometric Methods. Analytical Letters, 41, 1592–1602.
[8] - Tauler, R. (1995). Multivariate curve resolution applied to second order data. Chemometrics and Intelligent
Laboratory Systems, 30, 133-146
[9] - de Juan; A., & Tauler, R. (2006). Multivariate Curve Resolution (MCR) from 2000: Progress in Concepts and
Applications. Critical Reviews in Analytical Chemistry, 36,163–176.