Food Chemistry 125 (2011) 1020–1027

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Investigation of the pH effect and UV radiation on kinetic degradation

of anthocyanin mixtures extracted from Hibiscus acetosella
Paulo Henrique Março a, Ronei Jesus Poppi a,⇑, Ieda Spacino Scarminio b, Romà Tauler c
a
Institute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil
b
Department of Chemistry, University of Londrina, P.O. Box 6001, 86051-970 Londrina, PR, Brazil
c
Department of Environmental Chemistry, Institute of Environmental Diagnostics and Water Studies, IDAEA-CSIC, Jordi Girona, 16, 08034 Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The major anthocyanin pigments extracted from Hibiscus acetosella flower were investigated by UV–Vis
Received 15 March 2010 spectroscopy and multivariate curve resolution-alternating least squares (MCR-ALS). Pure spectra and
Received in revised form 29 September kinetic of the species present at different pH values were recovered for anthocyanins transformation
2010
and degradation products, found with and without UV radiation exposure. In the absence of UV radiation,
Accepted 3 October 2010
up to seven different species were detected and by UV radiation exposure, this number increased to up to
nine. The species detected in the absence of radiation were also detected when pigments samples were
exposed to UV radiation, where degradation occurred faster and two new species appear. The kinetic pro-
Keywords:
Anthocyanin
files obtained at different pH values allowed the proposal of a reaction mechanism and pathway.
Hibiscus acetosella Ó 2010 Elsevier Ltd. All rights reserved.
Dyes
MPCA
MCR-ALS

1. Introduction (AH+) is the predominant species at low pH (<2), and it is easily

Anthocyanins are an important group of plant pigments, play-
ing the major role in the appeal of many flowers and fruits, being
considered as indicators of fruit ripening. Due to their biological
activities and potential benefits for human health (Netzel et al.,
2007), there is considerable interest on studying these pigments.
Some extracted anthocyanins have been used as colourants for
food (Markakis, 1982) and beverages (Rosso & Mercadante,
2007), but because of the colour fade in neutral pH solutions, their
use has been limited. The anthocyanins are structurally based on
the flavylium nucleus, which in nature is found to be bonded to hy-
droxyl groups, sugars and organic acids (Dougall & Baker, 2008).
The main structures are shown at Fig. 1. Their colours are easily af-
fected 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
(Lewis, Walker, & Lancaster, 1995). A particular aspect of these
products is the pH influence on their colour. Depending on the
acidity or alkalinity, anthocyanins can form different chemical
structures. According to McClelland and Gedge (1980) and
Brouillard, Iacobucci, and Sweeny (1982), in aqueous solution, sev-
eral species can be involved in complex equilibria. Flavylium cation
⇑ Corresponding author.
E-mail address: ronei@iqm.unicamp.br (R.J. Poppi).
deprotonated to form the quinoidal base (A) or the colourless
pseudobase carbinol (B). The carbinol form does not absorb at
the visible range and it is in tautomeric equilibrium with cis-chal-
cone (CC). The equilibrium between carbinol and trans-chalcone is
always described as being a very fast equilibrium and sometimes
the two forms cannot be distinguished, being described as B + CC
or showed as only one form (Asenstorfer & Jones, 2007). Colour
intensity increases at basic medium but the colour and maximum
wavelength (kmax) are different when compared to solutions at
pH < 2 (Brouillard & Dubois, 1977; Brouillard et al., 1982). cis-Chal-
cone also isomerizes in trans-chalcone (Ct) via a cis–trans isomeri-
zation reaction. According to Maestri et al. (Maestri, Pina, Roque, &
Passaniti, 2000; Melo, Moura, Maestri, & Pina, 2002), this transfor-
mation 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 photodegra-
dation takes place can provide important information about the
evolution and mechanism of the kinetic process under study. How-
ever, experimental spectra of anthocyanin raw materials provided
by UV absorption are strongly overlapped and, most often, no
selective wavelengths are expected to be present. To solve this
problem, mathematical multivariate curve resolution techniques
can be applied (Mas, de Juan, Lacorte, & Tauler, 2008).

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.10.005
 P.H. Março et al. / Food Chemistry 125 (2011) 1020–1027 1021

R R R
OH OH O-

HO O - O O -
O O
OH R' OH
R' R'
+ +
H H
O-Sugar O-Sugar O-Sugar
OH pH 1 - 2 OH pH 6,5 - 8 OH pH > 8
Flavylium Cation (AH+) Quinoidal Anidrobase (A) Ionized Quinoidal (A-)
- H2O
-
OH
+ H2O
-
H+ OH
R OH
-
R H+ OH- R
- H2O + H2O
OH O- O-
OH HO OH O
HO O - OH
- O O
R' OH R' R'
+
H
O-Sugar O-Sugar O-Sugar
OH pH < 6 OH pH > 9 O- pH > 10
OH
- Di Ionized Quinoidal (A2-)
Carbinol (B) Cis-Chalcone ( CC )
H+
R
O-
R R
-
O - O- O
HO
O-Sugar O- O-Sugar O- O
OH
- R'
H+
R' R' O-Sugar
-
OH O O O -
pH > 9 pH > 12 O pH > 12
Trans-Chalcone ( C t ) Ionized Trans-Chalcone ( C t- ) Ionized Cis-Chalcone ( CC- )

Fig. 1. Main anthocyanins structures and pH dependent speciation equilibria suggested in this work for anthocyanin species.

Using traditional methods 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 chemo-
metric methods is proposed (Levi, Scarminio, Poppi, & Trevisan,
2004; Schiozer, Março, Barata, & Poppi, 2008). A general approach
involving the identification of a model from numerical and statis-
tical analysis of spectrometric data is proposed to solve the chem-
ical 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 (Taul-
er, 1995). Among the computational and statistical methods used
to investigate mixture analysis problems, factor analysis (FA), prin-
cipal 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 (Mendieta, Díaz-Cruz, Esteban, & Tauler, 1998). Phys-
ically meaningful solutions can be obtained by applying multivar-
iate curve resolution (MCR) methods (Tauler, 1995). The
multivariate curve resolution-alternating least squares method
(MCR-ALS) can be also used as a second-order calibration method,
since it can provide both qualitative and quantitative information
about the analytes in the sample, in the presence of unknown
interferences. Moreover, MCR-ALS works adequately with analytes
presenting several equilibrium species where the expected bilinear
structure of the data is achieved if the suitable number of compo-
nents is selected to be equal to the true number of spectrally differ-
ent species at equilibrium (Carneiro, Braga, Poppi, & Tauler, 2008).
The objective of this work is to investigate the effect of pH var-
iation and light exposure on anthocyanin degradation by using
multivariate curve resolution methods. The analysis were carried
out using anthocyanin pigments extracted from Hibiscus acetosel-
la flower, measuring their UV–Vis spectra with and without expo-
sure of UV radiation (in order to check the influence of radiation) at
different pH values over 2 h. The results obtained from this work
may help on the development of an analytical methodology for
the determination of the number and nature of species involved
in degradation processes and in particular, to elucidate the mech-
anism and pathways of the anthocyanin kinetics degradation
investigated in this work.
2. Materials and methods
2.1. Samples and materials
H. acetosella samples were 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. The calices of the flowers were crushed and the anthocya-
nins extracted with acidified ethanol (HCl 0.01 mol/L). The extract
was concentrated with a rotary evaporator connected to a
high-vacuum pump at 35 °C during 1 h. After completely ethanol
evaporation, the concentrated extract was dissolved with hexane
followed by dichloromethane. The process was repeated three
times under agitation at room temperature and so the later extract
was heated at 35 °C in order to evaporate the solvents. In sequence,
the extract was cleaned up through a XAD-7 column (50  4 cm)
by passing ultra pure water followed by methanol, where the
anthocyanins were eluted by. The methanol was evaporated and
the concentrated extract was dissolved with HCl 0.1 mol/L, being
then stored in refrigerator at 0 °C. Also 12 buffer solutions were
prepared following the methodology described by Perrin and
Dempsey (1974) and the pH values from 1.9 to 12.7 were certified
by pH metre measurements.
UV–Vis analyses were carried out using a diode array Agilent
8453 spectrophotometer with quartz cuvette (length = 1 cm). Tem-
perature was hold on 25 (±0.1) °C at constant agitation. UV–Vis
absorption spectra at different pH values from 190 to 1100 nm
were obtained for 30 lL of anthocyanin sample extracts diluted
to 1 mL with each of the buffer solutions previously prepared. Con-
centration values were selected for a suitable maximum absor-
bance around 0.8, at the kmax on the visible region. The same
procedure described above was adopted to the experiments using
UV radiation where a Xenon lamp of 9.9 W emitting from 220 to
750 nm was used, working at 220 Hz, attached to the cuvette with
1022 P.H. Março et al. / Food Chemistry 125 (2011) 1020–1027

Fig. 2. Data matrix augmentation and bilinear decomposition using either MPCA or MCR-ALS (see Section 2.2).

the radiation incidence being directly to the sample. The spectra
were monitored during 2 h, under radiation exposure and at dark,
and then the data were rearranged in individual and column aug-
mented data matrices (Fig. 2) to perform the chemometric analysis.
The strategy of column augmentation will be explained in Section
2.2. Each individual data matrix contains 239 spectra, recorded at
30 s each during 120 min (the last spectrum was discarded).
The strategy utilised to process this large amount of informa-
tion (a total number of 2868 spectra – 239 spectra  12 different
pH values – measured at 531 wavelengths each) was to arrange
all the spectral data in a large column-wise augmented data matrix
and appropriate constraints during the alternating least squares
optimisation of the parameters (species pure spectra and concen-
tration kinetic profiles at different pH values).
2.2. Data arrangement and modelling
In this work (Fig. 2), the individual k data matrices of dimen-
sions 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,
see also Table 1) were concatenated one on top of the other in
the new augmented matrix Daugment, giving a new matrix with
dimensions of (239  12,531). This method of matrix augmenta-
tion allows the analysis of the variability among pH batches or
samples (Ruckebusch, de Juan, Duponchel, & Huvenne, 2006) in
Daugment and summarises the information in the data with respect
of their spectra variation (in Fig. 2, the vectors defining the column
vector space of all Dk matrices (or of ST) are the same for all the
simultaneously analysed matrices).
According to the bilinear model described in Fig. 2, the aug-
mented data matrix Daugment is decomposed in the product of
two matrices, one augmented matrix describing respectively the
different row spaces Caugment of the original individual data matri-
ces, and one matrix describing the common column spectra space
ST of them. Eaugment gives the spectral variation not explained by
the bilinear model. Fig. 2 can be written in a more compact way
using the following equation:
Daug ¼ Caug ST þ Eaug ð1Þ

2.3. Multiway PCA

Table 1
Number of principal components at different pH values for the time evolving UV Multiway principal component analysis, MPCA (Pereira-Filho,
spectra of the Hibiscus acetosella samples not exposed (s) and exposed (d) to the UV
Pérez, Poppi, & Arruda, 2002), is equivalent to perform principal
radiation.
components analysis (PCA) on the augmented data matrix, formed
Buffer solution Not exposed to the UV Exposed to the UV concatenating individual data matrices as it is shown at Fig. 2. In
radiation (s) radiation (d)
PCA and MPCA, the bilinear data decomposition shown in Fig. 2
pH 25 °C PCs pH 25 °C PCs is performed under the constraints of orthogonality (for vectors
0 1.90 2 – in both C and ST), normalisation of vectors in ST and in the direc-
1 2.50 2 2.42 3 tions of maximum explained variances for the successively ex-
2 3.98 2 4.08 3
tracted components. Under these constraints, obtained solutions
3 5.12 4 5.12 4
4 6.13 2 6.12 3 are unique, but they lack direct physical meaning. They are useful
5 7.22 4 7.12 3 for data interpretation but not for species resolution.
6 8.12 4 8.16 2
7 8.67 2 8.47 2
8 9.32 2 9.32 2 2.4. Multivariate curve resolution
9 9.88 3 9.75 3
10 10.80 2 10.93 3 The main objectives of MCR are the isolation, resolution, and
11 11.83 2 11.70 3
relative quantification of the main sources of variation in a partic-
12 12.42 3 12.72 3
ular data set. The outstanding feature of this technique is that no a
 P.H. Março et al. / Food Chemistry 125 (2011) 1020–1027
priori chemical assumption about the contribution of the different
components is necessary (Izquierdo-Ridorsa, Saurina, Hernández-
Cassou, & Tauler, 1997). 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.
As it has ben shown in Fig. 2, the measured spectra data can be ar-
ranged in the augmented data matrix Daugment, which will contain
information about all of the system components at the different
experimental conditions investigated in each individual experi-
ment and data matrix. MCR performs the bilinear decomposition
of the data matrix Daugment, giving two data matrices which refer
to the matrices containing the pure concentration profiles Caugment
and species spectra ST (hence their names) of the different species
present in the system. However, MCR methods have been shown
also to work in many other diverse types of problems and situa-
tions (de Juan & Tauler, 2006). The bilinear decomposition is now
performed under a completely different set of constraints than
PCA, searching for solutions with physical meaning, for instance in-
stead of using orthogonality constraints like in PCA, non-negativity
constraints are applied. Those initial estimations of concentration
profiles Caugment and species spectra ST are optimised solving Eq.
(1) iteratively by alternating least squares (ALS) optimisation. At
each iteration of the optimisation a new estimation of the Caugment
and ST matrices is obtained. At each iterative cycle the following
constraints can be applied: (i) non-negativity, (ii) selectivity and
zero concentration windows, (iii) unimodality, and (iv) closure.
This constrained iterative optimisation is carried out until conver-
gence is achieved or until a preselected number of cycles are
reached (Tauler, 1995). Constraints, defined as chemical or mathe-
matical properties that should hold the resolved profiles, have
been and still are an active field of research. Improvements have
included the definition of new kinds of constraints and the pro-
gress in their implementation, trying to modify the profiles as
smoothly as possible (de Juan & Tauler, 2006).
The same procedure may be taken for the simultaneous analysis
of samples exposed and non-exposed to the UV radiation, giving a
super augmented data matrix Daugment data matrix which contains
now 24 individual matrices corresponding to the 24 experiments
performed at different pH values, with and without light exposure.
More details and recent examples about the implementation of the
multivariate curve resolution-alternating least squares, MCR-ALS
method, to augmented data matrices are given elsewhere (Carneiro
et al., 2008; de Juan & Tauler, 2006; Izquierdo-Ridorsa et al., 1997;
Mas et al., 2008; Tauler, 1995). MCR results will be shown as pure
spectra and as relative concentration profiles evolving with time
and at each pH.
3. Results and discussion
In order to have an initial estimation of the number of species,
the chemical or pseudorank (mathematical rank in absence of
experimental noise) of the data matrix obtained at each pH value
was estimated using principal components analysis (PCA) (Vande-
ginste et al., 1998) and singular value decomposition (SVD) (Linder
& Sundberg, 1998). In Table 1, pH values and the number of prin-
cipal components needed to describe more than 99% of data vari-
ance in each case are given in detail.
As it is shown in Table 1, the number of components depends on
pH value and also on whether samples were exposed to UV radia-
tion or not. In general, the system became more complex at inter-
mediate pH values and with light exposure. PCA individual analysis
of the different data matrices showed that different number of
coexisting species in equilibrium is present at the considered pH
value, between 2 species at low pH to 4 species at basic pH values.
1023
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 to find out
which of them were more similar. According to PC1, samples not
exposed to UV radiation (group I – ‘‘s” matrices in Fig. 3) were sep-
arated from samples exposed to UV radiation (groups II and III –
‘‘d” matrices in Fig. 3). According to PC2, the acidic and neutral
samples (group II) were also separated from those more basic
(group III). The pH values can be checked in Table 1. The not ex-
posed sample s3 (pH 5.12) behaved as an exposed sample because
at this pH, flavylium cation (AH+) is unstable, being transformed to
carbinol (B) and quinoidal (A) forms very fast. In the same way,
sample d5 (sample exposed to UV radiation at pH 7.22), can not
be seen in Fig. 3 because it shows exactly the same profile and
behaviour observed in sample d4 (pH 5.12), being located behind
it.
MCR-ALS was then applied to resolve the pure spectra and con-
centration profiles of all different species in equilibrium at the dif-
ferent pH and experimental conditions (with and without UV
exposure). The constraints applied during the ALS optimisation
were spectra and concentration profiles non-negativity and con-
centration profiles closure, besides the correspondent species
among different samples at different pH values. This information
about species correspondence was preliminary acquired from the
singular value decomposition (SVD) to the individual and aug-
mented data matrices and it was optimised afterwards during
the MCR-ALS resolution process. For brevity, only MCR-ALS final
results will be shown for the simultaneous analysis of all data
matrices at all pH values, 12 of them with UV radiation exposure
and 12 of them without UV radiation exposure. In order to better
interpret the different reaction steps, in the following paragraphs,
first the results will be shown for samples in absence of light radi-
ation exposure and then for samples exposed to the UV radiation.
MCR-ALS results for the H. acetosella samples at different pH
values without light exposure showed the presence of up to seven
different species. Fig.4(A) and (B) shows the best results achieved
by applying the MCR-ALS method considering seven species to
the whole set of samples not exposed to UV radiation. In
Fig. 4(A), the species spectra of the seven resolved species are gi-
ven. They are strongly overlapped and they could only be resolved
Fig. 3. PC1 versus PC2 scores obtained by MPCA. Group I contains the samples not
exposed to UV radiation; groups II and III contain the samples exposed to the UV
radiation.
1024 P.H. Março et al. / Food Chemistry 125 (2011) 1020–1027

Fig. 4. (A) Anthocyanin pure species spectra resolved by MCR-ALS simultaneous spectral analysis of 12 Hibiscus samples NOT EXPOSED to UV radiation at different pH values
during 120 min. (B) Anthocyanin time degradation species profiles resolved by MCR-ALS simultaneous analysis of 12 Hibiscus samples not exposed to UV radiation at
different pH values during 120 min. Species are referred to its spectra in (A) according to symbols. Each structure is given in Fig. 1.

by using MCR-ALS analysis of the augmented matrix, built by the
simultaneous analysis of the 12 kinetic experiments at different
pH values (not exposed to UV radiation). In Fig. 4(B), the resolved
concentration profiles of these seven different species evolving
with time and pH are given in details. Each species has been iden-
tified with a different line style to facilitate the interpretation of
the results. From this plot, it is possible to see what species were
present at each pH and how stable they were during the 2 h of
monitoring. It is clear that the speciation of this complex system
changes considerably with the pH of the experiment.
In absence of radiation, the two first experiments at pH 2.50 did
not show any change with time, indicating that the flavylium cat-
ion species (AH+) was stable at this medium (Figueiredo, Elhabiri,
Saito, & Brouillard, 1996). At pH 3.98 the formation of two species
was already detected, with the major one (AH+) showing a slight
transformation to a second species (B). At pH 5.12 the species B
was in equilibrium with species AH+. For experiments at pH 6.1
and higher, the species AH+ was not detected anymore because it
was completely converted into the species B. An additional new
deprotonated species, the quinoidal base (A) was also detected at
these pH values. As described before, the behaviour of the species
B and CC is similar (Brouillard et al., 1982), and their spectra are
very similar. However, the application of MCR-ALS allowed the res-
olution of both species (B and CC species) at Fig. 4(A) and (B). The
 P.H. Março et al. / Food Chemistry 125 (2011) 1020–1027
formation of the cis-chalcone species (CC) indicates that the break
at the aromatic ring of the anthocyanin structure (see structures
in Fig. 1) was already taking place at neutral pH values (Brouillard
& Dubois, 1977; Maestri et al., 2000; Markakis, 1982). This reaction
is usually considered to be irreversible and it was detected from
pH 7.1 to 9.3. In spite of that, the coloured deprotonated anhydro-
base species (A), which can adopt different structural forms (none
of them with their aromatic ring opened, see in Fig. 1 to the main
anthocyanin structures), were detected from pH 9.3 to 11.8, indi-
cating that either the reaction is reversible or that there is another
transformation to give this structure. In agreement with this pro-
posal, at basic medium, from pH 10.9 to higher than 12, another
pure spectrum with absorption band at visible range was resolved
by MCR-ALS, suggesting that this species could be the double ion-
ised quinoidal base (A2) (Brouillard et al., 1982; Dougall & Baker,
2008; Maestri et al., 2000). At stronger basic medium, at pH higher
than 11.83, another species increases its concentration with time.
At this basic medium, it is rather hard to find any report about
what could be the nature of this species and about its behaviour,
but following the reaction mechanism proposed until now, this
species could be the deprotonated opened ring species, and we
suggest therefore that it could be the ionised cis-chalcone (C C in
structures of Fig. 1).
For samples exposed to UV radiation, the best resolution was
achieved when MCR-ALS considered nine species, as showed in
Fig. 5(A) and (B). As saw to non-exposed samples, species spectra
were strongly overlapped (Fig. 5(A)) and could only be resolved
when MCR-ALS was applied simultaneously to the whole set of
samples at different pH values. Comparing Figs. 4(B) and 5(B),
the distribution of species previously identified as flavylium cation
(AH+) is now very unstable because of UV radiation, suggesting
that the radiation accelerated the initial transformation of species
AH+. This acceleration was also verified at higher pH values, where
other additional species not found previously were now detected
under UV radiation exposure. Even at the acidic pH of 2.4, two
additional species appeared immediately (see Fig. 5(B)), both
clearly formed from the species AH+ deprotonation. To the species
represented by the dashed line in pH 2.4, 4.0 and 5.2, Pina et al.
(Gomes, Diniz, Jesus, Parola, & Pina, 2009; Maestri et al., 2000;
Melo et al., 2002; Petrov, Gomes, Parola, & Pina, 2009) suggested
that this species could be a different conformation of B species
(see structures in Fig. 1), named as hemiketal B4, which can only
be detected when samples were exposed to light. By comparing
the spectra with the one identified in absence of radiation, the next
species detected in these pH values is attributed to carbinol species
(B), and now it is being formed at lower pH values. The quinoidal
base (A) is unstable under light exposition, and was detected at
the same pH range observed to the sample not-exposed to UV radi-
ation, indicating that the radiation has little effect on the behaviour
of this species. The cis-chalcone species (CC) was detected at a
wider pH region than the observed for non-exposed samples:
whereas for non-exposed samples the CC was detected at the pH
range from 7.1 to 8.5, and when exposed this range was from 6.1
to 10.9. Moreover, UV radiation seems to favour the CC structure
formation. At pH 6.1 the application of MCR-ALS achieved the res-
olution of B species from the CC species at very low concentration,
as it can be seem at Fig. 5(B) – pH 6.1. As explained before, the aro-
matic ring of CC structure is opened (Asenstorfer & Jones, 2007;
Brouillard & Dubois, 1977; Brouillard et al., 1982; Dougall & Baker,
2008; Gomes et al., 2009; Maestri et al., 2000; Melo et al., 2002;
Petrov et al., 2009). Nevertheless, the A species was also detected
up in samples exposed to UV radiation with similar behaviour, as
previously observed for A to the not-exposed samples. When
samples were exposed to radiation, A could not be detected at
pH 10.9. Ionised quinoidal species (Dougall & Baker, 2008; Maestri
et al., 2000; Melo et al., 2002) was also detected, and seems to have
1025
its stability reduced because of the influence of UV radiation. From
pH 11.8 to 12.6 a new species was detected. The suggestion is that
this new species refers to an ionised chalcone form or a quinoidal
base tautomer (C *). The ionised cis-chalcone was also de-
T or A
tected at exposed radiation conditions and does not seem to have
suffered significant radiation influence.
When the effect of radiation exposure on the distribution of the
different species is examined the following main trends are
observed:
(1) Fig. 1 summarises the pH dependent speciation equilibria
and kinetics proposed in this work described above which
also considers previous results from the literature.
(2) The evolution of the different kinetic profiles showed for the
well known flavylium cation (AH+) initial species (Brouillard
et al., 1982; Figueiredo et al., 1996) at low pH indicates that
UV radiation accelerated flavylium cation degradation, turn-
ing this species more unstable when pH is increased. This
kinetic behaviour is clearly in agreement with the expected
behaviour of flavylium cation, which is present only in acidic
to slightly acid medium (Asenstorfer & Jones, 2007; Brouil-
lard & Dubois, 1977; Brouillard et al., 1982; Maestri et al.,
2000; Melo et al., 2002). The flavylium cation is transformed
in two species which are in equilibrium; the colourless
pseudobase carbinol (B) and the coloured quinoidal base (A).
(3) At neutral pH values, between pH 4 and 6, the concentration
of carbinol species, B; increases considerably. When samples
were exposed to UV radiation, carbinol was already detected
at pH 2.4.
(4) The hemiketal species (B4) was formed from AH+ only for
samples exposed to UV radiation. This agrees with previous
results reported by Pina et al. (Gomes et al., 2009; Maestri
et al., 2000; Melo et al., 2002; Petrov et al., 2009).
(5) The quinoidal base, A, is a coloured species formed from AH+,
appearing in equilibrium with carbinol species at different
pH values (Dougall & Baker, 2008; Levi et al., 2004; Melo
et al., 2002; Schiozer et al., 2008). This species A is stable
and it was not affected by UV radiation.
(6) At slightly acidic and neutral pH values, the flavylium cation
AH+ was completely converted into the coloured quinoidal
base (A) together with some of its several tautomers, and
the colourless carbinol pseudobase species (B), which is at
fast equilibrium with other colourless cis-chalcone species
(CC), as proposed by Brouillard (Brouillard, Delaporte, &
Dubois, 1978; Brouillard & Dubois, 1977; Brouillard et al.,
1982) and McClelland (McClelland & Gedge, 1980; McClel-
land & McGall, 1982). CC spectrum was resolved by MCR-
ALS from pH 7.1 to 9.3 for samples non-exposed to radiation,
and from pH 6.1 to 10.9 when for exposed samples to
radiation.
(7) At slightly basic medium, the deprotonated quinoidal base
(A) was detected between pH 9.3 and 11.8 for non-exposed
samples. When exposed to radiation, A, appears to be in
equilibrium with the colourless chalcone CC species from
pH 9.3 to 10.9. Few studies have reported however the
anthocyanins behaviour in basic medium (Março & Scarmi-
nio, 2007; Schiozer et al., 2008). According to MCR-ALS
results and considering the sequence of deprotonation steps
described before, the most stable species around pH 9 would
be this deprotonated quinoidal base species (A).
(8) The colour observed for samples in the pH range from 9.3 to
10.9 starts to change at higher pH values. At this medium the
deprotonated quinoidal base (A) is transformed into
another new species. By applying MCR-ALS to the experi-
mental spectra of these samples, the pure spectrum and
kinetic profiles of a new species were resolved. Considering
1026 P.H. Março et al. / Food Chemistry 125 (2011) 1020–1027

Fig. 5. (A) Anthocyanin species pure spectra resolved by MCR-ALS simultaneous analysis of 12 Hibiscus samples EXPOSED to UV radiation at different pH values during
120 min. (B) Anthocyanin time degradation species profiles resolved by MCR-ALS simultaneous analysis of 12 Hibiscus samples exposed to UV radiation at different pH values
during 120 min. Species are referred to its spectra in (A) according to symbols. Each structure is given in Fig. 1.

that at this medium the major species initially present was
the deprotonated quinoidal base, we propose that this spe-
cies is transformed into either a new deprotonated quinoidal
tautomer or better into a further ionised species (A2). As it
can be seemed from Fig. 5(B), UV radiation exposure does
not seem to have any influence on the A2 behaviour.
(9) At more basic medium another new species (postulated as

ionised chalcone or ionised quinoidal tautomer, C T =A spe-
cies in structures of Fig. 1) was detected only when UV radi-
ation exposure was used. This new species starts to appear
at basic medium, at pH value 11.8, increasing its concentra-
tion with time. Because of the characteristics of the medium,
we suggest that this species could be another ionised quinoi-
dal tautomer or an ionised chalcone (Março & Scarminio,
2007; Petrov et al., 2009; Schiozer et al., 2008).
4. Conclusions
Multivariate curve resolution allowed the investigation of the
kinetic (photo)degradation study of antocyanins at different pH
 P.H. Março et al. / Food Chemistry 125 (2011) 1020–1027
values, with and without radiation exposure. Up to nine different
species were detected and resolved by the method; identified by
their pure spectra and kinetic profiles which allowed the final pro-
posal of a degradation pathway depending on pH and UV light
exposure (see Fig. 1). The following conclusions were derived:
(a) Speciation was more complex for samples exposed to radia-
tion than for samples non-exposed to UV radiation, with two
additional species (up to nine species compared to seven
species when samples were not exposed to the radiation).
(b) Acidic species resulted to be more unstable when samples
were exposed to UV radiation, in this case being faster
degradated.
(c) A reaction degradation mechanism and pathway is proposed
from the comparison between the pure species spectra
resolved by the MCR-ALS analysis of the flower extract sam-
ples at different pH values and known pure spectra of related
standards submitted also to changes of pH and light
exposure.
Acknowledgements
This research has been financed by FAPESP (project 04/09231-
6). Paulo Henrique Março acknowledges a Ph.D. grant from Santan-
der-Unicamp for International Mobility to perform a research stay
at the Institute of Chemical and Environmental Research (IIQAB-
CSIC) of Barcelona (Spain).
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