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Q-mode curve resolution of UV–vis spectra for structural transformation studies of anthocyanins in acidic solutions
Paulo Henrique Marco, Ieda Spacino Scarminio ∗ ¸
Laborat´ rio de Quimiometria em Ciˆ ncias Naturais, Departamento de Qu´mica, Universidade Estadual de Londrina, o e ı Caixa Postal 6001, CEP 86051-970 Londrina, Paran´ , Brazil a Received 13 May 2006; received in revised form 29 September 2006; accepted 29 September 2006 Available online 4 October 2006
Abstract Chemometric analysis of ultraviolet–visible (UV–vis) spectra for pH values 1.0, 3.3, 5.3, and 6.9 was used to investigate the kinetics and the structural transformations of anthocyanins in extracts of calyces of hibiscus ﬂowers of the Hibiscus acetosella Welw. ex Finicius for the ﬁrst time. Six different species were detected: the quinoidal base (A), the ﬂavylium cation (AH+ ), the pseudobase or carbinol pseudobase (B), cis-chalcone (CC ), trans-chalcone (Ct ), and ionized cis-chalcone (CC − ). Four equilibrium constant values were calculated using relative concentrations, hydration, pKh = 2.60 ± 0.01, tautomeric, KT = 0.14 ± 0.01, acid–base, pKa = 4.24 ± 0.04, and ionization of the cis-chalcone, pKCC = 8.74 ± 1.5 × 10−2 . The calculated protonation rate of the tautomers is KH+ = 0.08 ± 7.6 × 10−3 . These constants are in excellent agreement with those measured previously in salt form. From a kinetic viewpoint, the situation encountered is interesting since the reported investigation is limited to visible light absorption in acid medium. These models have not been reported in the literature. © 2006 Elsevier B.V. All rights reserved.
Keywords: Anthocyanins; Chemometric analysis; UV–vis spectrometry; Kinetic studies
1. Introduction Anthocyanins have been the subjects of many studies due to their importance as quality indicators in foods, important chemotaxonomic indicators for plants [1,2], as well as for their possible antioxidant action . It has long been known that anthocyanins bearing the same chromophoric structure can give rise to different colors. Their color is easily affected by a number of chemical and physical factors, such as temperature, pH, solvent, the structure of the pigment itself and the presence of other molecules, which can be generally described as copigments [4,5]. A particular problem is the pH inﬂuence on their color. Depending on their acidity or alkalinity, anthocyanins adopt different chemical structures. In acidic aqueous solutions , four species of the anthocyanin molecule may exist in equilibrium: the quinoidal base (A), the ﬂavylium cation (AH+ ), the pseudobase or carbinol pseudobase (B), and the chalcone (C), Fig. 1 .
Based on observation of a few relatively simple anthocyanins, the generally accepted chemical equilibria characteristic of structural transformation are shown in Scheme 1 [4,6,7]. According to Eqs. (1)–(3), the AH+ , B, and C species are interconverted with their relative amounts depending on pH. At a pH of approximately 3 or lower, anthocyanin exists mainly as a ﬂavilyium cation and the amount of quinoidal base is not signiﬁcant, thus the system can be described by Eqs. (1) and (2). AH+ B
(1) (2) (3)
ring/chain tautomeric equilibrium A + H+ acid–base equilibrium
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As the pH is raised, kinetic and thermodynamic competition occurs between the hydration reaction of the ﬂavylium cation and the proton transfer related to acidic hydroxyl groups of aglycone. While the ﬁrst reaction gives a colorless carbinol pseudobase, which can undergo ring opening to a chalcone pseudobase, the latter reaction gives rise to a quinoidal base. The
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Ka . The corresponding relaxation amplitudes are recorded by means of ultraviolet–visible (UV–vis) absorption spectrophotometry . KT and Kh are the equilibrium constants for.P. the hydration. Ka and Kh have been previously found to be 10−4 and 10−2 . the acid–base. .S. Scarminio / Analytica Chimica Acta 583 (2007) 138–146 c 139 different equilibrium constants are expressed as follows : Kh = aH+ KT = [C] [B] [A] [AH+ ] [B] [AH+ ] (4) (5) (6) Ka = aH+ Fig. R1 and R2 are usually H. Mar¸ o. 1. Molecular structures of ﬂavylium cation (AH+ ). and the ring chain tautomerism equilibria. quinoidal base (A). respectively at 25 ◦ C . Soundheimer  was the ﬁrst to measure the equi- Scheme 1. pseudobase or carbinol (B) and chalcone (C). where aH+ is 10−pH . Most of the studies about the measurement of the equilibrium constants associated with the structural transformations of anthocyanins in aqueous solution have been conducted by temperature-jump  or pH-jump  relaxation. R is a glycosil and R are usually H or sugar.H. OH. or OCH3 . I. respectively.
resolve the absorption spectra of the species. called cos θ.2. The contribution of each object is obtained by an eigen-analysis of a real symmetric matrix obtained from the data matrix. For such systems.  applied the pH-jump method to measure the equilibrium constants of one natural product (apigenidin) and three related 3-deoxyﬂavylium salts. I. Determination of ionization constants of natural anthocyanins by spectrophotometric methods is complicated by the fact that prior knowledge of the spectra of all the charged and uncharged species is required . The original matrix can be transformed to describe an index of proportional similarity. In this work. Considerable amounts of literature exist on the PCA method. Imbrie Q-mode factor analysis deﬁnes the similarity between samples based on the proportions of their constituents. orthogonal projection (OPA) . additional analysis is required to obtain detailed information about the structural transformation of anthocyanins. chemometric methods can be used to estimate the number of species present as well as extract the spectra of species involved and their corresponding concentration proﬁles. The data matrix A consists of n objects (spectra) represented by p variables (absorbencies at different wavelengths).28]. higher curve resolution was obtained with more accurate determinations of equilibrium and rate constants than with the PARAFAC model. cos θ is calculated by cos k = p j=1 akj a j p p 2 2 j=1 akj j=1 a j (8) . The number of principal components describing the major part of the data variance is represented by q. Also these studies. e. correlated spectral data by Procrustes rotation . therefore this section will provide only a summary of PCA. in contrast to other works measuring these constants in which the pigments are generally isolated and puriﬁed as ﬂavylium cations. PCA decomposes the matrix A into a product of two matrices. Imbrie and Purdy  deﬁne an index of proportional similarity. This method offers several advantages. 2. and heuristic evolving latent projection (HELP) .140 P. Theory 2. Curve resolution can also be used to deconvolute spectral features using models for equilibrium constant determination and kinetic information [16–25]. etc. pH equilibria. Curve resolution methods are a group of chemometrical approaches suitable for the treatment of multicomponent data matrices. Principal component analysis (PCA) Principal component analysis  is a multivariate technique used for reducing the dimensionality of a data set. in which case the original data matrix can be represented by the product of two smaller matrices that contain the concentration proﬁles and the pure spectra of each individual component present in the mixture .g. Imbrie Q-mode factor analysis followed by varimax and Imbrie’s rotations [27. q×p (7) where T is a score matrix. 2. interative target transformation factor analysis (ITTFA) . Imbrie Q-mode factor analysis followed by varimax and Imbrie’s oblique rotations and K matrix treatment were applied to the UV–vis spectral data. Their main purpose is the correct determination of the response proﬁles as spectral and concentration proﬁles of the component present in unresolved mixtures . Little research has been made using information from the full spectra for studying the structural transformation of anthocyanins.0. for complete UV–vis spectra measured between 240 and 748 nm for pH values between 1. and PT is the transpose of P. and E a matrix of residuals. Furthermore. var. regius maximus  and in the calyces of ﬂowers of the Hibiscus sabdariffa  species using the PARAFAC method to resolve the absorption spectral proﬁles as well as the kinetic concentration proﬁles.0 and 13. All of these methods assume Beer’s law holds. Mar¸ o. If the relative concentrations of the species (structures) can be shifted by changing factors that do not alter the individual spectral proﬁles. None of the spectroscopic methods is entirely suitable for elucidation of the structures of pigments. The major limitation in the identiﬁcation of pigments on the sole basis of the absorbance spectrum is overlapping absorbance bands of individual pigments present in the mixture. It is based upon eigen-analysis of the correlation or covariance matrix. dimerization. Q-mode analysis is especially convenient since it evaluates interrelationships between samples and concentration proportions are determined directly . we extend these studies to the kinetics of simultaneous structural transformations of anthocyanins in extracts of fresh calyces of hibiscus ﬂowers of Hibiscus acetosella Welw. determined equilibrium and kinetic rate constants of anthocyanin systems without isolation and puriﬁcation of the individual components. a direct spectral analysis is difﬁcult since the number of components contributing to the spectrum and their spectral proﬁles are usually unknown. Since individual pH curves were treated using this technique. simple to use interactive self-modelling mixture analysis (SIMPLISMA) . Brouillard et al. k and . in some cases the molecules studied are involved in complex equilibria such as tautomerism. A = Tn×q PT + En×p .H. The chemometric methods used here permit one to deduce the relative concentrations from observed spectral changes. A large number of curve resolution techniques have been published. as well as to obtain the equilibrium constants. For equilibrium constant determination. For two objects.S. ex Finicius. R-mode analysis is primarily used to evaluate correlations between variables although its scores can be used to determine concentrations. Recently we presented results of an investigation of anthocyanins from fresh petals of hibiscus ﬂowers of the Hibiscus rosa-sinensys L. Q-mode principal component analysis Paatero and Tapper have compared theoretical aspects of Q-mode and R-mode principal component analyses as general purpose methods .1. Scarminio / Analytica Chimica Acta 583 (2007) 138–146 c librium constant of reaction 1 in the case of pelargonidin 3-monoglucoside. Therefore. The common approach has been single point measurements at a wavelength where one component dominates the spectral response and the contributions from the other components are neglected. P a loading matrix.
Thus. Universidade Estadual de Londrina. (19). H.00 mL) of the ethanolic extracts of anthocyanin. The pH of each solution was measured with a HANNA HI model HI9321 pH Meter. scaled by the squares roots of the eigenvalues. Experimental Plants were cultivated in the garden of the Chemistry Department of the Universidade Estadual de Londrina (UEL).S. the mathematical procedure starts by data normalization. Chemometric methods were applied to the data matrix. (12). T (10) The row normalized data matrix can be expressed as the product of a loading matrix and a score matrix. (13) (14) the diagonal matrix (15) The matrix of loadings is then the matrix of eigenvectors. The ethanolic extracts were passed through ﬁlter paper. The symmetric matrix H can be factored according to H = UΛ UT . The relationship between W. K matrix method The method (sometimes referred to as classical least squares. but TT T = Λ so that PT = Λ−1 TT W. 3. The matrix of scores may be deﬁned by W ≈ TPT Premultiplication by TT gives TT W ≈ TT TPT .1% HCl in ethanol. composed of 90 rows (time) and 1450 columns (wavelength). According to . I. the concentrations of the q species in the N samples (n > q) and E is the error matrix. Buffer solutions with four different pH (1.1) ◦ C. The loadings are used to describe the relative proportions of the constituents in the sample. The access numbers is FUEL 33816. CLS) is based on the well-known Beer–Lambert law .3. A voucher was deposited in the herbarium of the Universidade Estadual de Londrina. The raw data matrix A is normalized by W = D−1/2 A (9) these end member samples. Manuel RC Paiva. In this work K was estimated from C. This is accomplished by constructing a Vq×q matrix that contains the highest absolute values of the varimax weights in each object column. P and T is given by H = WWT = TPT PTT . which was obtained by Imbrie’s oblique rotation. Mar¸ o. a series of simultaneous equations may be utilized to describe mixture spectra. leads to the following equation from Eq. H = WW .80 g) of the hibiscus ﬂowers with 0. 5. Once obtained. The association matrix is deﬁned as the major product moment of the row normalized data matrix. The analyses were carried out using an Ocean Optics model CHEM2000 spectrophotometer with a quartz cuvette (d = 1 cm). to one. This normalization does not affect the proportionality relationship between variables but removes the effects of size differences among samples. each pH set (time versus wavelength) was characterized by 1450 absorbance values obtained in an interval from 245 to 748 nm. Scarminio / Analytica Chimica Acta 583 (2007) 138–146 c 141 For positive a elements. The temperature was ﬁxed at 25 (±0. (18) (17) (16) It must be emphasized that the Tn×q and PT matrices so q×p derived are not the same as those derived in R-mode analysis. H = TTT . The cos θ are calculated for all pairs of objects in the data set and then arranged in an n × n matrix of associations.0.H. (11) Here T is an n × q matrix and P is p × q. The 240–245 nm region was very noisy and eliminated from subsequent data treatment. The oblique projection matrix is given by C = BV−1 (19) where V−1 is the inverse of V. Brazil. Each row of B corresponds to an object or a linear combination of objects and each column represents a factor. The corresponding matrix equation is AT = Kp×q Cq×n + Ep×n p×n (20) where D = diag(AAT ). (12) where A contains the sample spectra C. For the chemometric study. T may be rotated to B by varimax rotation. Since The constraint that the PT P = I. Twenty milliliter of buffer solutions were added to the aliquots (3. H. in Londrina. and immediately the spectra were recorded every 20 s during a 30 min interval.P. PR. 2. The C matrix row vectors furnish the proportional contributions of all the objects in the terms of the end member samples. Pigments were extracted by maceration of fresh calyces (11. W ≈ TPT . Departamento de Biologia. If a mixture set of known composition is used as a calibration set. this index varies from zero. no similarity. Imbrie’s oblique rotation  is used to rotate the factor axes of the B matrix so that they coincide with the most divergent samples and then express all the other samples as proportions of .3. Eq. and 6. T = Uq 1/2 q .9) values were prepared with phosphate following the detailed procedure described by Levi et al.3. 3. identity. where U is the matrix of eigenvectors and of associated eigenvalues. . Ultraviolet–visible absorption spectra at different pH values were recorded from 240 to 748 nm. All reagents were of analytical grade. The K matrix is the spectra of the pure species for unitary concentration and path length. resulting in a 90 × 1450 matrix. where q is the approximate rank of W. or P = WT TΛ−1 . The plants were identiﬁed by Dr.
3. 3.0. The absorbance increase can be due to a decrease in water activity or to the copigmentation reaction. Computer programs The FORTRAN programs used for the K matrix and Q-mode principal component analysis. respectively.S. 2b. Mar¸ o. It is affected by several factors of which pH is an important one.H. 4. 2a. varimax and Imbrie’s oblique rotations calculations were developed in our laboratory. At a pH of 1.1. and (d) 6. Scarminio / Analytica Chimica Acta 583 (2007) 138–146 c Q-mode analysis is performed. The copigmentation reaction consists of an increase in the visible band intensity for the pigment (hyperchromic effect) and frequently in a shift of this very band towards longer wavelengths (bathocromic effect).3. as well as to calculate their relative concentrations. 2. Since the factors are linearly independent. I. a 90 × 90 matrix is diagonalized instead of a 1450 × 1450 matrix necessary for R-mode analysis. It has been demonstrated that the copigment effect occurs from pH values close to 1 to neutrality. These programs can be compiled on any digital computer and are available from the corresponding author. This result is in agreement with the literature and suggests that the copigment effect parallels the hydration reaction . the carbinol pseudobase. Imbrie’s Q-mode factor analysis showed that two factors explain 99% of the total spectral data variance. The absorptions at 281 and 330 nm correspond to a carbinol pseudobase (B) and chalcone (C).9. it was concluded that three main species are present in the structural transformation of the investigated system: the ﬂavylium cation. (c) 5. Since there are at least three species. and Fig. 2a. no statistical method was applied considering that there was no change in λmax but only in the intensity of the absorbance. Imbrie Q-mode factor analysis followed by varimax and Imbrie’s oblique rotations and K matrix calculations were carried out.0 are given in Fig.142 P. In order to investigate the effect of pH on the structural transformation more fully and thereby gain an insight into the species that may be present in solution. A shoulder found at 422 nm is characteristic of trans-chalcone. 330 and 522 nm wavelengths occur until attaining equilibrium at this pH value. Results and discussion Representative spectra of the ethanolic extract of anthocyanin at pH 1. shown in Fig. 2a the spectral proﬁles appear similar but not identical. Copigmentation reduces the extent of the hydratation reaction and therefore increases the stability of the colored species . Fig. the red solution fades and a decrease in intensity of absorption at 522 nm occurs. Spectra vs. In comparison with Fig. increases in absorbencies at the 281. . time matrices corresponding to the following pH values: (a) 1. When the ethanolic extract of anthocyanin is added to the buffer at pH 3. After the addition of buffer solution. it is not possible to calculate the true or relative concentrations from the absorption data by conventional chemical analysis. (b) 3. Spectral modiﬁcations occur which predominantly reﬂect the partial transformation of the ﬂavilium cation into carbinol pseudobase (B) and chalcone (C) during the period required to establish equilibrium.3. Absorption in the 522 nm region is attributed to the presence of ﬂavylium cation.
Therefore. At − A0 = α1 (1 − e−k1 t ) + α2 (1 − e−k2 t ) (21) Fig. The results for the kinetic parameters and their errors are shown in Table 1. These values are in good agreement with our results obtained by the PARAFAC method for Hibiscus sabdariffa species . Time-dependence of the absorbance at (a) 281 nm. Since this a critical step in the analysis . and pKc obtained in this study were 2. (19). 3.01. anthocyanin degradation is explained by one ﬁrst-order rate constant. . pKh = 2.02. and their proﬁles are shown in Fig. the absorption spectrum of the trans-chalcone isomer is practically identical to that of the cis-chalcone isomer due to band overlap of the latter with the carbinol pseudobase form . The relative concentrations for the three species were calculated by Eq. for the chalcone formation reaction starting from ﬂavylium cation AH+ . Scarminio / Analytica Chimica Acta 583 (2007) 138–146 c 143 Fig. alternative calculations were carried out with additional principal components. I. and 3. 422 nm (trans-chalcone) and 522 nm (ﬂavylium cation).14 ± 0.70.54 ± 0. Eq. The experimental values determined for pKh . Relative concentrations after equilibrium have been established allowing determination of the apparent acidity constants Kc = ([C]/[AH+ ])aH+ = Kh KT . and (d) 522 nm. this model is not sufﬁcient to describe and explain the whole transformation process.04 obtained by the pH-jump method for pure malvidin-3-glucoside chloride .54 ± 0.60 ± 0. 3. 330. Concentration proﬁles obtained by the model at pH 3.12. Generally. (b) 330 nm. AH+ is the ﬂavylium cation. and 3. Besides that. Mar¸ o.3 based on the deconvolution of UV–vis absorption spectra. and pKc = 3.01. 4. KT . with the goodness of ﬁt determined by chi-square. (21). At 281. and 422 nm the models for the kinetic behavior were ﬁtted to a sum of exponentials. Kinetic analysis was performed by utilizing plots of the dependence of the variation of absorption against wavelength monitored at 281 nm (carbinol pseudobase). as well as for the hydratation and tautomeric reactions associated with the structural transformations of anthocyanins.S.54. chalcone.02. At this pH it was not possible to obtain the relative concentration of the trans-chalcone. because it only appears in negligible quantities. 4a–d. 0. However. The variation of the absorption with time is shown in Figs. Here.60 ± 0. and the literature values of 2. KT = 0. increases in the number of rotated principal components resulted in increased noise lev- els for the factor loadings. (c) 420 nm. and C is the chalcone.H. B is the pseudobase or carbinol pseudobase. respectively. 330 nm (cis-chalcone).14.P. three factors were subjected to varimax rotation and oblique rotation to obtain the ﬁnal species proportions. 0.
This value agrees with that measured by the pH-jump  method of 4.80) × 10−4 – 14.4(± 1.9(± 2.0(± 3. i. regius maximus. var. (21).8) × 10−1 1.9(± 1. Although the above values cannot be compared directly with results in the literature.5) × 10−2 1. and A is the quinoidal base. (6). the amplitude associated with the B→C equilibrium can be affected by the presence of other species .69) × 10−3 57(± 8. Chemometric analysis shows that three factors explain 100% of the total spectral data variance.5) × 10−2 80(± 7.8(± 2. Concentration proﬁles obtained by the model at pH 5. 4d. and KT « 1. The relative concentrations for the four species were calculated by Eq.04 and 0. 4d.4(± 1.6) × 10−3 57(± 8. At this pH value. is not seen at 330 nm and 422 nm but only at 281 nm. Inspection of Table 2 clearly shows that the tautomeric interconversion between B and C cannot be seen as acid catalysis by the proton at this pH. show that it is possible to obtain the KT values without interference from other species.44) × 10−3 5.25 ± 0. Eq. suggests that the pseudo-equilibrated mixture of products is constituted essentially by the cis-chalcone (CC ).08 ± 7. 2d.29 × 10−6 422 nm 8. four factors were used in the rotations. Fig. 5.34 × 10−6 522 nm 2.64) × 10−3 23. the species monitored at 422 nm showed the presence of two separate linear behaviors.12 ± 5. 2.e. At − A0 = α1 e−k1 t + α2 t (23) At pH 5. (22).3.0) × 10−2 40(± 0.3 based on the deconvolution of UV–vis absorption spectra. When the jump relaxation method ← is used. Scarminio / Analytica Chimica Acta 583 (2007) 138–146 c Table 1 Kinetic parameters and their errors for the structural transformation of anthocyanins at pH 3.24 × 10−6 1171(± 3. AH+ is the ﬂavylium cation.72(± 1. with an increase in intensity.9(± 2. a signiﬁcant copigmentation effect also exists.S. the mechanism of tautomeric interconversion between the B and C species proceeds through the formation of an intermediate by acid catalysis by the proton . with good agreement with the previous results at pH 3. Fig. Eq. with different rates.1) × 10−2 – k1 k2 a 359(± 1.69) × 10−3 48. As observed at pH 3. the analysis of the spectral changes showed that the disappearance of AH+ causes a bathochromic shift of 522–560 nm due to the formation of the quinoidal base.6) × 10−3 1. shows a bathochromic effect at 600 nm due to the formation of the quinoidal base.144 P. Since the data in Table 1 conﬁrms the relations k1 =281 nm /k2 =330 nm ∼ 1. 2c. However.3 281 nm χ2a A0 1 2 330 nm 327(± 1. The ﬁrst obeys ﬁrst-order kinetics and the second indicates that the kinetics does not depend on the species concentrations anymore. and their proﬁles are shown in Fig. Fig.3. The results based on the calculated spectral proﬁles using K matrix methods. cis-chalcone (Cc ) and quinoidal base (A) at a pH of 5.28) × 10−3 48.6 × 10−3 . At this pH. the transformation is very fast and the trans-chalcone (Ct ) concentration cannot be calculated. . the spectra obtained. The behaviour at 522 nm. This equation shows that two separate kinetic processes occur at this wavelength. B2 is the pseudobase or carbinol pseudobase. (23). Mar¸ o. The kinetic behaviors for 330 and 522 nm are described by Eq. the presence of two pH-independent reactions. the kinetic behavior ﬁtted to a sum of exponentials. 5.24 ± 0. This equation is characteristic of consecutive ﬁrst-order reactions. The ﬁrst can be observed between 0 and 10 min and the second after 10 min. Relative concentrations after equilibrium has been established allowing determination of the values for pKh and KT .19) × 10−3 24.3 are shown in Fig.3.1(± 1.61 ± 0. respectively. The calculated protonation rate of the tautomers (KH+ ) is 0. The relative concentrations of the ﬂavylium cation (AH+ ). The results for the In the case of pH 6.28) × 10−3 80(± 7.H.6 × 10−4 . an increase in absorption at 240–350 nm was observed.1. using the PARAFAC model . our results. I.29 × 10−6 χ2 —Chi-square distribution with (n − 1) degrees of freedom. very small absorbance differences from the initial values occur until equilibrium has been established. Fig.9. CC is the cis-chalcone. by Eq. On the other hand. as well as their errors are presented in Table 2. carbinol pseudobase (B2 ).04. It is possible to observe that at 600 nm. Fig. obeys ﬁrst-order kinetics. At − A0 = α1 e−k1 t (22) kinetic parameters at this pH. therefore. On the other hand. the carbinol pseudobase (B) and small amounts of the quinoidal base (A). 5. The estimated value for pKa obtained in this study was 4.69) × 10−3 26. The quinoidal base formation allowed measuring Ka . we obtained the same behaviour for anthocyanin extracted from fresh petals of hibiscus ﬂowers of the Hibiscus rosa-sinensys L.44)× 10−3 26.10) × 10−4 230(± 6. using chemometric methods. (19).
the ﬂavylium cation (AH+ ).0) × 10−5 0.51) × 10−3 51.09(±3.4(±4. and for the ionization of the cis-chalcone.96(±7.49 522 nm 1.4(± 1.423 9.3 281 nm χ2a A0 1 2 145 330 nm 1.14 ± 0. Ct is the trans-chalcone. Here a decrease in cis-chalcone concentration and the formation of ionized cis-chalcone and trans-chalcone can be observed.1(±1.67) × 10−3 6. At pH 3.0) × 10−5 2160(± 9. and CC − is the ionized cis-chalcone. Mar¸ o. However. . the cis-chalcone (CC ). [C− ][H+ ]/[CC ] = 8. and ionized cis-chalcone (CC − ).12) × 10−3 92(± 5.93 .048 – 7.S.42 × 10−6 NO FIT χ2 —Chi-square distribution with (n − 1) degrees of freedom.7(±2.5 × 10−2 .046 7.097 ± 2.5 4.82) × 10−3 −25.7(± 8.94(±4.09) × 10−3 0.81 × 10−6 331. The estimated kinetic parameters are shown in Table 3.0) × 10−4 – 422 nm – – – – 1.44 × 10−6 96. the situation encountered is interesting since the reported investigations are limited to visible light absorption. Six different species were detected: the quinoidal base (A). From a kinetic viewpoint.9 281 nm χ2a A0 1 2 330 nm 1.18) × 10−3 6.H. Concentration proﬁles obtained by the model at pH 6.7 522 nm k1 k2 a 320. which closely C agrees with the literature value of 8. three factors were used in the rotations. 5. The relative concentration proﬁles are presented in Fig.01.3.16 ± 0.4(± 7.63) × 10−3 37(± 2.81) × 10−3 1.1) × 10−4 52.91 ± 0. pKh = 2.01. When the jump relaxation method is used.56(±6.85 ± 0. for acid–base equilibrium.99 χ2 —Chi-square distribution with (n − 1) degrees of freedom.97 ± 1. In order to obtain the relative concentrations. It was possible to identify the species involved at equilibrium and obtain their relative concentrations as well as the equilibrium constants.70) × 10−3 0.59) × 10−3 8.0) × 10−3 0.04.34 × 10−2 6.8(± 1. pKa = 4.6(± 1.63(±2. KT = 0. Conclusion Imbrie Q-mode factor analysis followed by varimax and Imbrie’s oblique rotations and the K matrix method were used to investigate the kinetics and the structural transformations of anthocyanins. for tautomerism.5 × 10−2 .96 × 10−2 422 nm 1. our results using chemometric techniques show that it is possible to obtain the pK values without interference from other species. These values are in good agreement with literature values.9 based on the deconvolution of UV–Vis absorption spectra.P.5 ± 1.27) × 10−3 −24. The calculated protonation rate of Fig. From this table it is obvious that slightly larger structural transformations occur compared to those observed at pH 5. the amplitude associated with ← the B→C equilibrium can be affected by the presence of the other species .60 ± 0. pKCC = 8. k1 k2 a Table 3 Kinetic parameters and their errors for the structural transformation of anthocyanins at pH 6. I. CC is the cis-chalcone. 6. Evidence for this assertion comes from the estimated acidity constant obtained in this study.3.56 × 10−6 48. Four equilibrium constant values were calculated by using the relative concentrations: for hydration.12 × 10−6 575(± 3.54) × 10−3 4.69 ± 1.99 × 10−6 315(± 3.7(±4.74 ± 1. Scarminio / Analytica Chimica Acta 583 (2007) 138–146 c Table 2 Kinetic parameters and their errors for the structural transformation of anthocyanins at pH 5.69) × 10−3 − 40.19) × 10−3 54.24 ± 0. 6. the pseudobase or carbinol pseudobase (B). the mechanism of tautomeric interconversion between B and C species proceeds through the formation of an intermediate by acid catalysis by the proton.74 ± 1. trans-chalcone (Ct ).
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