Anthocyanin equilibrium forms

Absorption spectra have also been used in the reexamination of pH-dependent color and structural transformations in aqueous solutions of some nonacylated anthocyanins and synthetic flavylium salts." ° In a recent study, the UV-Vis spectra of flower extracts of Hibiscus rosasinensis have been measured between 240 and 748 nm at pH values ranging from 1.1 to 13.0." Deconvolution of these spectra using the parallel factor analysis (PARAFAC) model permitted the study of anthocyanin systems without isolation and purification of the individual species (Figure 2.21). The model allowed identification of seven anthocyanin equilibrium forms, namely the flavylium cation, carbinol, quinoidal base, and E- and Z-chalcone and their ionized forms, as well as their relative concentrations as a function of pH. The spectral profiles recovered were in agreement with previous models of equilibrium forms reported in literature, based on studies of pure pigments. 

Fig. 9A.2 Anthocyanin equilibrium forms and bleaching reaction with bisulfite...

One of the best-established functions of anthocyanins is in the production of flower color and the provision of colors attractive to plant pollinators. Considerable effort has been made to give explanations for the color variations expressed by anthocyanins in plants. Various factors including concentration and nature of the anthocyanidin, anthocyanidin equilibrium forms, the extent of anthocyanin glycosidation and acylation, the nature and concentration of copigmentation, metal complexes, intra- and intermolecular association mechanisms, and influence of external factors like pH, salts, etc. have been found to have impact on anthocyanin colors. ... 

As frequently mentioned in the literature, anthocyanins co-exist in equilibrium in four different forms. The pH conditions shift this equilibrium toward a variety of structural forms, with the direct consequences of color changes of these pigments. As a rule, at pH above 4, yellow compounds (chalcone form), blue compounds (quinoid base), or colorless compounds (methanol form) are produced. Anthocyanins have the highest stabilities at a pH between 1 and 2 since the flavylium cation is the most stable predominant form. 

Four anthocyanin species exist in equilibrium under acidic conditions at 25°C/ according to the scheme in Figure 4.3.3. The equilibrium constant values determine the major species and therefore the color of the solution. If the deprotonation equilibrium constant, K, is higher than the hydration constant, Kj, the equilibrium is displaced toward the colored quinonoidal base (A), and if Kj, > the equilibrium shifts toward the hemiacetalic or pseudobase form (B) that is in equilibrium with the chalcone species (C), both colorless." - Therefore, the structure of an anthocyanin is strongly dependent on the solution pH, and as a consequence so is its color stability, which is highly related to the deprotonation and hydration equilibrium reaction constant values (K and Kj,). 

Attempts to stabilize anthocyanins by complex inclusion with a- and P-cyclo-dextrins failed on the contrary, a discoloration of anthocyanin solutions was observed.Thermodynamic and kinetic investigations demonstrated that inclusion and copigmentation had opposite effects. In the anthocyanins, the cw-chalcone colorless structure is the best species adapted to inclusion into the P-dextrin cavity, shifting the equilibrium toward colorless forms. "... 

Quantification of anthocyanins takes advantage of their characteristic behavior in acidic media anthocyanins exist in these media as an equilibrium between the colored oxo-nium ion and the colorless pseudobase form. Using an average extinction coefficient, the total content of anthocyanins may be estimated from the absorption of the total extracts at 520 nm (Moskowitz and Hrazdina 1981). 

The anthocyanins exist in solution as various structural forms in equilibrium, depending on the pH and temperature. In order to obtain reproducible results in HPLC, it is essential to control the pH of the mobile phase and to work with thermostatically controlled columns. For the best resolution, anthocyanin equilibria have to be displaced toward their flavylium forms — peak tailing is thus minimized and peak sharpness improved. Flavylium cations are colored and can be selectively detected in the visible region at about 520 nm, avoiding the interference of other phenolics and flavonoids that may be present in the same extracts. Typically, the pH of elution should be lower than 2. A comparison of reversed-phase columns (Ci8, Ci2, and phenyl-bonded) for the separation of 20 wine anthocyanins, including mono-glucosides, diglucosides, and acylated derivatives was made by Berente et al. It was found that the best results were obtained with a C12 4 p,m column, with acetonitrile-phosphate buffer as mobile phase, at pH 1.6 and 50°C. 

Additional compounds corresponding to anthocyanin dimers in which one of the anthocyanins is in the flavylium form and the other in the hydrated form were detected in the solutions incubated at pH 3.8. Such products arise from nucleophilic addition of the hemi-ketal onto the flavylium, confirming that, at this pH value, anthocyanins exist and react under both forms, as expected from their hydration equilibrium. 

Under weakly acid conditions the red oxonium form 9 is in reversible equilibrium with a colorless pseudo-base 10 the position of the equilibrium depends on the pH. In a test trial in a synthetic medium a solution of anthocyanins is six times more colored at pH 2.9 than at pH 3.9. 

The first method uses the fact that in acid media anthocyanins exist in a colored and a colorless form (9 and 10) in equilibrium, with the position of the equilibrium depending on pH. Consequently, the difference in color intensity between the two pH values (0.6 and 3.5 for example) is proportional to the pigment concentration. Since the phenol function is not affected by this variation, other phenolic compounds, especially tannins, do not interfere since their absorption at 550 nm is the same at both pH values. 

Fig. 13 Equilibrium distribution of four anthocyanin forms of malvidin-3-glucoside as a function of pH The red flavylium cation (AH+), the blue quinonoidal base (A), the colorless carbinol pseudobase (B), and the colorless chalcone (C). (From Ref. 138.)...

Substitution of hydroxyl and methoxyl groups influences the color of the anthocyanins. This effect has been shown by Braver-man (1963) (Figure 6-25). Increase in the number of hydroxyl groups tends to deepen the color to a more bluish shade. Increase in the number of methoxyl groups increases redness. The anthocyanins can occur in different forms. In solution, there is an equilibrium between the colored cation R+ or oxonium salt and the colorless pseudobase ROH, which is dependent on pH. 

In solution, the anthocyanins actually might exist in equilibrium with essentially four molecular forms - the flavylium cation, the quinoidal base, the hemiacetal base and chalcone [19]. The relative amounts of the four structure forms depends on both the pH and the difference in structure of the anthocyanins [20-22], Generally, anthocyanins exist primarily as the stable flavylium cation above pH 2. This uniqueness in the chemical structure is one of the important key factors affecting their absorption, metabolism, bioavailability and, consequently, the biological responses of the human body to anthocyanins. 

Unlike other subgroups of flavonoids with a similar C6-C3-C6 skeleton, anthocyanins have a positive charge in their structure at acidic pH. In solution, the anthocyanin actually occurs in equilibrium with essentially four forms the flavylium cation, the quinoidal base, the hemiacetal base, and chalcone (Cooke et al., 2005) (Figure 5.3). The relative amounts of these four forms vary with both pH and structure of the anthocyanins (Brouillard et al., 1977 Brouillard 1982 Mazza and Miniati, 1993). Anthocyanins exist primarily as the stable flavylium cation only when the pH is less than 2. 

Studies in model solution containing an anthocyanin and flavanol oligomers (up to tetramers) at pH 3 carried out at 50 °C demonstrated that temperature is another factor that affects the progress of direct condensation reactions (Malien-Aubert et al. 2002). At acidic pH and high temperature, the anthocyanin is in equilibrium with the colorless chalcone. Although breakage of the flavanol C-C bond occurred under these conditions, the formation of the chalcone impeded the synthesis of F-A products and only A-F adducts were formed (Malien-Aubert et al. 2002). 

Copigmentation is driven by hydrophobic vertical stacking between the anthocyanin and the copigment to form tt-tt complexes from which water is excluded. The flavylium cation as well as the quinonoidal base are planar hydrophobic structures and can be involved in such complexes whereas the hemiketal form cannot. The association thus results in displacement of the anthocyanin hydration equilibrium from the colorless hemiketal to the red flavylium form that can be easily measured by spectrophotometry. 

The anthocyanins are structurally dependent on the conditions and composition of the media where they are dissolved and suffer interactions among them and with other compounds that influence their structural equilibria and modify their color. Anthocyanins are usually represented as their red flavylium cation, but in aqueous media this form undergoes rapid proton transfer reactions, leading to blue quinonoidal bases, and hydration, generating colorless hemiketals in equilibrium with chalcone structures. The proportion of each form is determined by the pH... 

The pH of the medium plays a particularly important role in the equilibrium between these different anthocyanin forms, and consequently in color modification. In strongly acid solution, at a pH below 2, the red cation AH+ is the dominant form. As the pH is increased, a rapid proton loss occurs to yield the red or blue quinoidal base A, usually existing in two forms (Figure 9.3). On standing, a further reaction occurs, i.e., hydration of flavylium cation AH+, to give colorless carbinol pseudobase B. Relative amounts of forms AH+, A, B, and C at equilibrium vary with both pH and the structure of anthocyanins. For the common anthocyanin 3-glycosides... 

The anthocyanin-ethyl-flavanol adducts thus formed are purple pigments ( nax 5 0 nm), and much more resistant towards acid-catalyzed cleavage than the equivalent colorless ethyl-linked flavanol dimers (29). This presumably results from the displacement of the hydration equilibrium toward the flavylium cation form, due to self association, since similar synthetic pigments have been shown to be greatly stabilized by sandwich-type stacking (28). 

In summary, an adsorptive equilibrium between solids and liquid appears to be a major factor in limiting the concentration of pigments in young wine, and in the first few months of aging there are few losses of the original anthocyanin fragment, but significant transformation of them to different forms. 

Heating an anthocyanin solution to 100°C causes color fading that becomes more marked over time (Table 6.4). This result could be explained by a shift in the equilibrium towards chalcone and colorless forms. However, once it has been heated, the solution never returns to its original color, whatever the subsequent conditions (temperature, time, darkness, etc.). 

In aqueous media, most of the natural anthocyanins behave like pH indicators, being red at low pH, bluish at intermediate pH and colorless at high pH. It has been demonstrated that in acidic or neutral media, four anthocyanin structures exist in equilibrium the fiavylium cation (AH-I-), the quinoidal base, the carbinol pseudo base and the chalcone. In general, anthocyanins are present in the fiavylium form at... 

Concerning temperature, anthocyanins become paler after heating as the equilibrium is displaced toward the colorless carbinol and chalcone forms. The stability of anthocyanins (or other derived pigments) regarding pH and temperature variations and their degradation pathways are reported in the literature [21]. 

Fig. 3.1 Equilibrium between the different structural forms of anthocyanins at different pH values (adapted from Brouillard Lang, 1990).

The color of anthocyanins depends essentially on the different structural forms in which they can be found, these structures being strongly influenced by the pH value. At pH values between 4 and 6, the typical value for fresh and processed fruits, an equilibrium between four structural forms the red flavylium cation (I), the blue anhydrous quinoidal base (IV), the colorless carbinol pseudobase (II) and the yellow chalcone (III) occurs for all naturally anthocyanins (Figure 3). 

In solutions of pH 1.0 and lower, anthocyanins exist solely as red-coloured flavylium salts. When increasing pH, the equilibrium shifts in favour of colourless carbinol pseudo base and the red colour fades. Around the range of pH values of 4.0 to 4.5, anthocyanins are completely colourless. Another increase in pH is manifested by the purplish-red colour, which is caused by formation of a neutral quinoid base that requires the presence of free hydroxyl groups on one of C-5, C-7 or C-4 carbons. In solutions of pH 7 a blue coloured quinoid base is formed. After some time or following an increase in pH value, a gradual decrease of blue colour intensity occurs as a result of yellow chalcone formation. If the solution is acidified to around pH 1.0, the blue quinoid and colourless carbinol bases are converted back into red flavyhum cations. The transformation of chalcones is slower and not quantitative. 

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