Determination of dissociation constants of flavonoids by capillary electrophoresis

José M. Herrero-MartínezMeritxell SanmartinMartí RosésElisabeth BoschClara Ràfols

Departament de QuímicaAnalítica,Universitat de Barcelona,Barcelona, Spain

Determination of dissociation constants offlavonoids by capillary electrophoresis

Ionization constants of some flavanols (catechin and epicatechin) and flavonols(kaempherol, fisetin, morin, and quercetin) are determined by capillary zone electro-phoresis (CZE). This technique allows the determination of pKa values until about 12.The pKa values obtained are compared with those calculated by the SPARC compu-tational program. This program predicts the microscopic and macroscopic pKa valuesand the order of deprotonation of the different 2OH groups. While for catechin andepicatechin the first ionizable OH group occurs in ring 1 and the second ionizablegroup in ring 2, in flavonols the first deprotonation occurs in ring 2 and the second inring 1.

Keywords: Capillary electrophoresis / Dissociation constant / Flavonoids / pKa / PolyphenolsDOI 10.1002/elps.200410258

1 Introduction

1.1 General aspects

Flavonoids comprise one of the most numerous andwidespread group of natural products. These polyphenolderivatives accumulate in substantial amounts in vegeta-tive parts of plants and food products included in humandiet [1, 2]. Due to their phenolic structure (Fig. 1), thesecompounds are strong antioxidants and free radical sca-vengers [3–5]. The increasing interest in flavonoids is dueto the appreciation of their broad pharmacological activi-ty. Beneficial effects of flavonoids have been describedfor diabetes mellitus, allergy, cancer, coronary heart dis-eases, viral infections, and inflammations [3]. The anti-oxidant efficiency of flavonoids has been related to theirhydrogen radical donating abilities, and with the numberof hydroxyl groups in the molecule [6–9]. Therefore, theknowledge of dissociation constants (i.e., pKa values) offlavonoids is fundamental to understand their behavior inbioclinical and pharmacological research studies. Thesecompounds present several ionizable hydroxyl groupswith pKa values relatively close to each other, and, there-fore, the accurate determination of these highly alkalinedissociation constants is a difficult task.

Several classical methods, including spectrophotometry[10–14] and potentiometry [11, 12] have been used for thedetermination of dissociation constants of such weakacids. However, these methods often cannot be applied

properly to compounds that are sparingly soluble in water.Furthermore, these conventional procedures require rela-tively high amount of the compound (i.e., in potentio-metric studies the concentration is usually greater than1024 M) and stable and high-purity substances. Capillaryelectrophoresis (CE) has evolved considerably over thelast decade and is particularly effective in separating ionicspecies. In fact, CE has been applied to many pKa deter-minations [15–31]. CE has some advantages over theconventional methods for pKa determinations. It requiresvery small amounts of sample at low solute concentra-tions. In addition, it is very simple because only themigration time is measured and compounds with highpurity are not required.

Figure 1. Molecular structures of the studied solutes.

Correspondence: Dr. Clara Ràfols, Departament de QuímicaAnalítica, Universitat de Barcelona, Diagonal 647, E-08028 Bar-celona, SpainE-mail: [email protected]: 134-934021233

1886 Electrophoresis 2005, 26, 1886–1895

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2005, 26, 1886–1895 Determination of dissociation constants of flavonoids by CE 1887

In this work, we used CE for the pKa determination of fla-vonoids, some of them with low water solubility. Theresults have been compared with those obtained by acomputational prediction program (SPARC) [32–34],which predicts both macroscopic and microscopic pKa

values strictly from molecular structure using relativelysimple reactivity models. It predicts also the preferentialorder of deprotonation of the different hydroxyl moieties inthese compounds.

1.2 Theoretical background

The pKa determination of acids and bases by CE is basedon measuring the electrophoretic mobility of chargedspecies associated with the acid-base equilibria as afunction of pH [15–31]. CE has been also applied to thedetermination of pKa in compounds with multiple pKa

values [20, 21, 26].

The acid-base equilibria for a fully protonated species,HnA

z, can be expressed by:

HnAz ! Hn�1Az�1 þ Hþ Ka1

��Hn�ði�1ÞAz�ði�1Þ ! Hn�iAz�i þ Hþ Kai

��Hn�ðn�1ÞAz�ðn�1Þ ! Az�n þ Hþ Kan

where n is the total number of ionizable groups, z is themaximum positive charge (in the fully protonated species),and Ka1,. . .Kai. . ., Kan, are the thermodynamic dissociationsuccessive constants. Thus, the effective electrophoreticmobility, me, of a polyprotic compound, HnA

z, in a buffer at acertain pH is related to the mobility of the individual spe-cies present at this pH and it can be expressed by:

me ¼Xn

i¼0

aHn�iAz�imHn�iAz�i (1)

where a is the mole fraction and m is the mobility of thecorresponding species. The term corresponding to theuncharged species will have m = 0. The ionization degreeis related to the acidity constants, thus, the effective mo-bility for a polyprotic compound can be expressed as afunction of the pKa values of each species and the pH ofthe BGE by a general expression equivalent to thosereported in the literature [20, 26]:

me ¼mHnAz þ

Pni¼1

10ipH�

Pi

j¼1

pK0aj

mHn�iAz�i

1þPni¼1

10ipH�

Pi

j¼1

pK0aj

(2)

where K0ai is a conditional acidity constant defined as:

K0

ai ¼aHþ ½Hn�iAz�i�½Hn�ði�1ÞAz�ði�1Þ� (3)

and it is related with the thermodynamic acidity constant,Kai, by

Kai ¼ K0

aigHn�iAz�i

gHn�ði�1ÞAz�ði�1Þ(4)

or

pKai ¼ pK0

ai þloggHn�ði�1ÞAz�ði�1Þ �loggHn�iAz�i (5)

where g is the activity coefficient of the involved solutesthat corrects the effect of solution ionic strength on soluteionization. In dilute solutions, g can be calculated by theDebye-Hückel equation

logg ¼ � Az2ffiffiIp

1þ BaffiffiIp (6)

where z is the ion charge and I is the solution ionic strength(in molarity). The Debye-Hückel A and B parameters arefunctions of temperature and dielectric constant of themedium [35]. For aqueous solutions at 257C, A = 0.51 andB = 0.33. The a value depends strictly on the solvatedradius of the ions, but, since this is unknown in mostinstances and its value has a small effect in the activitycoefficient value, the Bates-Guggenheim convention isusually applied [34]. This convention assigns a value of a =4.56 Å for aqueous solutions, and thus, Ba = 1.5.

In this work, the compounds studied present severalionizable hydroxyl groups, with pKa values relatively closeone to each other. Taking into account the relevant dis-sociation constants over the pH range studied (2–12) andEq. (2) the following equations can be derived for two (Eq.7) or three (Eq. 8) pKa values:

me ¼10ðpH�pK01ÞmH2A� þ 10ð2pH�pK01�pK02ÞmHA2�

1þ 10ðpH�pK01Þ þ 10ð2pH�pK01�pK02Þ(7)

me ¼10ðpH�pK01ÞmH2A� þ 10ð2pH�pK01�pK02ÞmHA2� þ 10ð3pH�pK01�pK02�pK03ÞmA3�

1þ 10ðpH�pK01Þ þ 10ð2pH�pK01�pK02Þ þ 10ð3pH�pK01�pK02�pK03Þ(8)

where mHA2, mA

22, mH2A22, mHA

22, and mA32 are the electro-

phoretic mobilities of the corresponding charged species.

The effective mobilities of analytes are determined at dif-ferent buffer pH using Eq. (9), where V is the applied volt-age, Ld is the effective capillary length to the detector, Lt isthe total capillary length, tm is the migration time of theanalyte, and teof is the migration time of the neutral markerdue to the electroosmotic flow (EOF).

me ¼LDLt

V1tm� 1

teof

� �(9)


CE

and

CE

C

1888 J. M. Herrero-Martínez et al. Electrophoresis 2005, 26, 1886–1895

2 Materials and methods

2.1 Reagents and chemicals

All chemicals used in the preparation of buffers were ofanalytical reagent grade unless otherwise noted. Sodiumdihydrogenphosphate, hydrogenphosphate and phos-phate, sodium acetate, ammonium chloride, ammonia,boric acid, sodium hydroxide, and hydrochloric acidwere from Merck (Darmstadt, Germany). Tris(hydroxy-methyl)aminomethane (Tris) was purchased from J. T.Baker (Deventer, Holland). Methanol from Merck was ofHPLC-grade. Catechol, (1)-catechin, (2)-epicatechin,kaempferol and quercetin dihydrate were from Fluka(Buchs, Switzerland), resorcinol and fisetin from Sigma(St. Louis, MO, USA). Morin was purchased from Riedel-de Haën (Seelze, Germany). The chemical structures ofthese compounds studied are shown in Fig. 1. All solu-tions were prepared with deionized water (Milli-Q deioni-zer, Millipore, Bedford, MA, USA).

2.2 Instrumentation and operational conditions

The CE experiments were carried out with a Beckmaninstrument P/ACE 5500 (Palo Alto, CA, USA) equippedwith a diode-array spectrophotometric detector. Theseparations were performed on an uncoated fused-silicacapillary (47.0 cm 6 50 mm ID 6 375 mm OD) obtainedfrom Polymicro Technologies (Phoenix, AZ, USA). Withthe Beckman cooling system, the temperature of thecapillary was kept at 25.07C (6 0.17C), basically with aliquid fluorocarbon coolant. Before first use, a new capil-lary was conditioned at 257C as follows: 10 min with 1 M

NaOH, 10 min with 0.1 M NaOH, 10 min with water, andfinally 30 min with the running buffer. Between runs, thecapillary was rinsed with 0.1 M NaOH (1 min), water(1 min), and running buffer (3 min). At the end of eachworking session, the capillary was rinsed with deionizedwater for 10 min and N2 for 1 min. Standards were in-jected hydrodynamically at 0.5 psi for 2 or 5 s (1 psi= 6894.76 Pa). UV detection was performed at 214 nm.Unless otherwise indicated, the applied voltage was 20 kVof positive polarity. Mobility measurements were done intriplicate. pH measurements were taken with a Rosscombination electrode Orion 8102 in a Crison micropH2002 potentiometer with a precision of 6 0.1 mV(6 0.002 pH units). The computational predictions of pKa

values were made using SPARC software [34].

2.3 Buffer and sample preparation

For CE, several background electrolytes (BGEs) wereprepared at pH values covering the range 2.0–12.2, andthe pH values of these solutions were adjusted by adding

the required amount of NaOH or HCl. The ionic strengthwas kept constant at 0.05 M. The buffers composition arelisted in Table 1. The buffers were filtered through a0.45 mm pore size nylon filter (Whatman, Maidstone, Kent,UK) and stored in the refrigerator at 47C until use. Stocksolutions of analytes were prepared at 1000 mg?mL21 inwater. Test injection solutions were made at a concentra-tion of 50–100 mg?mL21 in water. In the case of low aque-ous solubility of some flavonols (quercetin, fisetin, andkaempherol), they were initially dissolved in methanol toincrease their solubility and prepare the stock solution.After that, they were diluted with water or aqueous bufferat least 20 times to prepare the test injection solutions of50 mg?mL21. Benzyl alcohol (100 mg?mL21) and methanolwere used as EOF markers for the calculation of theeffective mobilities. Stock solution of these analytes werestored in brown bottles and kept frozen at 2207C untiltheir use.

Table 1. Composition of running buffers employed inCZE at 0.05 M ionic strength

pH range Constituent Stock solution

2.0–3.2 Phosphate 0.05 M NaH2PO4, 2 M HCl3.6–5.6 Acetate 0.05 M CH3COONa, 2 M HCl5.6–7.2 Phosphate 0.025 M NaH2PO4, 0.025 M

Na2HPO4, 2 M HCl7.6–9.2 Tris 0.2 M Tris, 2 M HCl9.6–10.8 Ammonium 0.1 M NH3, 0.1 M NH4Cl, 1 M

NaOH11.2–12.2 Phosphate 0.015 M Na2HPO4, 0.010 M

Na3PO4, 1 M HCl

3 Results and discussion

3.1 Choice of BGE

The most important factors for the determination of pKa

by CZE are pH and ionic strength of the buffer employedsince the reliability of the experimental measurements (ormobilities) depends largely on these factors. Thus, theproper choice of the buffer in CE is not a trivial task, takinginto account involved factors, such as pH, buffering ca-pacity, complexation, etc. In this study, a wide pH rangecomprised between 2.0 and 12.2 has been required. Thisrange can not be covered by a unique buffering systemand thus different buffers were selected. It should bementioned that borate buffer was eliminated from thepreliminary list of BGEs selected for this study (Table 1).Although it is widely used in the pH region from 8 to 10, itswell-known ability to complex vicinal diol-compounds,like the solutes under study, and others, such as carbo-hydrates [36–38], would produce wrong pKa values.



Buffers of constant ionic strength 0.050 M were used inthis study. The ionic strength must be constant through-out the buffer series, in order to eliminate the influence ofthis parameter on raw effective mobilities [39]. In thisinstance, the inflection point of the sigmoidal curvesobtained when mobilities are plotted versus pH, providesthe conditional pKa (pKa

0) value of the solute that can beeasily converted to the thermodynamic constant by cor-rection of the value by the activity coefficient (Eq. 4),which is ionic strength-dependent (Eq. 6). Thus, for anionic strength of 0.050 M, such as the one used in thiswork, the correction of the activity coefficient for mono-protic weak acid is log gA2 = 2 0.09, and therefore, this isthe difference between pKa and the inflection point of theplot (pKa

0). However, these differences could be relevantin the case of compounds with several ionizable groups,where the contribution of the activity coefficient can notbe ignored. For instance, the correction term of the activ-ity coefficient is about 0.26 and 0.43 (in the 50 mM bufferused) for pKa2 and pKa3 values, respectively.

Although pH and ionic strength are important factors inthe pKa determination of a solute, attention should also bepaid to other additional aspects, such as heat dissipation(i.e., minimizing the Joule heat production) and bufferingcapacity of the BGEs employed. Buffers of ionic strength0.050 M were selected as a reasonable compromise be-tween the need to minimize the Joule heat productioninside the capillary and the requirement for enough buf-fering capacity [19, 21, 40]. Selected buffers allow anacceptable current intensity between 10 and 55 mA, with-out Joule effect.

Furthermore, the influence of the electrolysis of buffers(caused by CE separation voltage) and the dissolution ofCO2 (from air) on the final buffer’s pH, especially signifi-cant in the case of buffers of alkaline pHs, has beenstudied. For checking the stability of the BGEs employed,the CE running buffers at the anode and cathode, afterrunning the analytes in a sequence of 24 h, were tested forpH changes. The buffer vial volume was approximately4 mL. Small changes (0.01–0.05) occurred for buffersbelow pH 10 due to electrolysis. For pH 12 buffer, thebuffer pH dropped 0.1 units after this period. For com-parison, a control vial that hold pH 12 buffer was placed inthe buffer tray but no voltage was applied. The controlbuffer showed similar drops to those used for CE runs,which confirms that the pH drop was mainly due to CO2

absorption from air. Overall, the pH buffers showed anadequate buffering capacity, which will allow a reliablemobility and pKa measurements of the analytes.

3.2 Determination of pKa values by CE

The chemical structure of flavonoids (flavanols and flavo-nols), given in Fig. 1, implies a low acidity of the OHgroups. Consequently, the determination of their highlyalkaline dissociation constants is a difficult task. A pre-liminary study was done with resorcinol and catechol,which constitute the main skeleton moieties of the com-pounds studied. Catechol and resorcinol show only twoionizable groups. The plot of electrophoretic mobility ver-sus pH for these compounds (Fig. 2) shows two inflectionpoints in the case of resorcinol, but only one for catechol

Figure 2. Electrophoretic mobilities of catechol and resorcinol versus the pH of the BGE, with curves fitted according toEqs. (8) and (9), respectively. Conditions are described in Section 2.



Table 2. Macroscopic CE pKa0 values of the studied flavonoids

Analyte pKa10 pKa2

0 pKa30 mH

2A2 mH2 A2 mA32 SD

Catechola) 9.32 6 0.06 . . . . . . 22.35 . . . . . . 0.053Resorcinola) 9.36 6 0.05 11.43 6 0.08 . . . 22.39 24.33 . . . 0.063Catechinb) 8.68 6 0.23 9.70 6 0.24 11.55 6 0.20 21.32 23.07 24.13 0.048Epicatechinb) 8.91 6 0.23 9.93 6 0.32 11.76 6 0.26 21.55 23.13 24.25 0.053Kaempherolb) 6.96 6 0.09 8.78 6 0.11 10.60 6 0.12 21.34 23.15 24.43 0.058Fisetinb) 7.27 6 0.09 9.44 6 0.07 . . . 21.67 24.33 . . . 0.093Morinb) 4.97 6 0.03 8.37 6 0.08 10.18 6 0.12 21.95 23.46 24.50 0.053Quercetinb) 7.10 6 0.12 9.09 6 0.11 11.12 6 0.36 21.42 24.14 24.90 0.094

a) Values at 100 mM

b) Values at 50 mM

which can be attributed to its high pKa2 value (13.7,according to the literature data [11]) that precludes its esti-mation by CE. Figure 2 shows the best fits obtained bynonlinear regression with the experimental data for cate-chol and resorcinol. The calculated pKa values of resorcinoland catechol are given in Table 2 (at 0.1 M ionic strength)and in Table 3 (after correction by the ionic strength).

The flavonoids studied present four or five ionizable OHgroups. In the case of flavanols, ring 1 and ring 2 are notconjugated, i.e., the deprotonation of OH groups of onering system should not appreciably affect the ionization ofOH groups of the other. Hence, ionizations of OH groups ofring 1 could be considered independent and distinguish-able from those of ring 2, and thus, catechol and resorcinolwere used as models for ring 1 and 2, respectively. Fromthe pKa values obtained for catechol and resorcinol (Table

2), we should expect at least three pKa values for flavanols,the first two corresponding to the deprotonation of OHgroups from resorcinol and catechol moieties, respec-tively, while the third corresponds to the pKa2 of resorcinol.However, Fig. 3 shows only two inflection points, the firstone (ca. pH 9) includes the first two pKa values of ionizableOH groups, since the difference of the consecutive pKa

values is less than one unit, while the second jump corre-sponds to the deprotonation of the third OH group in theflavanol molecule. Thus, the best fit with the experimentaldata was obtained by using Eq. (8). The pKa valuesobtained for catechin and epicatechin are given in Tables 2and 3. In contrast, flavonols exhibit conjugation betweenthe ring 1 and ring 2, which will facilitate the deprotonationof hydroxyl groups due to the charge delocalization. Thus,we should expect for these compounds more acidic pKa

values than those obtained for flavanols.

Figure 3. Dependence of electrophoretic mobility of catechin and epicatechin on the pH of the running buffer. Conditionsare described in Section 2.



Table 3. Macroscopic pKa values of the studied flavo-noids at I = 0

SPARC CE Mainaciditygroupa)

Catechol pKa1 9.19 9.43pKa2 13.09 . . .

Resorcinol pKa1 9.63 9.47pKa2 11.67 11.77

Catechin pKa1 8.85 8.77 DpKa2 10.23 9.97 B and ApKa3 12.16 11.99 A and B

Epicatechin pKa1 8.85 9.00 DpKa2 10.23 10.20 B and ApKa3 12.16 12.20 A and B

Kaempherol pKa1 7.11 7.05 ApKa2 9.09 9.04 EpKa3 11.33 11.04 BpKa4 13.26 – C

Fisetin pKa1 7.31 7.36 ApKa2 8.59 9.71 EpKa3 12.11 – CpKa4 13.85 – D

Morin pKa1 6.93 5.06 ApKa2 8.32 8.64 FpKa3 9.98 10.62 EpKa4 11.98 – B

Quercetin pKa1 7.04 7.19 ApKa2 8.55 9.36 EpKa3 11.26 11.56 BpKa4 13.06 – C

a) Main functional group of Fig. 1 that contributes to thepKa value

Figure 4 shows the plot of electrophoretic mobility dataversus pH for flavonols, and the best fits obtained bynonlinear regression with the experimental data in eachcase. The sigmoidal curves derived from the nonlinearregression based on Eq. (8) for triprotic acidic com-pounds give good fits to experimental data for quercetin,kaempferol, and morin, while fisetin shows a better fitwith Eq. (7). The pKa values obtained are listed in Tables2 and 3.

It should be noted that although the solutes studied havesimilar structures, they exhibit different behaviors at var-ious pH values. In particular, all polyphenolic compoundsstudied have a ‘zero’ mobility at pH values below 5,except morin, which has pKa values more acidic withrespect to other flavonoids. This fact could be explainedon the basis of the higher conjugation present in thismolecule in comparison to the others. In contrast, fisetinshows only two pKa values because the third pKa wouldbe higher than 12 and it can not be determined by CE.

3.3 Estimation of pKa values by thecomputational program SPARC

Computational programs constitute a valuable tool toestimate the different pKa values of a molecule with sev-eral ionization groups and to assign these values to theionizable group present. In particular SPARC program[32–34] uses algorithms based on fundamental chemicalstructure theory to calculate ionization constants (pKa’s)of organic compounds. Molecular structures are brokenat each essential single bond into functional units withintrinsic properties.

Table 3 shows the macroscopic pKa values estimated bythe SPARC program for the studied compounds. Theestimated values are quite similar to those experimentallyobtained by CE for the molecules with the simpleststructure (catechol, resorcinol, catechin, and epica-techin). These thermodynamic values are also given inTable 3. However, SPARC can not differentiate betweenthe cis/trans OH position. This is the reason that the pre-dicted pKa values for catechin and epicatechin are equaldespite the experimental values for the trans-derivative(epicatechin) are about 0.2 units higher than those of thecis-derivative (catechin). For flavonols, the SPARC valuesagree only with the CE values for kaempherol, the uniquestudied flavonol with solely one OH group in ring 1. For theother flavonols some SPARC pKa values are quite differ-ent from those obtained by CE. The greater discrepancyis observed in pKa1 for morin. The SPARC programassigned a pKa1 value similar to the rest of the flavonolswhereas the experimental CE value is significantly lowerthan the rest of the molecules studied. The spectro-photometric pKa1 value for morin [12] is also lower thanthat found for the other flavonoids. Moreover, pKa2 andpKa3 CE values are slightly higher than the predictedSPARC values. For quercetin the second CE pKa value ishigher than the one predicted by SPARC. The same ten-dency is observed for fisetin. Since the experimental CEpKa2 value is higher than the predicted value by SPARC,the experimental CE pKa3 value is expected to be alsohigher than the predicted SPARC value of 12.11 and thusit is not detected by CE. Liu et al. [27] determined by CEthe dissociation constants of quercetin in water. The firstpKa value (7.26) was in good agreement with our first pKa

(7.19), while the second one (10.76) represents an aver-age between our second (9.36) and third (11.56) pKa. Thisis due to the fact the pKa values established wereobtained by fitting the experimental points to a nonlinearregression expression for two pKas, instead of three, likehas been done in this study.

The SPARC program [32–34] allows not only estimation ofthe macroscopic pKa values but also the microscopicconstants attributed to the different ionizable groups



Figure 4. Dependence of electrophoretic mobilities of kaempherol, fisetin, morin, and quercetin as a function of pH. Con-ditions are described in Section 2.



Table 4. Microspecies and microconstant pk values of flavonoids

Microcon-stants/micro-species

[Hn-1X2] [Hn-2X

22] [Hn-3X32] [Hn-4X

42]

Catechin/ 80/14 D 39% pkD = 9.11 DB 31% pkDB = 10.39 DBA 51% pkDBA = 11.94epicatechin E 18% pkE = 9.45 pkBD = 9.70 pkDAB = 11.83

B 8% pkB = 9.80 DA 24% pkDA = 10.50 EBA 26% pkEBA = 11.91A 6% pKA = 9.92 pKAD = 9.70 pkEAB = 11.81

EB 15% pkEB = 10.36 DBE 2% pkDBE = 13.31pkBE = 10.01 pkEBD = 12.99

EA 12% pkEA = 10.47 pkDAE = 13.31pKAE = 10.01 DAE 2% pkEAD = 12.99

Kaempherol 32/8 A 80% pkA = 7.13 AE 85% pkAE = 9.14 AEB 70% pkAEB = 11.42 AEBC 85% pkAEBC = 13.19E 3% pkE = 8.62 pkEA = 7.54 pkABE = 9.67 pkAECB = 12.49

AB 1% pkAB = 10.88 AEC 12% pkAEC = 12.21

Fisetin 32/12 A 56% pkA = 7.40 AE 86% pkAE = 8.61 AEC 58% pkAEC = 12.18 AECD 56% pkAECD = 13.73E 11% pkE = 8.10 pkEA = 7.80 AED 14% pkAED = 12.81 pkAEDC = 13.15D 2% pkD = 8.90 AD 10% pkAD = 9.47 pkADE = 11.90 pkADCE = 12.76

pkDA = 7.99 ADC 6% pkADC = 12.29

Morin 80/12 A 58% pkA = 7.02 AF 54% pkAF = 8.44 AFE 80% pkAFE = 9.90 AFEB 88% pkAFEB = 12.00F 10% pkF = 7.80 pkFA = 7.55 pkAEF = 9.54 pkAFBF = 10.42E 3% pkE = 8.28 AE 22% pkAE = 8.80 AFB 2% pkAFB = 11.45 pkAEBF = 10.18

pkEA = 7.47 AEB 1% pkAEB = 11.37 AFEC 2% pkAFEC = 13.58

Quercetin 80/15 A 66% pkA = 7.09 AE 81% pkAE = 8.60 AEB 62% pkAEB = 11.35 AEBC 40% pkAEBC = 13.22E 7% pkE = 8.08 pkEA = 7.52 AEC 8% pkAEC = 12.24 pkAECB = 12.42D 1% pkD = 8.87 AD 10% pkAD = 9.46 ADB 7% pkADB = 11.40 AEBD 29% pkAEBD = 13.37

pkDA = 7.69 AED 2% pkAED = 12.79 pkAEDB = 11.95pkADE = 11.86 ADBC 3% pkADBC = 13.32

present in the compound. In fact, the macroscopic con-stants are predicted from the crossing of the charge curvesof all the microspecies with the same charge. Table 4 indi-cates the number of microconstants calculated for the fla-vonoids studied and the number of microspecies inappreciable concentration (above 1%), in the pH range0–14. Table 4 also shows the percentage corresponding ateach microspecies at pH value where it is maximum, andthe microconstant associated to each one.

The notation employed for microconstants is similar tothat reported by Hill [40]. The ionized group of interest isindicated in the microscopic pk by the last symbol in thesubscript. Any previous alphabetic character denotesother groups in the molecule, which already exist in thebasic form when ionization under consideration is takingplace. For example, pkDB denotes the pk value corre-sponding to the ionization of the B hydroxyl group whenthe D hydroxyl group has been already ionized and theother hydroxyl groups are still protonated. The symbolscorresponding to each microspecies indicate the OHgroup ionizated.

Figures 5 and 6 shows the fraction of each micro-especies as a function of pH for catechin and quercetin,respectively. These distribution diagrams of micro-species are useful to predict the more reliable pre-ferential order of deprotonation of the different hydroxylgroups. For catechin and epicatechin the first OH groupsionizable are attributed to ring 1, the OH group labelledwith the letter D has more tendency to deprotonate thanthe OH group identified with the letter E (see Fig. 1). Asmentioned above, due to the fact that rings 1 and 2 arenot conjugated, the ionization of the OH groups of ring 1is independent and distinguishable from that of ring 2.Thus, the pKa1 for catechin and epicatechin should besimilar to the pKa1 of the catechol. In fact, the pkD andpkE values and the macroscopic pKa1 are similar to pKa1

of catechol. The following OH group to ionize corre-sponds to ring 2. The two OH groups present in this ring,A and B, have the same probability to ionize. The fourionization does not occurs below pH 14. This assignationagrees with that reported by Slabbert, who determinedthe pKa values of catechin by potenciometry and spec-trophotometry [11].



Figure 5. Fraction of the major microspecies of catechin.Each curve refer to one microspecies. Symbols in par-enthesis symbolize the deprotonated site (as in Fig. 1).(1) HnX; (2) HnX

2 (D); (3) HnX2 (E); (4) HnX

2 (B); (5) HnX2 (A);

(6) HnX22 (D and B); (7) HnX

22 (D and A); (8) HnX22 (E and

B); (9) HnX22 (E and A); (10) HnX

32 (D, B, and A); (11) HnX32

(E, B, and A).

Figure 6. Fraction of the major microspecies of querce-tin. Each curve refers to one microspecie. Letters in par-enthesis symbolize the deprotonated site (as in Fig. 1).(1) HnX; (2) HnX

2 (A); (3) HnX2 (E); (4) HnX

22 (A and E);(5) HnX

22 (A and D); (6) HnX32 (A, E, and B); (7) HnX

32 (A, E,and C); (8) HnX

32 (A, D, and B); (9) HnX42 (A, E, B, and C);

(10) HnX42 (A, E, B, and D); (11) HnX

52 (A, E, B, C, and D).

A very interesting 13C-NMR approach to measure themicroscopic acidity constants of catechin and epica-techin leads to the same deprotonation sequence. More-over, pKa values calculated by SPARC fit well the linearrelations between successive microscopic constantsproposed [42]. However, pk SPARC values (Table 4) areslightly higher than those given for catechin (9.02, 9.12,

9.43, and 9.58) [41]. In spite of these small differences, theresults ratify the ability of SPARC program to estimate pkvalues and to assign calculated pk to a particular ioniz-able group.

For flavonols, SPARC proposes the OH group labelledwith A, in ring 2, as the most acidic group. The rings ofthese flavonols present conjugation and the negativecharge due to the deprotonation of the OH group A can bebetter stabilized than in flavanols. This conjugation couldexplain that the pKa1 of these molecules is lower than thepKa1 of catechin and epicatechin. Experimentally, wefound pKa1 values for flavonols about 7–7.5 (Table 3)except for morin that has a pKa1 of 5.06. The second OHgroup to deprotonate is commonly in ring 1 (group E). ThisSPARC assignation for flavonols does not agree with thatreported by Sauerwald et al. [14]. These authors pro-posed a first ionization in ring 1 and a second ionization inring 2. However, they give pKa1 = 7.36 (I = 0.1 M KCl) fordaphnetin with skeleton similar to ring 2 of flavonols andpKa1 = 7.03 and pKa2 = 9.1 (in 21.4% of ethanol) for quer-cetin. Lemanska et al. [9] compared several pKa values ofthe monohydroxyflavones obtained from two differentbibliographic sources [10, 43] to asses their deprotona-tion order and the firsts deprotonations were attributed toE [10] and A [43] hydroxyl moieties, respectively. How-ever, the pKa values obtained by Thomson et al. [10] wereperformed in 50% dioxane at 0.1 M ionic strength, whilethe values reported by Wolfbeis et al. [43] were done inwater. Consequently, the values from both authors cannot be easily compared. When the pKa values of mono-hydroxyflavones were done in the same conditions [43],the ionization order for the first two pKa values (A and E,respectively) was in the rough agreement with the orderpredicted by SPARC.

13C-NMR studies [44] proposed the following deprotona-tion order to flavonols and flavanones, such as narigenin:A, E, B, and C. This order agrees with that given bySPARC for the compounds studied here. The onlyexception is morin where the OH in position F (ortho-position) is more acidic than the OH in position E (para-position). A higher acidity in group F is also observed by13C-NMR [44].

4 Concluding remarks

CE is a suitable and useful technique for the determina-tion of macroscopic pKa values of polyphenolic com-pounds. CE does not require pure samples and it allowsthe determination of pKa of low-water-solubility com-pounds. However, it does not allow the assignment of pKa

values to a specific ionizable group. Computationalmethods, such as SPARC, predict reliable microscopic



constant values and attribute each one to a specificionizable group. However, the predictions are not alwaysaccurate enough. The combination of experimental CEand SPARC pKa values provide accurate values that canbe attributed to particular ionizable equilibria.

We are thankful for the joint financial support fromMCYT of the Spanish Government and FEDER of EU(Project CTQ2004-00965/BQU) and Catalan Government(2001SGR00055).

Received October 15, 2004

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Determination of dissociation constants of flavonoids by capillary electrophoresis

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