Analytica Chimica Acta 471 (2002) 145–158

Study of solvent effects on the acid–base behaviour of adenine,adenosine 3′,5′-cyclic monophosphate and poly(adenylic) acid in

acetonitrile–water mixtures using hard-modelling andsoft-modelling approaches

Isabel Marquésa,∗, Gemma Fonrodonab, Anna Barób,Jacinto Guiterasb, José L. Beltránb

a Department of Chemistry, University of Girona, Campus Montilivi s/n, 17071 Girona, Spainb Department of Analytical Chemistry, University of Barcelona, Avda. Diagonal 647, 08028 Barcelona, Spain

Received 24 April 2002; received in revised form 4 July 2002; accepted 18 July 2002

Abstract

The acid–base behaviour of adenine, adenosine 3′,5′-cyclic monophosphate and poly(adenylic) acid was studied at 37◦Cby spectrometric titration in acetonitrile–water mixtures. Hard-modelling and soft-modelling approaches were used for thispurpose. The first one, which requires the assumption of a previous chemical model, was used for the study of monomericcompounds and the second one, an alternating least-squares multivariate curve resolution procedure, was used for the studyof poly(adenylic) acid, because it does not require any previous chemical model. The pKa values were correlated with eithermacroscopic or microscopic parameters, such as solvatochromic parameters. The influence of solvent composition on theintramolecular and solute/solvent interactions such as base stacking and hydrogen-bonding, affects the structure, solubilityand the number of species detected.© 2002 Elsevier Science B.V. All rights reserved.

Keywords:Poly(adenylic) acid; Adenine; cAMP; Multivariate curve resolution; Dissociation constants; Spectrometric techniques

1. Introduction

The analysis of nucleotides and of their degrada-tion products (nucleosides, bases, etc.) is an importantissue in many areas of research, including, for exam-ple, clinical analysis and food analysis. These com-pounds are the building blocks in both DNA and RNAand are involved in a wide variety of processes, suchas cellular metabolism, cell bioenergetics and signal

∗ Corresponding author. Tel.:+34-972-418276;fax: +34-972-418150.E-mail address:isabel.marques@udg.es (I. Marques).

transduction pathways[1]. The level of nucleosidesand nucleotides has been proposed as a cancer marker,and a diagnostic marker of the human immunodefi-ciency virus (HIV). It can also be used to establishmyocardial cellular energy status, and this is the rea-son why these substances are useful in the study ofenergy metabolism in cardiac tissue[2].

Homopolynucleotides are macromolecules that areused to mimic the behaviour of nucleic acids in liv-ing organisms[3]. They have also found widespreaduse as therapeutic agents, owing to their antiviral[4,5], antitumoral [6,7] and antiimmunostimulatory[8] properties. More recently, they have been used

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0003-2670(02)00711-0

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for the production of pharmaceutical drugs contain-ing the so-called nucleotide blockers or countersensemolecules, which are able to interact with geneticmaterial that is at the base of many human diseases;these blockers are small fragments of a DNA or RNAchain, designed to join either a problematic gene ora genetic messenger. The synthesis of polynucleotideanalogues can also be useful in chemotherapy, whenthe natural nucleic acids are deteriorated by the hy-drolysis of phosphate linkages[9]. Therefore, theanalysis of these compounds can be very useful in thediagnosis of several serious diseases and metabolicdisorders[2].

Focusing on food analysis, some purine and pyrim-idine bases have been related to off-flavours in food,being a marker for its freshness on the one hand; onthe other hand, the end product of purine catabolismin the human body is uric acid, a substance known tobe a major trigger for gout, a common disease in de-veloped countries[10]. Separation, identification anddetermination of these compounds have been of greatinterest to researchers in the field of modern sepa-ration technologies. Several analytical methods havebeen used for the determination of these solutes in-cluding liquid chromatography (LC)[11–14], micellarelectrokinetic chromatography (MEKC)[15,16], cap-illary zone electrophoresis (CZE)[1,10,13,17–19]andcapillary electrochromatography (CEC)[2].

A good knowledge of the acid–base behaviour ofnucleotides, nucleosides, bases and polynucleotidesin acetonitrile–water mixtures is essential, in a morefunctional sense, to deduce the speciation and the pos-sible conformational changes with pH or percentageof organic solvent[20–23]and, in a more applied an-alytical sense, to predict the influence of pH on sev-eral fundamental parameters in separation techniques,as selectivity, retention, electrophoretic mobility, etc.and, consequently, to establish and optimise analyticalprocedures for the separation of these analytes[24].

In order to elucidate the influence of a change in themedium on the systems in study and on retention inLC, the values of the dissociation constants can be re-lated with macroscopic parameters (cosolvent percent-age, the molar fraction of acetonitrile (XACN) and thedielectric constant (ε)), and with microscopic parame-ters (Kamlet and Taft’s solvatochromic parameters (α,β, π∗) [25–27] and either Reichardt’sE30

T parame-ter [28] or its normalised form,EN

T ), which define the

characteristics of this medium[29]. Theπ∗ parameteris used to evaluate solvent dipolarity/polarisability andα andβ scales evaluate solvent hydrogen-bond acidityand solvent hydrogen-bond basicity, respectively.

The study of equilibria involving macromolecularligands, such as polynucleotides, is hindered by thefact that the law of mass action ruling the complex-ation equilibria is valid only for each of the reac-tion sites of the macromolecule separately and thatseveral additional or secondary effects must be con-sidered: (a) polyfunctional effects, derived from dif-ferences in chemical nature and in electrostatic andsteric environments of the co-ordination sites in themacromolecule, (b) conformational changes, causedby the modifications in chemical variables such as pH,solvent polarity, ionic strength of the medium, etc.,(c) polyelectrolytic effects, caused by the ionisationof major sites of the macromolecule, which leads tochanges in the local electric field at the surface of themacromolecule, and (d) co-operative effects in whichthe formation of a given bond facilitates that of theneighbouring site. All these effects contribute to thestability of the species formed; their relative impor-tance is difficult to define since it varies with the de-gree of site occupation (complexation or protonation).In polynucleotides, the most important effects are theco-operative and conformational ones, and their influ-ence is the opposite from what is usually understoodas poyelectrolytic effect: an increase in the chargeson the surface of a polymer would make the proto-nation/deprotonation process more difficult, whereasco-operative effects would facilitate it. For that reason,the interpretation of the experimental data concern-ing poly(adenylic) acid using traditional least-squarescurve fitting approaches (hard-modelling), in whichthe prior postulation of a chemical model is needed, israther cumbersome and unreliable, and makes it neces-sary to apply new approaches, free from the constraintsimposed of the law of mass action and by the previouspostulation of a chemical model (soft-modelling). Theabove mentioned effects, however, have no influenceon the acid–base and metal ion complexing behaviourof monomeric units, and the study of these equilib-ria can be carried out by means of the hard-modellingapproaches.

Taking into account the previous considerations,this work has been carried out by combining the exper-imental data obtained by three independent analytical

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methods: UV-Vis spectrometry, circular dichroism(CD) and fluorescence spectrometry, because spectro-metric data offer the following advantages:

• low concentration levels can be used;• they give a very useful information about the struc-

tures of the compounds;• they provide data with a structure that facilitates the

use of multivariate curve resolution procedures, spe-cially when different data matrices obtained throughdifferent independent spectroscopic methods can besimultaneously analysed, since it greatly enhancesthe resolving power of these methods.

The aim of the present work is to determinate thedissociation constants values of adenine, adenosine3′,5′-cyclic monophosphate and poly(adenylic) acid inpure water and in acetonitrile–water mixtures. The re-sults obtained will be discussed in terms of averagemacroscopic and microscopic properties of the mixedsolvents. Additionally, the values obtained will be ofinterest regarding further chromatographic or elec-trophoretic separation studies of similar compounds.

2. Materials and methods

2.1. Reagents

Adenine (Fluka, Buchs, Switzerland), adenosine3′,5′-cyclic monophosphate (sodium salt) (cAMP)(Sigma, St. Louis, MO), poly(adenylic) acid (potas-sium salt) (polyA) (Sigma) and acetonitrile, HPLCgrade (Merck, Darmstadt, Germany) have beenused without further purification. Hydrochloric acid,sodium chloride, sodium hydroxide, potassium hydro-genphthalate and tris(hydroxymethyl) aminomethanewere of analytical grade quality (Merck). All solutionswere prepared with de-ionised and CO2-free waterand, after the amount of acetonitrile corresponding tothe percentage (in weight) of the solvent mixture tobe used was added, their ionic strength was adjustedto 0.15 mol l−1 with NaCl. When not in use, theywere stored at 4◦C.

2.2. Apparatus

UV-Vis absorbance spectra were recorded with aPerkin-Elmer Lambda 19 spectrophotometer, circular

dichroism spectra were recorded with a Jasco J-720spectropolarimeter and fluorescence spectra wereobtained by means of a SLM Aminco AB-2 spec-trofluorimeter; all these instruments were controlledby personal computers. An Orion 8103 Ross combi-nation pH electrode and an Orion 720 potentiometer(±0.1 mV) were used to obtain the emf readingsleading to pH values. Test solutions were placed ina double-walled vessel, maintained at 37± 0.1◦Cby circulating water from an external thermostat, andwere magnetically stirred. A Watson Marlow 505 DUperistaltic pump was used to circulate the solutionsfrom the titration vessel to the spectrometer cell, andvice versa, through PTFE or Tygon tubes.

2.3. Procedure

The dissociation constants of the different sub-stances were determined by means of the dataobtained from spectrometric titrations in differ-ent acetonitrile–water mixtures at 37± 0.1◦C and0.15 mol l−1 ionic strength (NaCl). Each compoundwas studied by as many spectrometric techniquesas possible and in as wide a range of experimentalconditions as allowed by its physico-chemical char-acteristics. InTable 1, experimental conditions foreach substance are detailed. The working procedure,detailed later, was similar in all cases, with only smallchanges to adapt it to the different compounds instudy.

In a first step, the electrode system was calibrated byGran’s method. For this purpose, a measured amountof an acidic solution, at the same conditions of tem-perature, ionic strength and solvent composition to beused in later experiments (hereafter to be called thebackground solution), was placed in a double-walled,thermostated vessel, the electrode was immersed in itand it was titrated with a strong base. The potentialwas allowed to stabilise after each addition of titrantand its value was then used to obtainE◦. Usually, about10 or 12 additions suffice forE◦ to be accurately de-termined, provided that the pH of the background so-lution changes from the initial pH 2 to a value abouttwo units lower than the pKa of the compound to bestudied.

In a second step, a suitable amount of a solutioncontaining the compound to be analysed at the re-quired conditions of temperature, ionic strength and

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Table 1Experimental conditions of spectrometric titrations of adenine, cAMP and polyA

Substance Technique λ range (nm) %Acetonitrile Concentration(×10−5 mol l−1)

pH range

Adenine UV-Vis 220–300 0 5.72 0.94–11.7610 5.13 1.10–10.9920 5.69 1.94–12.0930 6.02 1.19–11.2040 5.71 2.08–11.3950 4.47 1.31–12.10

cAMP UV-Vis, circular dichroism 220–300 0 10.1 1.32–6.4910 10.1 1.23–6.1720 5.77 2.07–4.7530 10.1 1.26–6.4340 5.91 2.08–6.9650 10.1 1.32–6.71

PolyA UV-Vis, circular dichroism 220–300 0 4.97 3.53–7.27Fluorescence emission (λ (exc) = 279 nm) 310–500 10 5.05 3.62–7.58

30 4.96 4.49–7.55

solvent composition, was added to the pre-titratedbackground solution and small amounts of sodiumhydroxide or hydrochloric acid solutions were thenadded. These amounts should be high enough toprovoke a measurable change in the pH of the test so-lution, but also small enough to allow the increase ofvolume to be neglected. After each addition, the po-tential was allowed to stabilise, its value was used, incombination withE◦ calculated in the calibration step,to calculate the pH of the solution, and a spectrum wasrecorded.

In the UV-Vis spectrometric titrations, the test so-lution was pumped to a spectrometric flow-cell bymeans of a peristaltic pump, and the whole processwas automated: after each addition of titrant, and afterwaiting for the emf reading to be stable, a spectrumwas recorded, all relevant data were stored, and a newvolume of titrant was added to restart the cycle.

In order to be able to treat simultaneously the dataobtained from the different techniques employed, insome experiments discontinuous procedure was used,because of the limitations of the particular CD spec-trometer used.

In Fig. 1, the UV-Vis spectra of adenine in 50%acetonitrile–water, the UV-Vis and CD spectra ofcAMP in 30% acetonitrile–water and the UV-Vis, CDand fluorescence spectra of poly(adenylic) acid, alsoin 30% acetonitrile–water, are shown.

2.4. Data treatment

Data generated by a spectrometric titration havea linear structure. Their mathematical representationis a matrix formed by spectrometric measures of thesystem (vectors) obtained at different pH values. Thematrix of experimental data,D, hasm×n dimension,wherem, rows, correspond to the number of pH valuesstudied andn, columns, to the number of wavelengthsof the spectrum. The main objective of the differentcurve resolution procedures used in this paper is thedecomposition of matrixD into three new ones, aconcentration profile matrix (C), a pure spectra matrix(ST) and an error matrix (E), related by the equation

D = CST + E

This decomposition can be performed either withthe assumption of a previous chemical model (hard-modelling) or without it (soft-modelling). Both ap-proaches have been used in this case, hard-modellingby means of program stability constants by absorbancereadings (STAR)[30] and soft-modelling by meansof the alternating least-squares (ALS) multivariatecurve resolution procedure[31].

The program STAR requires a previous model ofthe chemical equilibria, based upon the existenceof certain chemical species, to be postulated. From

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Fig. 1. UV-Vis spectra of adenine in 50% acetonitrile–water mixture (a). UV-Vis and CD spectra of cAMP in 30% acetonitrile–water,respectively (b1 and b2). UV-Vis, CD and fluorescence spectra of poly(adenylic) acid in 30% acetonitrile–water (c1, c2 and c3, respectively).

this model and from additional chemical information,such as the total concentration of the components,the pH of the solutions and the experimental spec-tral data, the refined equilibrium constant and thecorresponding pure spectra of each species can beobtained. The optimisation is performed by means of

a non-linear least-squares procedure, based upon theGauss–Newton algorithm, aimed towards the minimi-sation of the differences between the experimentallymeasured absorbances and those calculated from therefined model. This program has been used wher-ever a previous chemical model can be selected. The

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original version has been kindly modified by its au-thor to make it possible for it to be used with circulardichroism spectra.

The ALS program allows the experimental data ma-trix containing the spectra formed by the contributionsof the different components to be decomposed into theindividual concentration profiles and the pure spec-trum of each absorbent species in the system withoutthe need of any previous chemical model. The only re-quirement is for the instrumental response to be linearversus the concentration of the different active con-stituents. This interactive procedure for multivariatecurve resolution makes use of information generatedby a previous factor analysis. ALS has been used forpoly(adenylic) acid, because in this case no previousmodel can be postulated.

3. Results and discussion

3.1. Determination of the dissociation constants

The experiments with solutions of monomeric com-pounds, adenine and cAMP, containing amounts ofacetonitrile higher than 50% could not be carried outbecause of phase separation phenomena. Adenine isneither fluorescent nor has any chiral atom, which pre-vented the use of fluorescence spectrometry or circulardichroism. cAMP shows no fluorescence, but it has achiral atom and, therefore, in this case both UV-Visspectrophotometry and circular dichroism could beused for the determination of its dissociation con-stants by spectrometric titration. Results obtained, forboth substances, with the program STAR are shownin Table 2.

Poly(adenylic) acid is fluorescent and has a chiralatom; for this reason, all three spectrometric tech-niques could be used in the determination of itsdissociation constants, in the same working condi-tions as the other two compounds. However, onlyacetonitrile–water mixtures containing less than 30%(w/w) of the organic solvent could be used, because,at higher percentages, some solubility problemsappeared. For this reason, titrations were started in ap-proximately neutral medium and stopped when a pre-cipitate was observed. The actual pH value dependedon solvent composition, as an increase in the percent-age of acetonitrile, leads to a less polar medium, to

Table 2Experimental pKa values for adenine and adenosine 3′,5′-cyclicmonophosphate (cAMP) in different acetonitrile–water mixtures

%Acetonitrile Substance

Adenine cAMP

pKa1 (S.D.)a pKa2 (S.D.) pKa (S.D.)

0 4.16 (0.02) 9.45 (0.01) 3.62 (0.02)10 3.947 (0.006) 9.343 (0.003) 3.42 (0.01)20 3.88 (0.02) 9.52 (0.02) 3.37 (0.01)30 3.87 (0.04) 9.65 (0.02) 3.33 (0.01)40 3.96 (0.01) 9.615 (0.006) 3.39 (0.03)50 3.99 (0.01) 10.129 (0.006) 3.44 (0.03)

a The values of the standard deviations (n = 3) are given inparentheses.

a worse solvation of the charged species and, conse-quently, to precipitation at a higher pH value. More-over, in this case, there is no possibility for a previouschemical model to be postulated, which has requiredthe use of ALS to obtain the pKa values from theexperimental data. These experimental data matrices,obtained in the different titrations using the three spec-trometric techniques, were set-up together for eachacetonitrile–water mixture, so that, the new augmenteddata matrices contain an amount of information aboutevery species much higher. Additionally, the set ofpossible numerical solutions for the matrix equationsis highly constrained, and consequently, the resolvingpower of the method is considerably increased. Theresults shown in this work were obtained from theALS treatment of these augmented data matrices.

When the poly(adenylic) acid system was studied inpure water and in a 10% acetonitrile–water mixture,the singular values analysis algorithm (SVD)[32] indi-cated the presence of three different absorbent speciesat the pH range studied. At higher percentages of ace-tonitrile precipitation started at about pH 5, and inthese conditions it was not possible to reach com-plete protonation of poly(adenylic) acid; consequently,SVD indicated only two different species, as the thirdcould not be detected. The number of different speciespresent was confirmed making use of Evolving FactorAnalysis (EFA)[33], which also allowed a first esti-mation of concentration changes along the titration tobe obtained.

Additionally, the fixed-size moving window(FSMW) procedure[34] was used to decide which

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species coexist along the titration, as the knowledgeof the nature of the chemical system and of the pHallows the number of species present according tothe FSWM to be correlated with actual species. Thismethod works by performing principal componentsanalysis (PCA) on fixed-size submatrices and gives alocal rank map of the original matrix (i.e. it indicateshow many species are present in the various regionsof the original matrix). For example, at a neutralpH value there is only one species in solution and,obviously, this must be the neutral molecule; in con-sequence, the protonated forms are not present andtheir values in the matrix will be zero. Two speciesare present in acidic medium, the two different formsof the protonated molecule, while the neutral form isabsent and its value in the selectivity matrix will bezero.

With data obtained from linear estimation orfrom the selectivity matrix, the ALS program canbe executed. Some restrictions were imposed: uni-modality, selectivity and closed system; for UV-Visor fluorescence spectra, the additional restriction ofnon-negativity was also imposed (this is not validfor circular dichroism spectra, as they have negativebands). The abstract distribution plots and the purespectra of the corresponding species for water, 10and 30% acetonitrile–water mixtures are shown inFigs. 2–4, respectively.

The intensity and the extent of base-to-base inter-actions lead to the apparition of more or less orderedstructures in polynucleotides. Two stacking bases cou-ple their chromophoric groups and, therefore, the in-tensity of the corresponding UV-Vis spectrum is lowerthan what would be obtained if the bases were isolated.Consequently, a decrease in the intensity of UV-Visspectra suggests an arrangement. The optical phe-nomenon associated with changes in the orientation of

Table 3Linear dependence of calculated log(Kapp)prot values with respect to the degree of protonation values (α) of polyA for differentacetonitrile–water mixtures

%Acetonitrile Na log(Kapp)prot (S.D.)b Slopeb %Variance explained by regression

0 12 4.40 (0.07) 1.87 (0.1) 99.4510 8 4.36 (0.08) 2.03 (0.2) 99.630 12 4.56 (0.05) 1.68 (0.1) 98.7

a N is the total number of points (log(Kapp)prot/α) subjected to the analysis.b The values of the standard deviations (n = 3) are given in parentheses.

the ribose ring and of the nitrogenated base, shown bythe CD spectra, is opposite to the UV-Vis effect pre-viously described: the higher the arrangement of themolecule, the better defined is the orientation of theN-glycosidic bond. Circular dichroism spectrometryis particularly useful for the study of conformationalchanges in solution. In fluorescence spectrometry, ahyperchromic effect is detected as a consequence ofan increase in the arrangement of the molecule. Theoptical effect is similar to that described for circulardichroism. The structural arrangement leads to nitro-genated bases being linked by hydrogen-bonding in aplane perpendicular to the axis of molecular stacking.

On basis to these considerations, if three differ-ent species exist in solution, one of them must bethe neutral poly(adenylic) acid (species 1), whichhas a random coil, single-stranded structure, whilethe remaining two species can be attributed to theprotonated molecule, which has the structure of adouble helix and may exist in two different confor-mations (species 2), predominant at higher pH valuesand with a higher degree of stacking between basesand (species 3), more compacted and predominant atlower pH values. The results obtained in this workagree with data found in the literature[22,35].

Concentration profiles allow the polyelectrolytic ef-fect to be detected as a strong change of profile alonga narrow pH range in the protonation zone. From theconcentration profile and from the pH, the protona-tion constant of poly(adenylic) acid can be calculated.If the logarithms of these apparent constants are plot-ted versus the degree of protonation and the plots areextrapolated to zero degree of protonation, the appar-ent constant for these conditions can be deduced. InTable 3, the values of log(Kapp)prot at zero degree ofprotonation of poly(adenylic) acid and for the severalmedia tested are shown.

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Fig. 2. Abstract distribution plot (a1, b1 and c1) and pure species spectra (a2, b2 and c2) obtained for poly(adenylic) acid in water, fromthe ALS simultaneous analysis of UV-Vis, CD and fluorescence titrations, respectively. For all plots: deprotonated species (dashed line, 1)and protonated species (solid line, 2) and (dashed line, 3).

I. Marques et al. / Analytica Chimica Acta 471 (2002) 145–158 153

Fig. 3. Abstract distribution plot (a1, b1 and c1) and pure species spectra (a2, b2 and c2) obtained for poly(adenylic) acid in 10%acetonitrile–water, from the ALS simultaneous analysis of UV-Vis, CD and fluorescence titrations, respectively. For all plots: deprotonatedspecies (dashed line, 1) and protonated species (solid line, 2) and (dashed line, 3).

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Fig. 4. Abstract distribution plot (a1, b1 and c1) and pure species spectra (a2, b2 and c2) obtained for poly(adenylic) acid in 30%acetonitrile–water, from the ALS simultaneous analysis of UV-Vis, CD and fluorescence titrations, respectively. For all plots: deprotonatedspecies (dashed line, 1) and protonated species (solid line, 2).

I. Marques et al. / Analytica Chimica Acta 471 (2002) 145–158 155

3.2. Influence of the medium

The dissociation process is ruled by electrostatic in-teractions, as well as by specific solute-solvent inter-actions.

3.2.1. Adenine and cAMPThe different behaviour observed in the study of

the acid–base properties of adenine can be attributedto the fact that the amino groups are not equivalent.Thus, when the N(1) group is deprotonated, there isno change in the number of charges involved in theprocess (H2A+ � H+ + HA), because the changeis from a positively charged species to a neutral oneplus an hydrogen ion, and, therefore, a change inthe polarity of the medium has a minor influenceon the dissociation process, which depends only onthe solvation of the different species by the solventsof the mixture. This behaviour explains why the in-fluence of the percentage of organic solvent on thepKa value is small; on the contrary, in the deproto-nation of the N(9) group charges are created becausethere is a change from a neutral species to two ionicspecies: a negatively charged one plus a hydrogen ion(HA � H+ +A−) and, therefore, any variation in thepolarity of the medium exerts a strong influence in thedissociation process, which leads to a considerablechange in the pKa value.

The values of the dissociation constants have beenrelated with the cosolvent percentage, the mole frac-tion of acetonitrile (XACN), the dielectric constant (ε),the Kamlet and Taft’s solvatochromic parameters (α,β, π∗) and either Reichardt’sE30

T parameter or itsnormalised form,EN

T (values of all of them obtainedfrom [36]).

The variation of pKa values as a function of thepercentage of acetonitrile in the solvent is representedin Fig. 5. The pKa values are plotted versus the ac-tual percentage of acetonitrile, its molar fraction, theinverse of the dielectric constant of the mixed solventor EN

T . In the case of the pKa corresponding to thedeprotonation of the purine (N(1)) amino group ofadenine and of cAMP, a non-linear behaviour canbe observed, the plot having a minimum when thepercentage of acetonitrile is about 30%; this can beexplained by the fact that in these conditions there is achange in the behaviour of the solvent, as it goes froma water-rich composition (in which the acetonitrile

molecules gradually occupy the cavities between thewater molecules without disrupting the water struc-ture) to an intermediate composition, where clustersof molecules of either water or acetonitrile are sur-rounded by regions containing both kinds of molecules[37,38]. For the second pKa of adenine, correspond-ing to the deprotonation of the purine (N(9)) group[39–41], a decrease of pKa is observed as the solventchanges from pure water to 10% acetonitrile–water,but from this point on, the pKa increases with increas-ing percentage of acetonitrile, and there is a linearrelationship between the pKa and the molar fractionof acetonitrile in mixtures containing between 10 and50% of the organic solvent. In this case, where chargesare created, the electrostatic interactions become im-portant and the corresponding dissociation constantvalue decreases with the increase in the percentage ofacetonitrile, because the polarity of the medium alsodecreases.

The dataset was not big enough to allow Kamletand Taft’s equation to be used in order to obtain amultiparametric expression. However, the correla-tionships with all possible binary combinations of thethree parameters (α, β, π∗) were tested using singlelinear regression for the datasets and the best resultswere obtained whenα andπ∗ were used. The resultsappear inTable 4, and it can be observed that thecoefficient forα is positive and that the coefficientfor π∗ is negative. In the cases of the first pKa ofadenine and of the single pKa of cAMP the coeffi-cients forα and π∗ are quite similar, and so is thebehaviour of the pKa, which decreases with increas-ing percentage of acetonitrile until a proportion ofabout 30% is reached and then increase. However, thepKa2 of adenine shows a different behaviour, as thepKa value starts to increase from a percentage of ace-tonitrile of about 10%; this can be explained by thefact that the coefficient forπ∗ is much higher than inthe cases of the other pKa, which simply reflects thecircumstance that the solvent polarity exerts a stronginfluence over the second dissociation equilibrium ofadenine.

3.2.2. Poly(adenylic) acidEven though cAMP is a monomer of poly(adenylic)

acid, the polymer shows a polyelectrolytic effect thancan considerably modify its behaviour as comparedwith that of the monomer, with the additional difficulty

156 I. Marques et al. / Analytica Chimica Acta 471 (2002) 145–158

Fig. 5. Plot of pKa values of adenine (pKa1 (�), pKa2 (�)) and cAMP (pKa (�)) vs. the percentage of acetonitrile (a), molar fraction(b), inverse of dielectric constant (c) andEN

T (d).

Table 4Expressions of Kamlet–Taft equations obtained by multiple regression applied to pKa values of adenine (pKa1, pKa2) and cAMP inacetonitrile–water mixtures of different composition

Substance Linear solvation energy relationships Correlation coefficienta

Adenine pKa1 = 3.5 (±0.1) + 2.5 (±0.3)α − 1.9 (±0.3)π∗ 0.983pKa2 = 13.2 (±0.3) + 2.5 (±0.6)α − 5.7 (±0.6)π∗ 0.994

cAMP pKa1 = 2.8 (±0.2) + 2.1 (±0.3)α − 1.4 (±0.4)π∗ 0.974

a n = 6.

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that species having a single helix conformation do notbehave in the same way as those having a double helixconformation.

The influence of solvent composition on the dif-ferent species of poly(adenylic) acid can be deducedfrom the UV-Vis, circular dichroism and fluorescencespectra represented inFigs. 2–4. For the single helixform, an increase in the percentage of acetonitrile inthe solvent leads to an increase of the absortivity andfluorescence intensity of the neutral species and to adecrease of ellipticity, all of which clearly indicates ahigher degree of disorder in the molecules. This canbe explained by the fact that a decrease in the polar-ity of the medium reduces the stability of the exter-nal charges of the phosphate groups, which inducesa repulsion effect among them. Moreover, the nitro-genated bases, which have a lower polarity, are sta-bilised because of the lower repulsion with the solventand, therefore, the tendency towards stacking of thebases is substantially reduced. Additionally, the fluo-rescence intensity is enhanced by an increase in sol-vent viscosity.

The influence of the solvent composition on thespecies with double helical conformation is lessmarked. This can be attributed to the higher rigidity ofthese molecules, stabilised by the stacking of the ade-nine bases, which results in the linking of the differenthelical filaments by hydrogen-bonding, and also bythe electrostatic interactions between the charges ofthe phosphate groups and of the protonated sites.

The differences in spectral intensity between thesingle-helical deprotonated species (species 1) andthe first double-stranded helical species (species 2)of poly(adenylic) acid become more accentuated asthe percentage of acetonitrile in the solvent mixtureincreases: the hypochromism in the UV-Vis spectraand the hyperchromism in the CD and fluorescencespectra, which appear as a consequence of the changeto a more ordered structure caused by the protonationprocess, have a greater magnitude. This is coherentwith the different influence that the solvent mixturesstudied exert on the two species.

The solvent composition also determines the num-ber of different species in solution in the working con-ditions. In pure water and in a 10% acetonitrile–watermixture, two conformations for the double helicalstructure can be observed before precipitation of theprotonated form starts (at about pH 3.7), but only one

is observed in a 30% acetonitrile–water mixture. Inthis case, the lower stabilisation of the charged speciesprovoked by the increase in the percentage of organicsolvent leads to the precipitation of the protonatedform at a higher pH value (about pH 4.8), thus, pre-venting the formation of other protonated structures.

The pKa value of poly(adenylic) acid was affectedby an associated error that was higher than desirable,but also unavoidable, as the pKa was obtained byextrapolation and without the fulfilment of the massaction law, two conditions that lead to a higher uncer-tainty of the results. This, combined with the fact thatonly three different solvent compositions were usedfor the study of poly(adenylic) acid, has prevented toobtain any correlation between the pKa values and thesolvent parameters descriptors. Anyway, it is evidentthat the pKa value decreases when the percentage oforganic solvent is decreased, reaching a minimum ina 10% acetonitrile–water mixture.

The polymer and the monomer show a differentbehaviour when the solvent composition is changed(for cAMP the minimum is reached in a 30%acetonitrile–water mixture), because of the poly-electrolytic effect. Thus, the protonation sites ofpoly(adenylic) acid remain in the external layer of thebases and, therefore, are under the influence of phos-phate chains, while in cAMP the protonation sites arewell separated from the negative charges of phosphategroups and, consequently, are not influenced by them.This different behaviour of polymer and monomerhas not been observed either in polynucleotides thatdo not show a polyelectrolytic effect or in polynu-cleotides that, while showing this effect, have all theirprotonation sites in the basic rings, as is the case withpolyU and polyC[42].

The knowledge of the acid–base equilibria of ion-isable compounds at different solvent compositionare subjects of main interest in the development andoptimisation of separation methods using LC[43] orCE [44]. Under this scope, it is remarkable that thedissociation constants of adenine and cAMP do notsuffer important changes when changing the com-position of the medium (acetonitrile–water mixturesup to 50% acetonitrile) except for pKa2 of adenineat the upper limit studied. Therefore, its retention ormobility behaviour in LC or CE experiments can beeasily modelled. On the other hand, the behaviourof poly(adenylic) acid is strongly dependent of the

158 I. Marques et al. / Analytica Chimica Acta 471 (2002) 145–158

medium, despite its relatively small change in the ap-parent dissociation constant (up to 30% acetonitrile),due to changes in their conformational structure. Thismeans that, in the optimisation of separations by LCor CE, this characteristic must be taken into account,especially in the pH range 3–5.5, where three speciescoexist in equilibrium, while at higher acetonitrile con-tents (30%) only two of these species remain present.

Acknowledgements

This research has been supported by DGICYT,Spain (Project no. PB96-0377).

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