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Talanta 71 (2007) 1061–1067

Dimerization of thymol blue in solution: Theoretical evidence

Patricia Balderas-Hernandez a,b, Rubicelia Vargas a, Alberto Rojas-Hernandez a,Ma. Teresa Ramırez-Silva a, Marcelo Galvan a,∗

a Universidad Autonoma Metropolitana-Iztapalapa, Departamento de Quımica, Apdo. Postal 55-534, CP 09340, Mexico D.F., Mexicob Universidad Autonoma del Estado de Mexico, Facultad de Quımica, Paseo Colon interseccion Paseo Tollocan S/N,

CP 50120, Toluca, Estado de Mexico, Mexico

Received 5 March 2006; received in revised form 30 May 2006; accepted 30 May 2006Available online 11 July 2006

bstract

The possibility of dimerization of thymol blue was addressed by ab initio and force field calculations. In agreement with experimental information,dimer forming symmetrical chemical environments for hydrogen bond formation was determined. This dimer is stable in vacuum and aqueousedia and corresponds to the same protonated state proposed by the experiment. A comparison of the CVFF and MM3 force fields and ab initio

esults shows the suitability of CVFF to qualitatively describe this system.2006 Elsevier B.V. All rights reserved.

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eywords: Thymol blue; Dimerization; Hydrogen bonds; Molecular mechanic

. Introduction

Thymol blue is an acid–base indicator from the family of theulphonephtalein that is used extensively for end-point volumet-ic determinations. It also has many other field of applications:n the quantitative determination of proteins (�-globulin, bovineerum albumin) [1], in the study of the effects of the surfactantsn proteins [2], in proton exchange in organic reactions [3], in theanufacturing of biosensors used in optic fibers for determining

rganophosphate pesticides and carbamates [4], in the determi-ation of active substances in pharmaceutical preparations [5],nd in the elaboration of culture media for some microorganismss Escherichia coli [6].

Thymol blue (TB) structure, as the indicators of the sameamily, is formed by three benzene rings bonded to a centralarbon, with a sulphonic group bonded to one of the rings, andeto-enol groups bonded to the other rings. The molecule hashree sites whose protonation state depends on the pH of thenvironment. Fig. 1 shows the full protonated structure of TB.

In a previous work [7], we reported some experimental resultsoncerning the determination of the chemical and electrochem-cal behavior of TB in solution. It was found that a chemical

∗ Corresponding author. Tel.: +52 55 58046413; fax: +52 55 58046415.E-mail address: mgalvan@xanum.uam.mx (M. Galvan).

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039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2006.05.084

ulations; Density functional theory calculations

odel that describes properly the spectrophotometric, conducti-etric, potentiometric and voltammetric information is a model

hat implicates dimeric structures of the indicator, however, nourther details for dimer formation have been provided.

Analysis of the fully protonated TB structure indicates thathree usual acidic hydrogen atoms can be recognized; this facteads to consider that the TB dimer may be stabilized by hydro-en bonds between –OH or –SO3H groups. Further, it has beenroved that theoretical methods are useful to properly describeeometrical parameters and binding energies of hydrogen bondomplexes. There are many examples where ab initio methods,uch as Moller–Plesset perturbation theory and density func-ional theory (DFT), are used to describe the electronic structuref hydrogen bond adducts. It is evident from these studies,hat high quality basis sets are necessary to study this kind ofomplexes [8–12]. Then, the size of the systems that could betudied for ab initio methods is limited by the computationalost. Molecular mechanics (MM) methods are a good alterna-ive for large systems, as TB dimer, where applying ab initio

ethods is not computationally viable.It is well known that MM methods are successful if the func-

ional form and the parameters used in the force field, describe

roperly the main interactions in the system under study. Ineneral, protein force field functions (CHARMM, GROMOS,mber, CVFF) are simpler than more accurate approaches for

maller molecules (MM3), and consequently cheaper. It is not

1062 P. Balderas-Hernandez et al. / Ta

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tinguish a symmetric dimer from a monomeric species [7].

Fig. 1. Schematic representation of the H3L+ monomeric structure.

lways easy to decide what force field is more suitable for aarticular system. In the case we are considering, TB dimer-zation, we must look for a compromise between accuracy andeasibility.

The main goal of this paper is to use theoretical methods suchs MM and ab initio calculations in the study of the thymol blueimerization, because these methods provide information to sup-ort experimental findings in this problem. In the next section, anverview of the experimental facts supporting the dimer forma-ion is presented. Section 3 explains the assortment of techniques

sed in this work to address the question of dimerization. Sec-ion 4 is divided in monomer, dimer and solvent effects on dimerormation. Section 5 gives an overview of the main conclusionsbtained.

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Fig. 2. Representative tautomeric structures for different protonat

lanta 71 (2007) 1061–1067

. Dimerization of thymol blue, experimental facts

It has been experimentally determined that an acid–basequilibria model involving TB dimers, properly describe spec-rophotometric, potentiometric, conductimetric and voltammet-ic information [7]. The equations that represent the chemicalquilibria are

4L2 � H3L2− + H+; (1)

+ + OH− � H2O; (2)

3L2− + OH− � 2HL− + H2O; (3)

L− + OH− � L2− + H2O; (4)

he first titration reaction corresponds to proton neutralization,q. (2), because the first dissociation (Eq. (1)) is strong. Eqs. (3)nd (4) correspond to the second and third titration reactions.he scope of this paper only deals with the study of the H4L2imer.

The dimer H4L2 could be formed by different combinationsetween two of the monomeric structures represented in Fig. 2.ue to the fact that the global structure contains four acid pro-

ons, the possible combinations are: I–V, I–VI, II–II, I–III, II–IV,II–III, III–IV and IV–IV. The structure IV is discarded, sincehis molecule is not present in aqueous solution [13].

Previous NMR studies show that it is not possible to dis-

herefore, in this work, the possibility of a dimer of the com-inations II–II and III–III was assessed because these are goodandidates to form symmetric dimers.

ion states of TB. L represents the non-protonated structure.

. / Talanta 71 (2007) 1061–1067 1063

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Fig. 3. (a) Comparison of the H3L+ monomer structure, obtained by molecularmechanics (CVFF force field) and ab initio calculations (LDA/DZVP). (b) Com-parison of the same structure using MM3 force field and ab initio (LDA/DZVP)calculations The molecular mechanics structures are colored by atom: C in green,O in red, S in yellow, H in light blue, and the ab initio structure is colored in blue.(For interpretation of the references to color in this figure legend, the reader isr

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P. Balderas-Hernandez et al

. Methodology

To get some insight about the molecular mechanics perfor-ance in the geometry description of this kind of systems, two

ifferent force fields were tested in the TB monomer: CVFF andM3. The MM3 calculations were carried out using MM3(96)

rogram with standard atoms [14]. Previous works have shownhat C–H· · ·O weak hydrogen bonds can be important in thetabilization of some systems [15–18], the default MM3(96)arameter set was used after the parameters ε* and r* were mod-fied for the corresponding atoms in the hydrogen bond to betterescribe these weak interactions, the values used for the param-ters were obtained from prior calculations [19]. The parameterssed in the CVFF are the standard ones [20]. Both force fieldalculations were also compared with the ab initio density func-ional theory results at the local level [21] and using a polarizedouble-� basis set (LDA/DZVP) as a basis set [22].

For the dimer, the CVFF force field was used to minimize thetructures II–IV and also to explore the energy surface for theimer formation of II–II, III–III and IV–IV structures depictedn Fig. 2. This exploration of the energy surface for the dimernteraction energy includes several simulated annealing steps,tarting at 700 K and finishing with a 0 K full geometry opti-ization. We observed in this procedure that the formation of

imers II–II and IV–IV is possible, but in contrast we did notbtain any III–III stable dimer.

With the same force field, CVFF, a molecular dynamics sim-lation for the possible II–II dimer was performed for 220 ps at98 K within the NVT ensemble. To consider solvent effects,ur model includes the presence of a surrounding layer of waterolecules 20 A thick, which corresponds to a simulation includ-

ng 908 water molecules of the first solvation sphere. The first0 ps were considered as equilibration; therefore the analysis ofhe molecular dynamics was done on the last 200 ps of the simu-ation. The time step for integration of the movement equationsas 1 fs within a Leapfrog–Verlet scheme.Ab initio full geometry optimizations were performed on

he three molecules corresponding to the structures of the H2Lpecies in Fig. 2. The level of theory for the optimization wasDA/DZVP. To get a better description of the energy differ-nces between them, single point calculations using a hybridxchange-correlation functional within DFT (B3LYP) [23–25],ere done with the same basis set. All the ab initio calculationsere performed with the NWChem program [26].

. Results and discussion

.1. Monomer

The comparison between the minima obtained with forceeld and ab initio methods is shown in Fig. 3. In general, one mayay that MM3 provides better agreement with respect to LDAalculations. However, CVFF force field maintains the hydro-

en bond donors and acceptors in similar positions as MM3 andDA. This is important because those groups are responsible for

he stabilization of the dimer and consequently CVFF is able toescribe the dimer formation in our study.

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eferred to the web version of the article.)

The first two columns in Table 1 display energy differencesetween the fully protonated species I and structures II and IV,his corresponds to the protonation energies. The energy differ-nces between II and IV tautomers are 11.15 and 14.84 kcal/molor B3LYP and LDA, respectively, indicating that in vacuum theV structure is more stable than species II. It is interesting to

ote that the order of stability is not triggered by the use of LDApproximation and that the protonation energy is in agreementith the order of magnitude for this kind of quantities in vacuumrocesses.

1064 P. Balderas-Hernandez et al. / Talanta 71 (2007) 1061–1067

Table 1Relative energies in kcal/mol and B3LYP/DZVP HOMO–LUMO gaps in a.u.for three monomer species of TB

Structure B3LYP/DZVP LDA/DZVP HOMO–LUMO gap

IV 232.95 225.53 0.1720II 244.10 240.37 0.1142I 0.00 0.00 0.1053

Fig. 4. Representation of possible dimers, linked by hydrogen bonds, formedwith H2L monomers.

Fig. 5. Comparison of the dimer structure in vacuum optimized using molecularmechanics with MM3 force field and with CVFF force field. The MM3 structureis colored by atom (C in green, O in red, S in yellow, and H in light blue), whereasthe CVFF structure is colored in blue. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of the article.)

Fig. 6. Three different views of the H4L2 dimer configuration in the presenceof the solvent (the water molecules are not shown). In the top panel the arrowsindicate the three orientations of the views. The dashed yellow lines and numbersindicate the hydrogen bonds formed. (For interpretation of the references to colorin this figure legend, the reader is referred to the web version of the article.)

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P. Balderas-Hernandez et al

To predict the order of appearance in the UV–vis spectra forpecies II and IV, the HOMO–LUMO gaps reported in Table 1re used to estimate the first electronic transitions. Even whenhis is a crude estimation, it is well known that B3LYP gaps areseful in this context [27] and therefore we only use this level ofheory to perform such evaluation. By transforming the gap toavelength, for structures IV and II, one gets a 265 and 399 nm,

espectively. These results indicate that structure IV wavelengthorresponds to the UV region and that structure II wavelength isn the visible region. More important for our study is the relativerder indicating that structure IV must be colorless.

.2. Dimer

According to force field simulations, there are two possibil-ties for dimer formation: II–II and IV–IV. However, it is wellnown that structure IV is stable in some non-aqueous solutionsnd that its absorption spectrum does not present any peak in theisible region [13], this is in agreement with the estimated firstlectronic transition for this species. Also, all the experimentsn aqueous media, at the pH in which species II–IV may exist,ndicate the presence of a colored solution, consequently, as our

ork focuses in aqueous media, all dimer simulations were per-

ormed on the II–II species. For this dimer, constructed by using2L(II) monomers (see Fig. 2), the conformational space was

ampled with the CVFF force field, according to the following

ig. 7. Fluctuations of the angle O–H· · ·O and distances O· · ·O and H· · ·O, for the staorresponds to the simulation at 298 K with a layer of solvent built from 908 waterond formation for these parameters [28]. Panels (a–c) correspond to hydrogen bond

4

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rocedure: (1) the monomer was optimized; (2) several possi-ilities for the dimer structure were tested according to Fig. 4;3) for each dimer structure a short molecular dynamics (3 ps) atigh temperature (700 K) was run to identify local minima; (4)nally, a full minimization with CVFF force field was done forach minimum. Seven structures were found that correspond toocal minima.

The most stable dimer among these minima is shown in Fig. 5,he comparison with the structure obtained using MM3 forceeld is also displayed. From Fig. 5 one can conclude that theimer is stabilized by two O–H· · ·O hydrogen bonds formedetween by –C=O and –C–OH groups. Also, it is observedhat the relative positions of the functional groups implied inhe hydrogen bond formation are equivalent for both methods.he interaction energy for this minimum was obtained at theLYP/DZVP level of theory including the basis set superposi-

ion error (BSSE) correction. This calculation was performedsing single point calculations in the geometries obtained byhe CVFF method for the dimer and monomer. The calculatedinding energy of the dimer is −13.5 kcal/mol, which clearlyndicates the formation of two O–H· · ·O hydrogen bonds [28].

bilizing hydrogen bonds named 1 and 2 in Fig. 6, of the dimer H4L2. This figuremolecules. The horizontal dashed lines show the standard limits for hydrogen1 and (d–f) are related to hydrogen bond 2.

.3. The solvent effect on the H4L2 dimer formation

The experimental evidence indicates that the dimer is stablen aqueous media. As the molecular mechanics results show, the

1066 P. Balderas-Hernandez et al. / Talanta 71 (2007) 1061–1067

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ig. 8. Schematic representation of typical structures for the H4L2 dimer obsorrespond to regions where one or the two hydrogen bonds are broken. In thisroup identification.

imer is also stable in vacuum. In order to study the dynamicaltability of the dimer in aqueous media, a molecular dynamicsas performed according to what is described in the method-logy section. A typical low energy structure, taken from theolecular dynamics results obtained for the dimer in a layer ofater molecules, was fully relaxed to reach a minimum with

he CVFF force field. This relaxation includes all the waterolecules in the layer; Fig. 6 shows three views of the molecule.

t can be seen, by comparing Figs. 5 and 6, that this minimum,hich is representative of the structure in the presence of the sol-ent, is similar to those obtained in vacuum. What is remarkablen Fig. 6 is the “symmetry” of the dimer: the functional groupsesponsible for the hydrogen bond formation are in equivalentositions in both molecules. Indeed, this “symmetry” implieshat equivalent chemical environments surround both hydrogenonds. This is in agreement with NMR experiments.

A dynamical picture of the hydrogen bonds in the dimer cane extracted from Figs. 7 and 8. These figures show the geomet-ical fluctuations in the hydrogen bond moiety. From Fig. 7, ones able to see that during the simulation there are periods in whichoth hydrogen bonds are formed, at approximately 140 ps; dur-ng some time only one of the hydrogen bonds is formed, see forxample the zone around 160 ps. Also, there is one area, closeo 170 ps in which both hydrogen bonds are lost. During theimulation it is seen that some molecules of the solvent formydrogen bonds with the active groups of the dimer.

The representative structures of these interactions are dis-layed in Fig. 8; structures A, B, and E represent the situationsn which the dimer is stabilized by at least one hydrogen bondithout intrusion of water molecules. The structures C, D, I–L

orrespond to bridge situations where a water molecule formydrogen bonds with both monomers; sometimes these interac-ions involve a position of the water molecule in which it is actings a double donor. Also, some structures show that the presence

f water breaks the hydrogen bonds that stabilize the dimer (C,, K, and L). The water molecules also interact with the sul-honic group as it is indicated in structures G and H. Finally,t is possible to identify the structures such as F, in which both

during the simulation in the presence of solvent at 298 K. These structurese the bold angular solid line indicates the water molecule; see Fig. 5 for other

ydrogen bonds are broken. The above discussion shows theariety of hydrogen bonds interactions of the dimer in the sol-ent and that the dimer is stable for most of the time, which isn agreement with the experimental evidence.

. Conclusions

The comparison of CVFF with MM3 force field indicateshat the former represents appropriately the structure of the rel-vant hydrogen bond environments for TB molecule and dimer.owever, MM3 force field gives a better-detailed descriptionith respect to LDA structure, particularly in the weak hydro-en bonds environments.

In relation to the TB monomers, it can be concluded that,mong the species having the possibility of forming dimers, theost stable in vacuum corresponds to structure IV depicted inig. 2. Nevertheless this structure is not able to form a dimer

n aqueous media. In contrast, the structure II forms a sta-le dimer in vacuum and aqueous media. The dimer foundn our theoretical study generates symmetric hydrogen bondnvironments, which is in agreement with experimental facts.herefore, according to our results, the stable dimer proposedy experimental data is of the II–II form. In vacuum, the dimer-zation energy calculated by ab initio methods corresponds towo O–H· · ·O hydrogen bonds.

cknowledgments

This research was partially supported by C01-39621 and6482-E CONACYT projects. PB acknowledges CONACYTor a PhD scholarship.

eferences

[1] Y. Wei, K. Li, S. Tong, Anal. Chim. Acta 97 (1997) 341.[2] J. Eley, G. Halbert, A. Florence, J. Pharm. Pharmacol. 41 (1989) 858.[3] R. Maggini, F. Secco, M. Venturini, J. Phys. Chem. A 101 (1997) 5666.[4] R. Andres, R. Narayanaswamy, Talanta 44 (1997) 1335.

. / Tal

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[5] N. Acar, F. Onur, Anal. Lett. 29 (1996) 763.[6] K. Chuang, D. Fung, Proceedings of the IFT Annual Meeting E.U., 1995,

p. 253.[7] P. Balderas-Hernandez, M.T. Ramırez, A. Rojas-Hernandez, A. Gutierrez,

Talanta 46 (1998) 1439;P. Balderas-Hernandez, A. Rojas-Hernandez, M. Galvan, M.T. Ramırez-Silva, Spectrochim. Acta A 60 (2004) 569.

[8] I.R. Gould, P.A. Kollman, J. Am. Chem. Soc. 116 (1994) 2493.[9] W. Han, S.J. Suhai, J. Phys. Chem. 100 (1996) 3942.10] S.S. Xantheas, J. Chem. Phys. 102 (1995) 4505.11] R. Vargas, J. Garza, R.A. Friesner, H. Stern, B.P. Hay, D.A. Dixon, J. Phys.

Chem. A 105 (2001) 4963.12] J. Sponer, J. Leszczynsky, P. Hobza, J. Phys. Chem. 100 (1996) 1995.13] I.M. Kolthoff, M.K. Chantooni, S. Bhowmik, Anal. Chem. 39 (1967)

315.14] MM3 (96) may be obtained from Tripos Associates, 1699 S. Hanley Road,

St. Louis, MO 63144, for commercial users, and it may be obtained fromthe Quantum Chemistry Program Exchange, Mr. Richards Counts, QCPE,Indiana University, Bloomington, IN 47405, for noncommercial users.

15] R. Vargas, J. Garza, D.A. Dixon, B.P. Hay, J. Phys. Chem. A 104 (2000)5115.

[

[

anta 71 (2007) 1061–1067 1067

16] R. Vargas, J. Garza, D.A. Dixon, B.P. Hay, J. Am. Chem. Soc. 122 (2000)4750.

17] S. Shceiner, T. Kar, Y.J. Gu, Biol. Chem. 272 (2001) 9832.18] W. Bing, J.F. Hinton, P.J. Pulay, J. Phys. Chem. A 107 (2003) 4683.19] R. Vargas, J. Garza, D.A. Dixon, B.P. Hay, J. Mol. Struct. (Theochem.) 541

(2001) 243.20] Discover version 4.0.0, msi, San Diego, CA, USA.21] S.H. Vosko, L. Wilk, M. Nusair, Can. J. Phys. 58 (1980) 1200.22] N. Godbout, D.R. Salahub, J. Andzelm, E. Wimmer, Can. J. Chem. 70

(1992) 560.23] A.D. Becke, J. Chem. Phys. 98 (1993) 5648.24] A.D. Becke, J. Chem. Phys. 98 (1993) 1372.25] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 78.26] D.E. Bernholdt, E. Apra, H.A. Fruchtl, M.F. Guest, R.J. Harrison, R.

Kendall, R.A. Dutteh, X. Long, J.B. Nicholas, J.A. Nichols, H.L. Taylor,A.T. Wong, G. Fann, R.J. Littlefield, J. Nieplocha, Int. J. Quantum Chem.

Symp. 29 (1995) 475.

27] U. Salzner, J.B. Lagowski, P.G. Pickup, R.A. Poirier, J. Comp. Chem. 18(1997) 1943.

28] G.A. Jeffrey, An Introduction to Hydrogen Bonding, Oxford UniversityPress, New York, 1997, p. 12.