Journal of Colloid and Interface Science 392 (2013) 281–287

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Journal of Colloid and Interface Science

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23Na and 35/37Cl as NMR probes of growth and shape of sodium taurodeoxycholatemicellar aggregates in the presence of NaCl

Fioretta Asaro a,⇑, Luigi Feruglio a, Luciano Galantini b, Alessia Nardelli a

a Department of Chemical and Pharmaceutical Sciences, University of Trieste, via L. Giorgieri 1, 34127 Trieste, Italyb Department of Chemistry, University of Rome ‘‘La Sapienza’’, P. le A. Moro 5, 00185 Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 July 2012Accepted 20 September 2012Available online 13 October 2012

Keywords:T1

DQFQuadrupole splittingBile saltsNaClNaBrAnisometry

0021-9797/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jcis.2012.09.045

⇑ Corresponding author. Fax: +39 0405583903.E-mail addresses: fasaro@units.it (F. Asaro), luigi.fe

l.galantini@caspur.it (L. Galantini), giallotweety@liber

The growth of the aggregates of the dihydroxylated bile salt sodium taurodeoxycholate (NaTDC) uponNaCl addition and the involvement of the counterion were investigated by NMR spectroscopy of mono-atomic ionic species. 23Na T1 values from 0.015, 0.100, and 0.200 mol kg�1 NaTDC solutions in D2O, at var-iable NaCl content, proved to be sensitive to the transition from primary to secondary aggregates, whichoccurs in the former sample, and to intermicellar interaction. Some 79Br NMR measurements were per-formed on a 0.100 mol kg�1 NaTDC sample added by NaBr in place of NaCl for comparison purposes. The23Na, 35Cl, and 37Cl double quantum filtered (DQF) patterns, from the 0.100 mol kg�1 NaTDC sample, and23Na ones also from the 0.200 mol kg�1 NaTDC one, in the presence of 0.750 mol kg�1 NaCl, are a clearmanifestation of motional anisotropy. Moreover, the DQF spectra of 23Na and 37Cl, which possess closequadrupole moments, display a striking similarity. The DQF lineshapes were simulated exploiting the Sci-lab environment to obtain an estimate of the residual quadrupole splitting magnitude. These results sup-port the description of NaTDC micelles as cylindrical aggregates, strongly interacting at high ionicstrengths, and capable of association with added electrolytes.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Bile salts are a class of anionic amphiphiles of natural origincharacterized by a rigid steroidal nucleus, which bears a few OHgroups and a short lateral chain ending with a charged head-group.The convex side of the condensed ring system, termed b, with twomethyl groups, corresponds to the polar moiety. The sickle shapeendows the molecules with three axial chirality [1].

They are involved in many physiologically and pathologicallyrelevant processes [2–6] in vivo and the biomedical significancedrove the intense research of the early times, the second half of lastcentury. At present, the interest toward such compounds is notfading, stimulated by the increasing technological relevance andtheir use [7–13], also as semisynthetic derivatives [14–17], in thematerials of future.

The peculiar self-aggregation behavior of these molecules,which remarkably differs from that of common surfactants, playsa paramount role in the practical application. It is phenomenolog-ically well characterized, owing to the many investigations, carriedout by a broad array of techniques. The correspondingly copiousliterature has been summarized in various reviews [2,18], alsorecently [19,20]. In spite of this, the supramolecular arrangement

ll rights reserved.

ruglio@yahoo.it (L. Feruglio),o.it (A. Nardelli).

in bile salts micelles remains not fully understood. It is describedby means of various widely different models, which imply corre-spondingly different morphologies. The most popular, and com-monly used, ones [20] range from the first model, proposed bySmall, of elongated secondary micelles [21] to those of disklikeaggregates [22] and of elongated helical geometry [23]. Theoreticalmethods, as well, were invoked in order to get insight into the sub-tle interplay of polar and hydrophobic interactions and hydrogenbonds, certainly quite different than in micelles of classical surfac-tants, ruling the micellar structure. The molecular dynamics calcu-lation outcomes are in line with the Small model and suggest anirregular morphology [24]. However, owing to the high complexityand delicacy of the matter, the comparison with reliable experi-mental results of micellar morphology is mandatory. Here, weaim at providing experimental information on the morphology ofbile salts assemblies, in particular at high ionic strength, at whichthe aggregates are rather large, from a new, unexplored point ofview, namely the one of the sodium counterion and of the co-ionsof the added electrolyte, in order to improve the structural model-lization of these peculiar self-assemblies.

Indeed, NMR spectroscopy of the nuclei of sodium and of ha-lides, conventional counterions, has frequently been employed inthe study of structure and dynamics of micellar and biopolyelec-trolytes systems [25–27]. Besides relaxation times, we have em-ployed double quantum filtered (DQF) experiments, suited to soft

282 F. Asaro et al. / Journal of Colloid and Interface Science 392 (2013) 281–287

matter [28–32], because they are capable of enlightening in astraightforward manner, just through the spectral pattern, slowmotions’ contributions to relaxation [33,34] and, even unresolved,quadrupolar splitting [28]. The latter is a distinctive feature ofslight motional anisotropy in incompletely disordered systems.

Sodium taurodeoxycholate (NaTDC), considered in the presentstudy, belongs to the class of dihydroxylated bile salts, well knownto give rise to aggregates that grow to a great extent upon increas-ing both concentration and medium’s ionic strength [35–37].

2. Experimental section

2.1. Preparation of samples

NaTDC purchased by Sigma was crystallized twice from a mix-ture of water and acetone. D2O 99.9% (CIL) was employed assolvent.

Three solutions, at different NaTDC concentration, namely0.015, 0.100 and 0.200 mol kg�1, were prepared by weight. TheNaCl concentration was increased by adding the appropriateamounts of solid NaCl. A 0.100 mol kg�1 NaTDC solution was stud-ied also in the presence of NaBr instead of NaCl.

2.2. NMR measurements

The NMR measurements were performed, at 303 K, on a JEOLEclipse 400 spectrometer (9.4 T) operating at 400 MHz for 1H and105.75 MHz for 23Na, 39.17 MHz for 35Cl, 32.60 MHz for 37Cl and100.16 MHz for 79Br, equipped with a JEOL NM-EVTS3 variabletemperature unit.

The 23Na longitudinal relaxation times, T1, were determined byinversion recovery using 19 intervals, accumulating 64 transientsfor each interval, employing a spectral width of 600 Hz over 1 Kcomplex data points.

The 79Br transverse relaxation rates (R2 = 1/T2) were determinedfrom the line-width at half-height (Dm1/2) of the signals present inthe spectra, acquired using a spectral width of 30 kHz over 8192complex data points, from the very same solution used for the23Na R1 measurements, by the relation R2 = pDm1/2, since the mag-netic field inhomogeneity contribution to line-width is negligiblefor 79Br.

The 23Na double quantum filtered (DQF) spectra were per-formed on two samples, containing NaTDC at 0.100 and0.200 mol kg�1 concentration, respectively, and 0.750 mol kg�1

NaCl, using the pulse sequence reported in the literature [34]employing two p/2 pulses for the filter. 288 transients for each sinterval were accumulated, and the s values for the former samplewere 3.2,6,20, and 60 ms along with a spectral width of 600 Hzover 1 K complex data points, while for the latter, they were1.6,3.2 and 6 ms, and the spectral width 2644 Hz over 8 K datapoints.

For chlorine nuclei, the DQF experiment was carried out on theformer sample employing a spectral width of 600 Hz and 1 K com-plex data points, with 128 scans and s values of 2,2.9,4.2, and10.8 ms for 35Cl and with 256 scans and s values of 3,5,7, and19 ms for 37Cl.

The DQF can be briefly and efficaciously described, making refer-ence to the expansion of the density operator in terms of irreducibletensor operators [33,34,38,39] and to the Redfield theory of relaxa-tion [40]. Accordingly, the initial p/2 pulse converts the equilibriumlongitudinal magnetization into single quantum coherences (SQCs)of the first rank. During the following s interval, they evolvepartially without changing rank, according to either f11 or f011 coef-ficient [39], and partially are converted into SQCs of higher rank.SQC of the third rank may be originated for 3/2 spin quantum

number nuclei, like those here considered, by both biexponentialtransverse relaxation and residual quadrupolar splitting, f31 or f031

[39], while SQCs of the second rank are exclusively due to the pres-ence of residual quadrupolar splitting, f21 or f021 [39]. The analyticalforms of the relevant coefficients depend on the relative magnitudeof the residual quadrupolar splitting, xQ, and of the spectral densityat twice the Larmor frequency, J(2x0), namely [39]:

If J(2x0) < xQ

F11ðtÞ ¼35

cosðxeff tÞ þ Jð2x0Þxeff

sinðxeff tÞ� �

expð�R2f tÞ þ 25

� expð�R2s tÞ ð1Þ

f21ðtÞ ¼ f12ðtÞ ¼ i

ffiffiffi35

rxQ

xeffsinðxeff tÞ expð�R2f tÞ ð2Þ

f31ðtÞ ¼ f13ðtÞ

¼ffiffiffi6p

5cosðxeff tÞ þ Jð2x0Þ

xeffsinðxeff tÞ

� �expð�R2f tÞ

�ffiffiffi6p

5expð�R2s tÞ ð3Þ

where xeff ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffix2

Q � Jð2x0Þ2q

On the contrary, if J(2x0) > xQ

f 011ðtÞ ¼35

coshðleff tÞ þ Jð2x0Þleff

sinhðleff tÞ" #

expð�R2f tÞ þ 25

� expð�R2s tÞ ð4Þ

f 021ðtÞ ¼ f 012ðtÞ ¼ �i

ffiffiffi35

rxQ

leffsinhðleff tÞ expð�R2f tÞ ð5Þ

f 031ðtÞ ¼ f 013ðtÞ

¼ffiffiffi6p

5½coshðleff tÞ þ Jð2x0Þ

leffsinhðleff tÞ� expð�R2f tÞ

�ffiffiffi6p

5expð�R2s tÞ ð6Þ

with leff ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiJð2x0Þ2 �x2

Q

qThe two transverse relaxation rates, R2, denoted by the sub-

scripts f and s, suggestive of fast and slow, can be expressed bythe values taken at various frequencies, namely 0,x0 and 2x0, ofthe spectral density function, J, of the motional processes involvedin relaxation.

R2f ¼ Jð0Þ þ Jðx0Þ þ Jð2x0Þ ð7Þ

R2s ¼ Jðx0Þ þ Jð2x0Þ ð8Þ

The p pulse at the middle of s interval refocuses chemical shifteffects. At the end of s, the double quantum filter (DQF), made oftwo pulses coupled with the opportune phase cycle, blocks theSQCs of the first rank letting through those of second and thirdranks. During the following acquisition period, the higher rankSQCs evolve back into first rank ones, the very NMR observable,according to coefficients f21 and f31. The eventual signal in the fre-quency domain is the Fourier transform of the sum of f12 and f13 intime domain, weighed by the factors acquired due to evolutionduring the s interval.

2.3. Fitting of DQF spectra

The DQF spectral patterns were calculated according to Eqs. (2),(3) and (5), (6), Fourier Transformed to the frequency domain andweighted by intensity factors due to evolution during s, which

F. Asaro et al. / Journal of Colloid and Interface Science 392 (2013) 281–287 283

were determined by means of the same formulas [39]. AGaussian distribution was considered for the quadrupole splitting(mQ, which corresponds to xQ/(2p)). The calculated data were fittedto the experimental one by non-linear least squares minimizationcarried out by the function leastsq of the software Scilab 5.3.3 [41].The relevant Scilab script is reported in SupplementaryInformation.

3. Results

3.1. NMR relaxation measurements

Fig. 1 displays the 23Na longitudinal relaxation rates (R1 = 1/T1)measured both from NaTDC and from sodium decylsulfate solu-tions in D2O at various surfactant concentrations. Sodium decylsul-fate is a common anionic surfactant useful for comparison. In thesimplifying ‘‘two site approach’’ for sodium, that is, the sodium cat-ion in fast exchange between two limit situations, labelled as‘‘free’’ and ‘‘bound’’, the NMR parameters are weighted averagesof the values relevant to the two situation, and specifically for R1

[25]:

R1 ¼ R1free þ XboundðR1bound � R1freeÞ ð9Þ

where R1free and R1bound are the R1 values in the two sites, that is,bulk water and close proximity to aggregates.

For conventional micellar solutions, the plot of R1 data vs. in-verse surfactant concentration ðC�1

0 Þ is a straight line [25], due tothe constancy of the free surfactant concentration, which is equalto the critical micellar concentration (CMC). Thus, we plotted R1

data vs. C�10 to better appreciate the difference in behavior between

the two kind of molecules. Indeed, the 23Na R1 data above the CMCof sodium decylsulfate lie on a straight line. The CMC value of0.039 mol kg�1, in agreement with literature data [26,42,43], wasobtained from the parameters of the regression line, takingR1free = 18.8 s�1, value measured in most diluted (5.5 � 10�3

mol kg�1) examined sodium decylsulfate solution.On the contrary, the R1 values for NaTDC were slightly higher

than that of free sodium already at very high dilution, in line withthe extremely low critical aggregation concentration of bile salts.Then, they increased steadily and smoothly, without exhibitingeither any clear linear trend or any slope discontinuity. Thus,23Na NMR indicates a progressive growth of the aggregates; how-ever, in pure bile salts solutions, it is not suited to evidence thetransition from ‘‘primary’’ to ‘‘secondary’’ micelles, which is betterrevealed by other techniques, for example, by the abrupt change ofslope occurring at about 0.040 M in the electric dipole moment, perNaTDC monomer, plot vs. NaTDC concentration [44].

18.8

23.8

28.8

33.8

38.8

0 10 20 30 40 50 60 70 80 90 100

23N

a R

1(s

-1)

C0-1 (kg mol-1)

Fig. 1. 23Na R1 data plotted vs. inverse amphiphile concentration for: NaTDC(triangles) and sodium decylsulfate (circles). The straight line corresponds to thelinear regression of the R1 data at sodium decylsulfate concentrations above theCMC.

The growth induced by NaCl was followed by 23Na R1 measure-ments on three different samples: the former of ‘‘primary’’ micelles(0.015 mol kg�1 NaTDC) and the latter two of ‘‘secondary’’ micelles,containing 0.100 and 0.200 mol kg�1 NaTDC, respectively.

The trend of R1 with NaCl concentration is not monotone (Fig. 2)for the 0.015 mol kg�1 NaTDC solution. The negative slope of thesegments at low and high NaCl concentrations can be rationalizedwithin the two-site frame, that is, sodium partitioned betweenbulk and aggregates, by postulating that the aggregates presentat low and high NaCl content are somewhat different, under the re-spect of R1bound and amount of bound sodium. It may be supposedthat the pristine NaTDC micelles, ‘‘primary’’, are capable to bind so-dium only weakly, while at higher NaCl content, the aggregates arelarger and able to bind sodium more efficiently. The positive slopeat intermediate NaCl concentrations, on the contrary, cannot be ac-counted for by coexistence of only two sodium species; therefore,both kinds of aggregates, with relative populations dependent onNaCl concentration, must be contemporarily present along withfree sodium.

For the 0.100 mol kg�1 NaTDC sample, a nonmonotonic trendcould be envisaged, as well, with the minimum occurring at higherNaCl content than in the former case. In the frame of the two-siteapproach, it may be explained by two kinds of secondary micellesin addition to free sodium. On the contrary, the trend for the mostconcentrated NaTDC sample, 0.200 mol kg�1, is monotone.Although it must be considered that a lower number of pointsalong the NaCl concentration coordinate was sampled, probablyjust the latter aggregates were present on the first NaCl addition,so that the two-site approach again holds.

This hypothesis can be proved by recording on the very samesample also the relaxation of another nucleus that resides predom-inantly in bulk solution, specifically that of the anion of added salt.For this reason, NaBr was added, instead of NaCl, to a0.100 mol kg�1 NaTDC sample. Bromine isotopes, both possessingI = 3/2, are well suited to NMR measurements due to the highermagnetogyric ratios. Moreover, 79Br is a more sensitive magnifierof static and time-modulated changes of the quadrupole couplingthan 81Br, owing to the stronger nuclear quadrupole moment[45]. Indeed, in a 0.2 mol kg�1 NaBr solution in D2O, R2, which isequal to R1, holding the extreme narrowing regime, is 1880 s�1,that is, one hundred of times higher than 23Na R1, 18 s�1. Such a ra-tio between 23Na and 79Br R1s is maintained in a simple solventupon viscosity changes, because the correlation times of the mo-tions determining relaxation of the two nuclei are equally affected.Interestingly, the 79Br R2 values of our sample increased monoton-ically upon increasing NaBr concentration (Fig. 3), supporting the

1819202122232425262728

0 0.2 0.4 0.6 0.8

23N

a R

1(s

-1)

NaCl concentration (mol kg-1)

Fig. 2. 23Na R1 data plotted vs. added NaCl concentration for NaTDC samples atconcentrations: 0.015 (diamonds), 0.100 (circles), and 0.200 (triangles) mol kg�1.The lines were drawn taking into account the two-site approach as described in thetext.

2500

3000

3500

4000

4500

22.5

23.0

23.5

24.0

24.5

0 0.5 1

79Br R2 (s -1)23

Na

R1

(s-1

)

NaBr concentration (mol kg-1)

Fig. 3. 23Na R1 data (filled square) and 79Br R2 data (empty squares) plotted vs.added NaBr concentration. The line was drawn analogously to Fig. 2.

284 F. Asaro et al. / Journal of Colloid and Interface Science 392 (2013) 281–287

view that the peculiar behavior of 23Na R1 was not caused by sol-vent viscosity variations, but rather it was due to the co-presenceof NaTDC aggregates differing as far as counterion associationand R1bound value were concerned. Moreover, the 79Br R2 magni-tude, much higher than in the NaBr solution and increasing withNaBr concentration, would suggest an interaction of the co-ionwith the aggregates, rather than its exclusive localization in thebulk. The change of slope in the plot of 79Br R2 data vs. NaBr con-centration taking place soon before the minimum in the 23Na R1

trend suggests that the interaction of the halide with the aggre-gates depends on the kind of micelle. The 23Na R1 trend is slightlydifferent than in the presence of NaCl (not widely different: inFig. 3, the 23Na R1 range is smaller than in Fig. 2). This also pointsto an association of the co-ion with the aggregates.

3.2. DQF NMR spectra

Spin 3/2 nuclei actually exhibit two relaxation rates for bothlongitudinal and transverse relaxation [46]. Under extreme nar-rowing conditions, that is, when the motional processes responsi-ble for relaxation take place on a timescale faster than the Larmorfrequency, the rates are equal and one observes monoexponentialdecays. On the contrary, they differ when slow motions participateto the modulation of quadrupole coupling. However, even in thelatter case, it is not usual to appreciate the biexponentiality ofthe longitudinal magnetization decay, because neither relaxationrate depends on very slow motions. Instead, it is more commonto observe biexponential decays for transverse magnetization be-cause one of the two relaxation rates, R2f, is affected by slow mo-tions [31,38]. Nevertheless, it is not always easy to fit the echodecay by means of two exponential functions or the frequency do-main signal by means of the sum of two Lorentzians possessing dif-ferent line-width and intensity. The most convenient method toreveal the biexponential decay consists in the use of multiplequantum filters [38]. In addition, it is able to reveal quadrupolarsplittings, diagnostic signatures of anisotropic motion, even whenthey are smaller than the line-width [28]. The DQF experimentswere not performed on 79Br because they are hampered by thehigh quadrupole moment [45], which, inducing very fast relaxa-tion, requires extremely small s values, on the order of pulselengths, and leads to very broad spectra.

The DQF measurements were successfully carried out on 0.100and 0.200 mol kg�1 NaTDC samples added by 0.750 mol kg�1 NaClthat insures the presence of well grown aggregates [44]. The spec-tral patterns, reported in Figs. 4 and 5 and S1–S2 of Supplementary

information, clearly revealed the simultaneous presence of SQCcoherences of second and third rank.

Indeed, they recall those observed for 23Na in cartilage, whichwere accounted for in terms of local order in that kind of tissues[28,29]. Thus, both samples presented a residual quadrupolar split-ting. It goes completely overlooked in the conventional 23Na NMRspectrum of the 0.100 mol kg�1 NaTDC sample (Fig. S5), whichcould be satisfactorily reproduced by the sum of two Lorentzianswith different line-width at half-height (Dm1/2), the narrower rele-vant to the central transition and the broader to the satellites(Fig. S5). On the contrary, the 1D 23Na NMR spectrum of the moreconcentrated sample, at a careful inspection, shows a broad hump,due to the satellites transitions, at the foot of the central one. Thefitting of the signal by means of three Lorentzian lines returned amQ value of 79 Hz (Fig. S6). However, the simulation by Lorentzianlines assigns the large width of the satellites transitions exclusivelyto high R2f. Instead, a distribution of mQ values, due to a distributionof orientations and, possibly, of residual electric field gradient val-ues, is highly probable in these samples that do not display bulkanisotropy. The fit of the 23Na DQF spectra (Figs. 4 and 5, S1 andS2) contributed in elucidating this point. We performed the fit ofthe experimental DQF results to Eqs. (2), (3) and (5), (6), makingvarious assumptions, already discussed in literature, in order tokeep the calculation as simple as possible. Namely, the spectraldensity values J(x0) and J(2x0) were set equal, thus consideringthat slow motions are exclusively affecting J(0). Indeed, for mono-atomic ions, the electric field gradient at the nuclear site is origi-nated by distribution of the nearby electric charges and electricdipoles, for example, those of hydration water molecules. Fluctua-tions in such distribution time modulate the quadrupole interac-tion and induce relaxation. In micellar systems, they occur atvarious timescales and spatial restrictions. An effective way tomodel this behavior is provided by the two step model [47,48],which represents the spectral density function as the sum of twodifferent Lorentzian functions, relevant to two processes occurringon two widely different timescales. The faster one is somewhatanisotropic, so that a residual quadrupole coupling is left for themodulation by slow motion. A Gaussian distribution was chosenfor the mQ values, in line with the appearance of the 1D spectra(Figs. S5 and S6), which look quite different from powder spectra,and because it is adequate to account for the signal shapes frompoorly ordered systems, such as biopolymer gels or tissues[29,49]. Exchange was not explicitly taken into account, assuminga fast exchange of sodium among the various sites, so that, actu-ally, the obtained parameters are mean values. These crudeapproximations together with lack of a wide array of experimentaldata prevent the exploitation of the best fit parameters (reportedcompletely in Supplementary Information) to the end of a rigorousand detailed motional analysis. Nevertheless, they provide valu-able insights about the overall behavior of bile salts micelles. Firstof all, the mean mQ values of 4 Hz and 80 Hz indicate motionalanisotropy, paying attention that, due to the contribution of freesodium, for which mQ is null, mQ of bound sodium must be signifi-cantly higher, by at least 4.75–8.5 times. The line-width of the sat-ellites is originated by the combined action of fast R2f and broad mQ

distribution, so that the Dm1/2 of the satellite resonances is not afaithful reporter of R2f.

The similarity between the DQF spectral pattern of 23Na and37Cl (Figs. 4 and S3) is really striking if one considers that 23Na sitsin the counterion of taurodeoxycholate, whereas chloride pos-sesses the same charge of the bile salt headgroup and thereforeis less prone to interaction. It indicates that both ions experiencethe same environment, and therefore, both are associated withthe micelles. This is a confirmative prove of the strong interactionof anions inferred previously from 79Br relaxation rates. Indeed, themagnitude of the quadrupole moment of 37Cl is somewhat lower

Fig. 4. Experimental (lower traces) and calculated (upper traces) DQF NMR spectra of: (from left to right): 23Na with s = 3.2 ms (red); 37Cl with s = 3 ms (green); 35Cl withs = 2.9 ms (cyan) for 0.100 NaTDC in the presence of 0.750 mol kg�1 NaCl. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.)

Fig. 5. Experimental (lower) and calculated (upper trace) 23Na DQF NMR spectrumfor 0.200 mol kg�1 NaTDC in the presence of 0.750 mol kg�1 NaCl, with s = 1.6 ms.

F. Asaro et al. / Journal of Colloid and Interface Science 392 (2013) 281–287 285

than that of 23Na, 6.435 vs. 10.4 fm2 [45], but it must be recalledthat the higher Stenheimer shielding factor and the different coor-dination of water amplifies the quadrupole coupling and its mod-ulation and the effect of the electric field gradients due to nearbycharged aggregates and molecular species in the case of the anion.Even in H2O at 25 �C, at infinite dilution the longitudinal relaxationrate, R1, is 25 s�1 for 35Cl [50] and therefore 16 s�1 for 37Cl, veryclose to that of 23Na 17.55 s�1 [50].

The different DQF pattern of 35Cl (Figs. 4 and S4) clearly evi-denced faster relaxation. The relaxation is faster for 35Cl than for37Cl because of the higher magnitude of the nuclear quadrupolemoment of the former, 8.165 fm2. The fitting of the DQF patternsof both isotopes (see Supplementary information Figs. S3 and S4),performed analogously to the 23Na case, returned values of thespectral densities for the two isotopes with ratios in line withthe ratio of the squares of the relevant quadrupole moments, inagreement with the square dependence of the relevant densityfunctions [39].

4. Discussion

The interpretation of the NMR spectroscopy results of monova-lent counterions’ nuclei in self-assembling systems is greatly aidedby the information on the size of the aggregates provided by scat-tering techniques [51]. Luckily, the growth of NaTDC micelles withionic strength had attracted the attention of previous investiga-

tors; thus, we can profit from information in the literature on sizeand tumbling rate of the NaTDC aggregates from scattering [35,52–54] and EPR techniques [53]. Quasi-elastic light scattering (QELS)revealed that the size of NaTDC aggregates increases with concen-tration and that their growth is boosted by the presence of NaCl[35,44] and enlightened the transition from small oblate micelles(primary micelles), to larger prolate micelles (secondary micelles).The aggregation number is 612 for the former and P15 for the lat-ter [44], which grow further with NaTDC concentration and associ-ate sodium to a much higher degree [55]. The better interactionwith counterion of larger micelles is in line with the good responseof 23Na NMR to the growth of the aggregates in the presence ofNaCl. The non-monotonic trend of R1 in the 0.015 mol kg�1 NaTDCsample can, thus, be safely attributed to the evolution form pri-mary to secondary micelles. The coexistence of primary and sec-ondary micelles, on the contrary, cannot be the origin of the non-monotonic trend with increasing NaCl concentration observed forthe 0.100 mol kg�1 NaTDC sample, because the starting solutionwas already containing secondary micelles. Instead, it can be ex-plained by a progressive increase in b(R1bound–R1free) (with b the so-dium association constant of self-assembled surfactant) of thesecondary micelles, which are elongating upon increasing NaClconcentration [35], at least up to 0.8 M NaCl, the highest concen-tration examined by QELS [53]. Scattering data from 0.2 M NaTDC[56] and from 0.1 M sodium glycodeoxycholate [57] solutions ledto discriminate between two growth regimes. At low NaCl concen-tration (60.2 M), the interaggregate interactions are exclusivelyrepulsive, electrostatic and excluded volume ones, while at higherconcentration, the electrostatic interactions are efficaciouslyscreened and the aggregates interact attractively due to van derWaals forces [52,56] and growth is favored. Indeed, the positiveslope of the 23Na R1 curve (Fig. 2) for 0.1 mol kg�1 NaTDC occursbetween 0.2 and 0.4 mol kg�1 NaCl, which may be recognized asthe cross over interval between the two regimes.

A further remarkable feature of these systems is the ion specificeffect of bromide on 23Na R1. The R1 data at low NaX concentrationsseem to suggest the presence of larger aggregates when NaBr isused in place of NaCl, while at the highest employed concentra-tions, the trends coincides (Fig. 3). The precocious appearance oflarger aggregates is probably the cause of the shift of the minimumto higher NaX concentration and of the reduction of its depth in thepresence of NaBr. After all, specific ion effects are well known for

286 F. Asaro et al. / Journal of Colloid and Interface Science 392 (2013) 281–287

halides even in the role of co-ion, like in the present case, for exam-ple, in the transition coil–helix of j-carrageenan [58], a negativelycharged biopolymer. The uptake of NaCl by the aggregates was al-ready invoked to explain the values of specific conductance mea-sured in, NaCl containing, NaTDC solutions [59,60]. The DQFsignals of 37Cl and 35Cl are a clear proof of this.

The 23Na, 37Cl and 35Cl DQF spectral patterns are a patent man-ifestation of motional anisotropy at high NaCl content. Residualquadrupolar splitting can be observed when quadrupolar mono-atomic ions are interacting with nonspherical objects, the freetumbling of which is prevented by the hindrance provided by thesurrounding like objects. However, the residual quadrupolar split-ting does not provide the kind of shape, whether rodlike or disk-like. The cylindrical shape for NaTDC aggregates in the presenceof NaCl was proposed on the basis of QELS measurements[35,52,56,57,61] and eventually assessed by the joint use of DLSand SAXS data [54]. Consistently, the 23Na mQ value is higher forthe more concentrated NaTDC sample, where the micelles arelonger and the intermicellar interactions stronger. The mQ distribu-tion is rather broad, in line with micellar polydispersity [52]. Theoccurrence of a distribution for the mQ values allowed to realizethat the width of the satellites transition is not entirely due toR2f. R2f relaxation rate is sensitive to slow motions, which, in micel-lar systems, modulate the quadrupole coupling survived to averag-ing by fast motions. In these systems, the slow motional processesmay be the tumbling of the whole aggregates, the sodium diffusionabout the charged surface, but also the exchange between micellarsites and bulk water, or a combination of them [62,63]. The R2f val-ues inferred from the DQF experiments suggest that 23Na nucleusdoes not experience as long correlation times as the whole aggre-gates, which are on the order of ls, according to the very largeprincipal values of the rotational diffusion tensor determined byESR spectroscopy of spin labelled cholestane, firmly embedded inthe aggregates, from 0.1 M NaTDC solutions containing sizeableNaCl concentrations [53]. Thus, sodium residence time in the mi-celles might contribute determinantly to the effective correlationtime ruling R2f, analogously, for example, to the case of sodiumtrapped in the minor groove of DNA [64]. The R2f values for 35Cland 37Cl are on the same order of magnitude and probably thesame slow motions the same as for 23Na R2f. Further investigationsare required to clarify this interesting point.

The results reported in this work afford information on theunconventional aggregation behavior of bile salts. In particular,they show important interactions between the TDC� aggregatesand the ions in solution and underline the anisometry of thegrowth induced by electrolytes.

5. Conclusions

The 23Na nucleus, and more interestingly the 37Cl and 35Cl ones,proved to be convenient NMR probes to study NaTDC systems,especially the micellar growth induced by the addition of electro-lytes, and the micellar shape. In particular, the DQF experimentswere very informative. Their spectral patterns are diagnostic of aresidual quadrupolar splitting, due to micellar assemblies under-going somewhat anisotropic motion, consistent with the cylindri-cal shape, previously inferred by means of scatteringmeasurements [35,52,54,56,57,61]. This is in line with the elon-gated micelles of the Small and helical models, while isotropicand irregular morphologies should be discarded. Furthermore,the results reported above stress not only the important roleplayed by the counterion in taurodeoxycholate self-assemblies,suggested by theoretical calculations [65], but also reveal a newpeculiar characteristics of these surfactants, namely the excep-tional interaction with co-ions, much higher, for instance, than that

found for dodecylsulfate in lyotropic liquid crystals [66]. The cylin-drical shape and the ability to interact as strongly with the cationsand the anions of the added electrolyte fit well to the helical model[23]. According to it, the taurodeoxycholate ions are arranged intrimers, which pile up as the aggregate grows. The inclusion ofNaX might be possible in the helix interior, which contains the in-ward directed, taurine side chains, the Na+ counterions, and water[60]. On the other hand, also the interaction of halides with theouter surface of the helix, which bears nonpolar groups [23], mayaccount for their fast relaxation, being the relaxation of large polar-izable ions dominated by the closeness to hydrophobic groups[67–69]. The helical model assumes that the supramoleculararrangement of the solid state and maintained along the sequenceof transitions: crystal – fiber (also from NaCl containing solutions)– fibril-lyotropic liquid crystal (NaTDC 40 wt%), as testified X-raydiffraction patterns [70], still holds upon further dilution to geland secondary micelles, as suggested by the coincidence of Rb+

coordination patterns in solid and in micelles signaled by EXAFS,in the case of substitution of rubidium for sodium [71].

It also appears that NMR spectroscopy of the quadrupolar nucleiof monoatomic ions may contribute, as a simple and not invasiveinvestigation tool, to the development of nanotechnologies thatemploy bile salts [8–10] and their derivatives [14–17]. It may beapplied, for instance, to monitoring the self-assembly changesdue to the interaction with carbon nanotubes and to the assess-ment of the role of sodium counterion in catanionic nanotubeswith tunable charge from cholate derivatives, in the presence ofan excess of the anionic partner. The DQF experiment may resultparticularly advantageous in systems like the above mentioned,which contain objects with high aspect ratio.

Acknowledgment

Italian MIUR (PRIN_2006039789_005) is gratefully acknowl-edged for financial support.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jcis.2012.09.045.

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