Journal of Colloid and Interface Science 357 (2011) 434–439

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Journal of Colloid and Interface Science

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Effect of sodium salicylate, sodium oxalate, and sodium chloride on themicellization and adsorption of sodium deoxycholate in aqueous solutions

Sujit Das, Jahar Dey, Teiborlang Mukhim, Kochi Ismail ⇑Department of Chemistry, North Eastern Hill University, NEHU Campus, Shillong 793 022, India

a r t i c l e i n f o

Article history:Received 21 November 2010Accepted 9 February 2011Available online 15 February 2011

Keywords:Sodium deoxycholateSalicylateOxalateCritical micelle concentrationCounterion binding constantSurface excess

0021-9797/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.jcis.2011.02.020

⇑ Corresponding author. Fax: +91 364 2550486.E-mail addresses: kismail@nehu.ac.in, kinehu@hot

a b s t r a c t

The salicylate ion increases the rate of bile flow (choleretic effect) and bile salts are known to affect thecolonic absorption of oxalate. Owing to this physiological relevance of salicylate and oxalate ions, criticalmicelle concentration (cmc) values of sodium deoxycholate (NaDC) were determined in aqueous sodiumoxalate, sodium salicylate, and sodium chloride solutions by using surface tension, fluorescence, and EMFmethods. The results indicate, besides a counterion effect, the influence of coanions on the cmc. In therange from 25 to 40 �C, cmc increases almost linearly with temperature. In the temperature range from30 to 40 �C, the counterion binding constant b of NaDC micelles has the same value (0.17 ± 0.01) in thepresence of sodium chloride and sodium salicylate. On the other hand, in sodium oxalate solutionb = 0.05 ± 0.02 when oxalate concentration is less than or equal to c� and b = 0.48 ± 0.04 above c�, wherec� � 0.038 mol kg�1. EMF measurements also supported this type of counterion binding to NaDC micellesin sodium oxalate solutions. In sodium oxalate solution, at c� a change in the shape of deoxycholatemicelles is expected to take place. Salicylate, oxalate, and chloride coanions have a similar effect onthe adsorption of NaDC. This study reveals that the choleretic effect of salicylate is not due to the influ-ence of salicylate ions on the micellization of NaDC.

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1. Introduction

Bile salts, which belong to the category of biosurfactants of an-ionic type, perform important physiological functions during thedigestion of food. The physiological functions of bile salts are re-lated to their ability to aggregate. The molecular structure of bilesalts, which have a rigid framework due to a steroid nucleus,is quite different from the conventional head-and-tail typesurfactants. The convex side of the steroid ring system acts as ahydrophobic surface, while the other side of the steroid ring sys-tem is concave and possesses hydrophilicity due to hydroxylgroups (Fig. 1). Owing to such a molecular structure, aggregationproperties of bile salts are different from those of conventional sur-factants. Bile salts form primary aggregates with a small number ofmonomers, wherein the hydrophobic surfaces face each other andthe hydrophilic groups (hydroxyl and carboxylate) point outwardtoward the solvent (Fig. 1). Bile salts are therefore called facialamphiphiles [1] and their aggregates may be called facial micelles.On increasing the bile salt concentration primary aggregates formsecondary aggregates of bigger size (Fig. 1). Accordingly, twocritical micelle concentrations (cmc) are reported for bile salts:first cmc and second cmc.

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mail.com (K. Ismail).

Electrolytes are known to affect the adsorption and aggregationbehaviors of ionic surfactants and this effect of electrolytes ismainly controlled by the influence of its counterions. Nevertheless,coions from the added electrolyte, if they have sufficient hydro-phobicity in their structure, may also influence the adsorptionand aggregation of ionic surfactants. In the case of bile salts, the ef-fect of NaCl on their micellization has been studied extensively andregarding the effect of other electrolytes we could find only threereports [2–4]. Shilnikov et al. [2] made density, ultrasound velocity,and NMR studies of sodium deoxycholate (NaDC) in the presenceof sodium salts of homologous fatty acids (C3–C18), which indicatedthat carboxylate ions of chain length 6C9 do not interact withdeoxycholate micelles, while those of chain length >C9 formedmixed micelles with NaDC. In the presence of these sodium saltsof fatty acids, cmc values of NaDC were, however, not reported[2]. Sesta et al. [3] measured density, viscosity, and microwavedielectric constant of NaDC in the postmicellar concentrationregion in the presence of sodium bromide, cesium bromide, andtetrabutylammonium bromide, and reported only the effect ofthese salts on the second cmc of NaDC. Jana and Moulik [4]reported the interaction of dodecyl sulfate anion with anions ofdifferent bile salts based on surface tension, conductance, and calo-rimetric measurements. They [4] also reported first and secondcmc values of mixtures of sodium dodecyl sulfate with various bilesalts as these mixtures formed mixed micelles.

OHCOO -

OH

O

O

O

O

OO

Hydrophobic convex surface

Hydrophilic concave surface

O

COO-

OH

O

O

O

O

OO

O

O

O

O

OO

A

BC D

O

Fig. 1. (A) Molecular structure, (B) schematic structure, (C) primary aggregate, and (D) secondary aggregate of sodium deoxycholate.

S. Das et al. / Journal of Colloid and Interface Science 357 (2011) 434–439 435

The secretion of bile by the liver into the gallbladder is calledcholeresis and salicylate ion is known to have a choleretic effectin rats, dogs, and humans [5–8]. Choleretic effect refers to increas-ing the rate of bile flow and the mechanism by which salicylateexhibits this effect is not clearly known. It is reported [5] thatthe influence of salicylate anion on the bile salt micelles mightbe one of the reasons for its choleretic effect. Further, there are re-ports of bile salts affecting the colonic absorption of oxalate andinhibition of calcium oxalate precipitation by bile salts [9,10].

The above reports reveal the physiological importance of salic-ylate and oxalate ions and how these two organic anions influencethe micellization of bile salts is, however, not known. Thisprompted us to take up this study and the objective of this studyis therefore to investigate the aggregation and adsorption behav-iors of NaDC in aqueous sodium salicylate (NaSa), sodium oxalate(Na2Ox), and sodium chloride (for comparison purpose) media.The effect of added counterion (same as that of the surfactant mol-ecule) on the cmc of ionic surfactant is well understood and re-cently a thermodynamic treatment was reported [11] to explainthe effect of mixed counterions also. However, the effect of coionson the micellization of ionic surfactants is yet to be understoodproperly and the present study will throw light on this aspect.

2. Materials and methods

NaDC (Fluka, 98%), NaSa (Fluka, 99.5%), Na2Ox (Himedia, A.R.grade, 99.9%), and NaCl (Merck, GR grade, 99.5%) were used as re-ceived. Solutions were prepared using Milli-Q grade water. Surfacetension measurements were made by the Wilhelmy plate methodusing a K11 Krüss tensiometer. EMF measurements were madeusing a Jenway 3345 ion meter and a Jenway 924-329 sodiumion selective combined electrode. Electrical conductance measure-ments were made using a B905 Wayne Kerr Automatic PrecisionBridge and a dip-type conductivity cell. Fluorescence emissionintensities (excitation wavelength = 335 nm) of pyrene (Fluka)

were recorded at 373 (I1) and 384 (I3) nm using Hitachi F4500 FLspectrophotometer.

3. Results

Experimental data of surface tension (c) of aqueous solutions ofNaDC in the presence of Na2Ox, NaSa, and NaCl at 25, 30, 35, and40 �C are shown in Figs. S1–S6 as plots of c versus surfactant con-centration. The values of cmc determined from the surface tensionisotherms are given in Table 1 and shown in Fig. 2.

Although we employed a conductance method to determine thecmc of NaDC in water, the plot of specific conductance versus sur-factant concentration did not show a change in slope over a widerange of concentrations, rendering the conductance method notsuitable for bile salts. The nonsuitability of specific conductanceversus concentration plots for determining the cmc of bile saltshas been discussed by Matsuoka and Moroi [12], which is attrib-uted partially to the low counterion binding constant of bile salts.

We also used the fluorescence method to determine the cmc ofNaDC in water. The values of the ratio I3/I1 of the emission intensi-ties of pyrene are plotted versus NaDC concentration in Fig. S7.From the plots given in Fig. S7 cmc was chosen as the concentra-tion at which the value of I3/I1 starts increasing sharply. The valuesof cmc thus obtained (Table 1) are found to be comparable withthose determined from surface tension data.

In Na2Ox solution, we measured the EMF (E) as a function ofNaDC concentration by using a sodium ion-selective electrode.The plots of EMF versus log([Na+]) are shown in Fig. 3. Up to0.03 mol kg�1 Na2Ox, the plots are linear in the entire range ofNaDC concentration and hence cmc could not be determined fromthe EMF data. Above 0.03 mol kg�1 Na2Ox, the plots of EMF versuslog([Na+]) show breaks and the concentrations of NaDC corre-sponding to these breaks are taken to be the cmc values, whichare listed in Table 1.

Table 1Critical micelle concentrations of NaDC.

[Salt], mol kg�1 Temperature

298 K 303 K 308 K 313 K

cmc (mmol kg�1)

Salt = Na2Ox0 2.34a (2.04)b 3.55 (2.33) 3.78 4.13 (4.03)0.005 2.25 3.28 3.59 3.800.01 2.17 3.16 3.43 3.570.02 2.16 3.05 3.30 3.540.03 2.14 2.92 – 3.440.05 1.92 2.43 2.70 (2.47)c 3.000.1 1.44 1.82 (1.88) 2.00 2.160.2 1.03 1.30 1.35 (1.47) 1.47

Salt = NaSa0.005 2.11 3.16 3.31 3.720.01 1.95 2.88 3.09 3.300.02 1.82 2.57 2.81 3.090.03 1.78 – 2.63 2.820.05 1.70 2.24 2.46 2.600.1 1.60 1.95 2.14 2.290.2 1.46 1.70 1.89 2.00

Salt = NaCl0.005 1.93 2.98 3.20 3.780.01 1.73 2.60 3.00 3.470.02 1.57 2.34 2.69 3.110.03 1.51 2.26 2.58 2.950.05 1.37 2.03 2.33 2.750.1 1.18 1.96 2.03 2.37

a From surface tension.b From fluorescence.c From emf.

436 S. Das et al. / Journal of Colloid and Interface Science 357 (2011) 434–439

Cmc values of NaDC in both buffered and unbuffered aqueousmedia have been reported [4,12–27] by different groups by usingvariety of techniques. Some of the reported values of first cmc inthe absence of added electrolyte are shown in Fig. 4 and comparedwith the present values of cmc of NaDC in water. It has generallybeen considered that the value of the first cmc of NaDC falls inthe range of 2–10 mM (M = mol kg�1). So much variation in thevalues of cmc of bile salts in contrast to the case with conventionalsurfactants is considered to be due to the small size of their aggre-gates. In view of such broad range for the cmc values of bile salts,the variation of cmc of NaDC with temperature is considered to bealmost linear as shown in Fig. S8.

However, from a fluorescence (using pyrene probe) study Mats-uoka and Moroi [12] reported first cmc at around 2 mM and secondcmc at 6 mM (at 25 �C), whereas Sen et al. [24] reported (using 2,6-p-toluidinonaphthalenesulfonate probe) first cmc at 7 mM and sec-ond cmc at 60 mM. Similar to the observation made by Matsuoka

0.0 0.2 0.4

1

2

3

4

0.0 0.2 0.4

1

2

3

4

[Na+]

cmc

/ mm

ol k

g-1

25 oC 30oC

Fig. 2. Variation of cmc of NaDC with sodium ion concentration. Sodium ion

and Moroi [12], we also noted from the fluorescence emission ofpyrene second cmc of NaDC at about 8 mM at 25 �C. Surprisingly,second cmc of bile salts is noted only in fluorescence emissionmeasurements. Jana and Moulik [4] also reported that surface ten-sion, conductance, and calorimetric data of pure bile salts do notexhibit second cmc, but for mixed surfactants containing bile saltseven conductance and calorimetric data showed second cmc. Inthis paper our discussion is, however, limited to first cmc of NaDConly.

4. Discussion

4.1. Dependence of cmc on salt concentration and counterion bindingconstant

From Fig. 2, it is clear that the decreasing trend in cmc of NaDCwith counterion (from the added salt) concentration is different inthe presence of different salts, thereby indicating the influence ofcoions on the cmc of NaDC. In the temperature range from 30 to40 �C, cmc values of NaDC in NaCl and NaSa solutions, however, be-come almost the same and hence chloride and salicylate coionshave a similar influence on the micellization of NaDC despite theirdifferent chemical identity. The dependence of cmc on counterionconcentration can be explained in terms of the Corrin–Harkins(CH) equation:

ln c0 ¼ A� blnðc0 þ ceNaÞ ð1Þ

In Eq. (1) A is a constant related to the standard free energy ofmicellization, b is the counterion binding constant, c0 is the cmcof NaDC in the presence of a particular concentration (ce) of elec-trolyte, and ceNa is the sodium ion concentration contributed byce concentration of electrolyte. The CH plots are shown in Fig. 5and values of b determined from these plots are listed in Table 2.At 25 �C, the values of b are found to be 0.18 in NaCl and 0.10 inNaSa solutions, but become equal to 0.17 ± 0.01 in both these solu-tions in the 30–40 �C temperature range. Therefore, the bindingbehavior of sodium ion to deoxycholate micelles is the same inthe presence of chloride and salicylate coanions at human bodytemperature. On the other hand, the binding behavior of sodiumion to deoxycholate micelles is different in the presence of oxalate.b is equal to about 0.05 ± 0.02 up to about 0.038 mol kg�1 oxalateconcentration (c�) and it is equal to about 0.48 ± 0.04 above c�. Asimilar counterion binding behavior has been reported [28] forAOT in different aqueous electrolyte solutions. As in the case ofcmc, a wide range of values have been reported for b of bile salt mi-celles also. The reported values vary from 0 to 0.7 [12,14–18,29,30].

0.0 0.2 0.4

1

2

3

4

0.0 0.2 0.4

1

2

3

4

/ mol kg-1

35 oo

C 40 C

concentration increased by adding Na2Ox (s), NaSc (�), and NaCl (h).

-2.0 -1.8

0

2

4

6

-1.70 -1.65 -1.6012

14

16

-1.40 -1.36

30

31

-1.22 -1.20 -1.18

24

25

26

-1.00 -0.99 -0.98

49

50

51

-0.696 -0.69262

64

66

-0.396 -0.392

54

55

56

-0.692 -0.688

63

64EMF/

mV

0.005 (35 oC)

log ([Na+] / mol kg-1)

0.01 (35 oC) 0.02 (30

oC) 0.03 (35

oC)

0.05 (35 oC) 0.1 (30

oC) 0.2 (35

oC)0.1 (40

oC)

Fig. 3. Variation of EMF with sodium ion concentration at different concentrations of Na2Ox. Sodium ion concentration increased by adding NaDC. Temperature and Na2Oxconcentration (in mol kg�1) are indicated in the insets.

280 290 300 310 320 330 340 3502

4

6

8

10

cmc

/ mM

Temperature / K

presentref. 22ref. 12, ref. 13ref. 27, ref. 4,16

Fig. 4. Reported and present values of first cmc (M = mol kg�1) of NaDC in aqueousmedium in the absence of added electrolyte.

S. Das et al. / Journal of Colloid and Interface Science 357 (2011) 434–439 437

The measured EMF data also confirm such a counterion bindingbehavior in the presence of oxalate coanion. The linearity of theplots of EMF versus log([Na+]) in the entire range of NaDC concen-tration up to 0.03 mol kg�1 Na2Ox (Fig. 3) can be explained as dueto negligible binding of counterions to deoxycholate micelles and

-6 -4 -2 0-7.0

-6.5

-6.0

-5.5

-6 -4-7.0

-6.5

-6.0

-5.5

Ln (c

mc

/ mol

kg-1

)

Ln ([Na+ ] /

Na2Ox NaSa

Fig. 5. Corrin–Harkins plots for NaDC at 298 K (h), 303 K (s), 308 K (D), an

this observation is in agreement with the very low value of b ob-tained from CH plots. Above 0.03 mol kg�1 Na2Ox, the EMF data be-low and above cmc fall on two straight lines (Fig. 3), which indicatebinding of sodium counterion to the micelle.

To estimate the value of b from the EMF data we adopted themethod reported earlier [28]. First, we least-squares fitted theEMF data lying below cmc to the equation of the form

E ¼ E0 þ B logðc þ ceNaÞ atc 6 c0 ð2Þ

The EMF data above the cmc were then least-squares fitted to an-other equation of the form

E ¼ E0 þ B logfbc0 þ ð1� bÞc þ ceNag at c P c0 ð3Þ

In Eqs. (2) and (3), E0 and B refer to empirical constants and c isthe concentration of NaDC. While least-squares fitting the EMFdata above cmc to Eq. (3), we substituted for E0 and B the valuesobtained from Eq. (2) and the best-fit value of b was evaluatedby an iteration process. The values of E0, B, and b are given inTable 2. These values of b are comparable to the b values obtainedabove from CH plots. Thus, oxalate coanion depending on its con-centration has a suppressing as well as enhancing effect on thebinding of sodium ion to deoxycholate micelles. In aqueousAOT + NaCl solution, the sudden increase in the value of b at about0.02 mol kg�1 NaCl was reported [31] to be due to the shapechange of AOT micelles and similarly at about 0.038 mol kg�1 of

-2 0 -6 -4 -2 0-7.0

-6.5

-6.0

-5.5

mol kg-1)

NaCl

d 313 K (�) in the presence of different salts (indicated in the insets).

Table 2Values of counterion binding constant, E0 and B.

T (K) Na2Ox b in NaSa b in NaCl Reported b

c�/mol kg�1 b from CH plot b from EMF

c < c� c > c� [Na2Ox] in mol kg�1

0.05 0.1 0.2

298 0.038 0.03 0.45 – – – 0.10 0.18 0.2a, 0.08b, 0.32c, 0.3d, 0.38e

303 0.030 0.07 0.44 – 0.45 – 0.18 0.18 0.7f

E0 = 455.5B = 562.7

308 0.034 0.06 0.47 0.36 – 0.5 0.17 0.18 0g

E0 = 159.8 E0 = 298.7B = 110.6 B = 616.2

313 0.039 0.07 0.52 – 0.48 – 0.19 0.17E0 = 264.7B = 291.0

E0 and B are in mV.a Ref. [12].b Ref. [16].c Ref. [17] (temperature not indicated).d Ref. [18] (temperature = 301 K).e Ref. [29].f Ref. [14].g Ref. [15].

Fig. 6. A schematic representation of hydrogen bonding between oxalate anion and hydroxyl groups of deoxycholate ions.

0.0 0.2 0.4

0.8

1.2

1.6

0.0 0.2 0.4

0.8

1.2

1.6

0.0 0.2 0.4

0.8

1.2

1.6

Γ cmc

x 10

6 / mol

m-2

[Na+ ] / mol kg-1

Na2Ox NaSa NaCl

Fig. 7. Surface excess at cmc of NaDC as a function of added sodium ion concentration at 298 K (h), 303 K (+), 308 K (s), and 313 K (�).

438 S. Das et al. / Journal of Colloid and Interface Science 357 (2011) 434–439

Na2Ox the shape of the deoxycholate micelles may be expected tochange from prolate ellipsoid to rod-like. At c�, oxalate ion may beundergoing hydrogen bonding with the hydroxyl groups of thedeoxycholate ions of the two neighboring primary aggregates,thereby inducing formation of secondary aggregates and henceshape change. This probable interaction between oxalate ion andaggregates of deoxycholate is shown in Fig. 6.

4.2. Free energy

The standard free energy of micellization per mole of NaDC wascalculated using the relation

DG0m ¼ RTð1þ bÞ ln c0: ð4Þ

In Eq. (4), R and T represent gas constant and absolute temperature,respectively. The values of DG0

m evaluated from Eq. (4) are listed inTable S1. From the values of DG0

m, it is apparent that in the temper-

ature range from 30 to 40 �C salicylate coanion has no special effecton the micellization of NaDC. In the temperature range from 25 to40 �C and below c�, oxalate coanion, however, has a hindering influ-ence on the micellization of NaDC in comparison to both chlorideand salicylate.

4.3. Adsorption

Surface excess of NaDC at the cmc, Ccmc, in the presence ofadded salts was evaluated using the relation [32]

Ccmc ¼ �1

RT1

1þ c0c0þceNa

" #dc

d ln c

� �cmc: ð5Þ

The computed values of Ccmc for NaDC in water and in the dif-ferent electrolyte solutions are shown in Fig. 7. It is clear fromFig. 7 that on adding electrolyte Ccmc initially increases, reaches

S. Das et al. / Journal of Colloid and Interface Science 357 (2011) 434–439 439

a maximum value, and then starts decreasing. The maximumin Ccmc occurs at about 0.03, 0.07, and 0.05 mol kg�1 of oxalate,salicylate, and chloride ion concentration, respectively. Since thesodium ion concentration at the maximum corresponds to0.06 ± 0.01 mol kg�1 in the case of all three salts, the counterionseems to control the position of maximum in the plot of Ccmc ver-sus salt concentration. Such a dependence of Ccmc on electrolyteconcentration was reported [33] for sodium dodecyl sulfate (SDS)in aqueous sodium butyrate. Adsorption of an ionic surfactant isinfluenced by salts because of a salting-out effect, which, in turn,is controlled by the hydration of the ions of the added salts. Inaqueous NaDC solution, it appears from Fig. 7 that the hydrationof salicylate, oxalate, and chloride coanions has a similar effecton the surface excess of NaDC.

5. Conclusions

Based on the present and reported data it has become clear thatin unbuffered water medium surface tension data and fluorescenceemission intensity data of pyrene probe provide similar values forthe first cmc of NaDC, which at 25 �C is equal to 2.5 ± 0.5 mM. Be-sides counterion effect, salicylate, oxalate, and chloride coanionsalso influence the cmc values of NaDC. The binding behavior of so-dium ion to NaDC micelles is similar in the presence of sodiumchloride and sodium salicylate (30–40 �C temperature range), butdifferent in the presence of oxalate, thereby indicating that an or-ganic coanion depending on its structure may have an influence onthe counterion binding behavior of deoxycholate micelles. By com-paring the present system with the AOT + electrolyte system inwhich a correlation between the shift in counterion binding con-stant and micellar shape change has been reported [31], a changein the shape of the deoxycholate micelles from prolate ellipsoidto rod-like may be expected at about 0.038 mol kg�1 of Na2Ox.

The general concept regarding the effect of coions on the micel-lization of ionic surfactants has been that only those coions withsufficient hydrophobicity can interact with ionic micelles. In con-tradiction to this general view, this study has revealed that evencoions having negligible or no hydrophobicity can interact with io-nic micelle. Our recent report [31] on the binding of salicylatecoanion to AOT micelle which inhibited a shape change of this mi-celle is another example of interaction of a coion having lowhydrophobicity with ionic micelle. Thus, it may be concluded thatin determining the effect of a coion on the micellization of an ionicmicelle their (coion and ionic micelle) structures play an importantrole, and hence hydrophobicity of the coion is not the only control-ling factor.

Since NaDC has similar micellization and adsorption behaviorsin aqueous NaCl and NaSa media at human body temperature,the present study rules out the influence of salicylate ion on the

micellization of NaDC as responsible for the choleretic effect ofsalicylate. The special effect of oxalate coanion on the binding ofcounterion to deoxycholate micelle may have relevance to bilesalts affecting the colonic absorption of oxalate.

Acknowledgments

S.D. and T.M. acknowledge the financial assistance receivedfrom the UGC, New Delhi, and J.D. acknowledges the financialassistance received from the DST, New Delhi.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jcis.2011.02.020.

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