JOURNAL OF COLLOID AND INTERFACE SCIENCE 200, 310–312 (1998)ARTICLE NO. CS975363

NOTEEffect of a Weak Electrolyte on the Critical Micellar Concentration of Sodium Dodecyl Sulfate

were always prepared using bidistilled water and used without any furthertreatment throughout experiments. The concentration of SDS stock solutionsThe critical micellar concentration of sodium dodecyl sulfate iswas well above the CMC (Ç8 1 1003 M in water) and ranged from 48 tostrongly altered by tris (hydroxy-methyl)methylammonium ions.200 1 1003 M. The concentration of 1,8-ANS was lower than Ç1.4 1The effect of buffer solutions containing this weak electrolyte as1003 M, as judged using a molar absorptivity coefficient of 6,800 M01

the counterion source has been studied using various concentra-cm01 at 374 nm (6). Aqueous buffer solutions, whose total concentrationtions of the acid–base system as well as modifying the pH. Resultsranged from 10 to 600 1 1003 M, were prepared using either tris(hydroxy-

show that counterion concentrations ranging from 0 to Ç340 1 methyl)aminomethane (Tris) or ammonium hydroxide (28.0–30.0% NH3),1003 M induce an appreciable diminution of the critical micellar both from Sigma Chemical Co. Their pH after dissolution was left un-concentration from Ç8 to Ç0.7 1 1003 M. The analysis of data changed. In a number of experiments the pH was adjusted by adding smallsuggests that the critical micellar concentration of sodium dodecyl aliquots of a concentrated solution of HCl, so that the only source ofsulfate depends on the concentration of weak electrolytes in a way counterions was the buffer itself. In any case, pH values were used to

evaluate the counterion concentration using a pKa of 8.21 and 9.40 at 207Cvery similar to that of strong electrolytes. q 1998 Academic Press

for Tris/TrisH/ and NH3/NH/4 , respectively. A pKa of 7.77 was used forKey Words: micellization, effect of pH on; sodium dodecyl sul-

Tris/TrisH/ at 377C. The pKa values were estimated by reported thermody-fate; charged surfactants; acid–base balance; micelle counterions;namic functions (7) .1,8-ANS fluorescence.

Fluorescence. Emission spectra were excited at 370 nm and then re-corded from 450 to 600 nm at either 20 or 377C using a Perkin–ElmerMPF 66B model spectrofluorometer equipped with a thermostatable cell

INTRODUCTION holder and a Grant model LTD 6 water circulating bath to control thetemperature. Equal excitation and emission bandwidths (5 nm) were used

Surfactants are largely used to characterize in vitro those proteins and/ throughout the experiments, with a scan speed of 120 nm/min and a timeor enzymes whose function needs anchorage to a membrane-like environ- constant of 2 s. Fluorescence titrations were performed as previously de-ment (1) . Since pioneering studies on the critical micellar concentration scribed (8), adding small aliquots of SDS to a fixed volume (2 1 1003

(CMC) of long-chain electrolytes it is well known that the aggregation of dm3) of aqueous or buffered solutions of 1,8-ANS, whose absorbance wascharged surfactants depends on the ionic strength (2–5). These reports lower than Ç0.05 at the excitation wavelength. This ensured fluorescencealso clarified that the logarithm of the CMC depends logarithmically on linearity. Lower concentration surfactant stock solutions were used whenthe univalent counterion concentration. This observation is usually under- the salt concentration was raised, in order to get enough experimental pointsstood to be the consequence of the fact that the Gibbs energy change of before reaching the CMC. Corrections were always made for the dilutionmicelle formation can be conveniently dissected into a hydrocarbon and an of the fluorophore to take into account only fluorescence changes originatedelectrostatic part (5) . This last contribution, in turn, depends on the charge by the interaction of the probe with micelles.of the micellar system as well as on the degree of counterion binding. It is Evaluation of the critical micellar concentration. The CMC of SDSalso affected, although to a limited extent, by the chemical nature of the was evaluated by linear least-squares fitting of the 1,8-ANS fluorescencecounterion (5). To our knowledge, investigations performed to date mostly intensity at 530 nm vs the surfactant concentration before and after theregard strong electrolytes. change of slope (8) . The CMC was calculated at the intersection of these

The biological activity of membrane proteins and/or enzymes is assayed lines as the negative ratio of the intercept difference (A) to the slopein vitro at pHs close to neutrality to mimic the condition of the living difference (B) , 0CMC Å A /B . Only plots with a correlation coefficientorganism. This achievement requires the use of pertinent buffer systems, of 0.9 or higher were considered. The error was estimated by propagation,which release charged species, thus controlling the acid–base balance. Of assuming that the CMC uncertainty (dCMC) is given by the sum of CMCcourse, the CMC of an ionic surfactant can depend on pH, if the released partial derivatives with respect to A and B , each multiplied by the pertinention acts as a micelle counterion. The presence of a strong electrolyte, which uncertainty (dA and dB , respectively) . This procedure results in the relation-does not participate in the buffer system, can obscure this occurrence, ship dCMC Å (1/B)[dA 0 CMC(dB)] , giving a relative error of 16.6 andalthough the onset of a cumulative effect is likely to be the case. Therefore, 1.4% in the worst and in the best case, respectively, and of 6.8% on theit seems interesting to investigate ionic surfactant aggregation in a buffered average. The CMC of SDS in water is in agreement with previous worksolution without strong electrolytes, in order to analyze the effect of pH (5). Data analysis was performed by the program Scientist for Windowsand buffer concentration. This study regards the effect of tris(hydroxy- version 2.0 by MicroMath Scientific Software.methyl)methylammonium (TrisH/) on the micellization of sodium n-dode-cyl sulfate (SDS).

RESULTS

MATERIALS AND METHODS Buffer concentration dependence of the CMC. The determination of theCMC of detergents hinges on sharp changes in physical properties of surfac-tant solutions. Several handy methods make use of appropriate reporterChemicals and solutions. Ultrapure sodium n-dodecyl sulfate was pur-

chased from USB. 1-Anilinonaphthalene-8-sulfonic acid magnesium salt molecules, whose spectral features undergo large alterations on interactionwith a micellar aggregate (5) . In this regard, it has been shown that a hugedihydrate (1,8-ANS) was from Fluka. Stock solutions of these substances

3100021-9797/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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311NOTE

TABLE 1Critical Micellar Concentration of SDS at 207C

CTris CTrisH/ CMC { dCMC

(M 1 1003) pH (M 1 1003) (M 1 1003)

0 — 0 8.06 { 0.3310 8.05 5.9 5.61 { 0.9310 7.87 6.9 4.72 { 0.4020 8.25 9.5 4.12 { 0.1130 8.25 14.3 3.38 { 0.1740 8.01 24.5 3.12 { 0.15

125 8.65 33.3 2.16 { 0.0380 8.25 38.2 2.09 { 0.13

125 8.41 48.4 1.69 { 0.12125 8.19 63.9 1.62 { 0.17125 7.25 112.6 1.36 { 0.09300 8.25 143.1 1.02 { 0.04600 8.25 286.2 0.68 { 0.09600 8.09 341.2 0.72 { 0.02

tion of 125 1 1003 M (pH 8.16 and 8.20, respectively) . At these pHs andFIG. 1. Critical micellar concentration of SDS in various media. Theequal total concentration of the buffer system the CMC of SDS is apparentlyfluorescence intensity of 1,8-ANS at 530 nm is plotted against the totalunaltered as compared to 207C. However, this effect masks the change ofconcentration of SDS. Small aliquots of SDS stock solutions were addedthe pKa of the buffer with temperature. If the actual ionic strength is consid-to 2 1 1003 dm3 of a water solution of 1,8-ANS, whose absorbance wasered, the CMC at 377C shows a modest decrease, which accounts for a°0.05 at the excitation wavelength (370 nm). Points before and after theslightly positive micellization enthalpy (Ç4.5 kJ/mol) .change of slope were fitted to two straight lines, whose slope and intercept

Treatment of data. CMC data vs counterion concentration are best fittedwere used to estimate the CMC as described under Materials and Methods.by double logarithmic plots, which are straight lines with a slight depen-Data at 207C regard water (squares) , 10 1 1003 M Tris, pH 7.87 (circles) ,dence on the nature of the counterion. The slope is usually in the range40 1 1003 M Tris, pH 8.01 (triangles) , and 80 1 1003 M Tris, pH 8.25from00.4 to00.6, and represents a rough estimate of the degree of counter-(diamonds). Data for 125 1 1003 M Tris, pH 8.16 (asterisks) , are at 377C.ion binding to the micelle (5) . The linear regression analysis of our datais shown in Fig. 2. Both regression parameters (slope Å 00.62 { 0.03,

increase of the 1,8-ANS fluorescence quantum yield in water occurs whenthis fluorophore binds SDS micelles (8) , although presumably this probeis not able to penetrate the micellar core (9, 10). The fluorescence intensityof 1,8-ANS depends almost linearly on the SDS concentration, but withdifferent slopes, reflecting the interaction of the probe with free and micellarsurfactant, respectively. The intersection of these two lines corresponds toa surfactant concentration which is usually taken as the CMC (8). We haveadopted this method (see Materials and Methods) to measure the CMC ofSDS as a function of the TrisH/ concentration. The concentration of thisspecies in turn depends on the total concentration of the buffer system aswell as on the pH. It can be estimated by

[TrisH/] Å [H3O/]CTris / (Ka / [H3O/]) , [1]

which is easily obtained by combining the mass action law for the dissocia-tion of TrisH/ , Ka Å [H3O/][Tris] /[TrisH/] , and the mass balance equa-tion, CTris Å [Tris] / [TrisH/] . Other univalent electrolytes can be treatedsimilarly. A few experiments have been performed using ammonia as thesource of counterions to explore higher pHs and also to check whether theCMC dependence on the counterion concentration displays any appreciableeffect linked to the chemical nature of TrisH/ .

As an example, Fig. 1 shows the fluorescence titration profiles of 1,8-ANS in various media at 207C. The value of (8.06 { 0.33) 1 1003 M forthe CMC of SDS in water well agrees with the previous estimate of (8.3

FIG. 2. Total counterion concentration dependence of the critical micel-{ 0.4) 1 1003 M (8), which was obtained by the same experimentallar concentration. The double logarithmic plot regards TrisH/ data at 207Cprocedure. This also seems to ensure good reproducibility as well as reliable

error evaluation. The whole set of experiments is summarized in Table 1. (gray squares) . Other points are for NH/4 (filled circles) and TrisH/ at

377C (filled squares) . Published log–log plots at 257C (3) are also shownAs expected, a diminution of the CMC from Ç8 to Ç0.7 1 1003 M takesplace on increasing the TrisH/ concentration up to Ç340 1 1003 M. Two for Li/ ( long dash), Na/ (medium dash), and tetramethylammonium

(short dash).determinations were also performed at 377C using a total buffer concentra-

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312 NOTE

intercept Å 03.50 { 0.04, r Å 00.986) are close to previous estimates of weak electrolyte concentration. As a consequence, the function of pro-teins and/or enzymes that need anchorage to a membrane-like environmentregarding strong univalent electrolytes (2, 3, 5) . It is well known that the

aggregation behavior of anionic detergents is less sensitive to the nature of strongly depends on the concentration of the buffer system as well as onthe pH. Of course, surfactants display different properties according to theirthe counterion than cationic surfactants (5) . Therefore, the effect of pH on

the CMC of SDS reflects mostly changes in the ionic strength, which in chemical nature. Preliminary results suggest that the behavior of tris(hy-droxy-methyl)methylammonium is very similar to that of strong electro-the present case is under the control of TrisH/ .lytes.

DISCUSSIONACKNOWLEDGMENTS

It should be interesting to compare our results with other relevant studiesThanks are due to Professor Harold C. Helgeson, whose computer andin the literature. Unfortunately, only in recent years interest has shifted

financial support was made available to R.R. during his stay at the Depart-from inorganic ions to organic ions. The vast majority of these studiesment of Geology and Geophysics, University of California at Berkeley.regards the aggregation behavior of cationic surfactants in the presence of

anions. Even if these ions are usually conjugate bases of weak acids, mostexperiments were aimed to analyze the effect of the chemical structure of REFERENCEScounterions on the CMC. They were therefore performed outside the pHrange where maximum buffer capacity occurs, i.e., outside the counterion 1. Tanford, C., and Reynolds, J. A., Biochim. Biophys. Acta 457, 133concentration range where pH effects can be appreciated. This is the case (1976).for the aggregation of decyltrimethylammonium micelles in the presence 2. Corrin, M. L., and Harkins, W. D., J. Am. Chem. Soc. 69, 683 (1947).of n-alkyl carboxylates (11–13), methyl-, chloro-, and phenyl-substituted 3. Schick, M. J., J. Phys. Chem. 68, 3585 (1964).acetates (14), and benzoates (15). Dodecyltrimethylammonium salts have 4. Emerson, M. F., and Holtzer, A., J. Phys. Chem. 69, 3718 (1965).been studied in the same context (16). 5. Kresheck, G. C., in ‘‘Water: a Comprehensive Treatise’’ (F. Franks,

These studies clarified that the aggregation behavior of surfactants does Ed.) , Vol. 4, p. 95. Plenum Press, New York, 1975.not display any peculiar dependence on small counterions, such as formate 6. Cantor, C. R., and Schimmel, P. R., ‘‘Biophysical Chemistry.’’ Free-and acetate (11). Unusual binding to the micelle surface occurs only when man, New York, 1980.the carboxylate moiety is linked to bigger hydrocarbon substituents. Such 7. Edsall, J. T., and Wyman, J., ‘‘Biophysical Chemistry.’’ Academiceffects are explained in terms of hydrophobic interactions. In the case we Press, New York, 1958.have examined the occurrence of interaction via hydrogen bonding between 8. DeVendittis, E., Palumbo, G., Parlato, G., and Bocchini, V., Anal.alcoholic functions of TrisH/ and oxygens of the sulfate moiety seems Biochem. 115, 278 (1981).most likely. This should favor the binding of TrisH/ to the negatively 9. Radda, G. K., Biochem. J. 122, 385 (1971).charged surface of SDS micelles. We presume that for this reason the 10. Lesslauer, W., Cain, J. E., and Blasie, J. K., Proc. Natl. Acad. Sci. USAassociation degree of TrisH/ , as estimated by the slope of the regressed 69, 1499 (1972).line in Fig. 2, is higher than that for tetramethylammonium (0.62 against 11. Anacker, E. W., and Underwood, A. L., J. Phys. Chem. 85, 24630.57). Even if this ion is smaller, nevertheless it cannot form hydrogen (1981).bonds with the micellar surface. 12. Jansson, M., and Stilbs, P., J. Phys. Chem. 91, 113 (1987).

However, our study was mainly aimed to show that counterions originat- 13. Jansson, M., and Jonsson, B., J. Phys. Chem. 93, 1451 (1989).ing from buffer systems of biological interest can be as effective as strong 14. Underwood, A. L., and Anacker, E. W., J. Colloid Interface Sci. 100,electrolytes in inducing micellar aggregation. This happens through pH 128 (1984).control. Other recent, and more specific, reports demonstrate that this evi- 15. Underwood, A. L., and Anacker, E. W., J. Phys. Chem. 88, 2390dence cannot be disregarded, although it is quite rare to have enzymes (1984).that need membrane-like environments to work and are even dissolved in 16. Brady, J. E., Evans, D. F., Warr, G. G., Grieser, F., and Ninham, B. W.,solutions whose ionic strength is mostly determined by a weak electrolyte. J. Phys. Chem. 90, 1853 (1986).It has been reported, for example, that the apparent hydrodynamic radius 17. Hofer, M., Hampton, R. Y., Raetz, C. R. H., and Yu, H., Chem. Phys.of lipid IVA is not sensitive to the nature of the buffer, but depends on the Lipids 59, 167 (1991).ionic strength and pH (17). Incidentally, this confirms that the aggregation 18. Amante, J. C., Scamehorn, J. F., and Harwell, J. H., J. Colloid Interfaceof anionic surfactants is scarcely affected by the nature of the counterion Sci. 144, 243 (1991).(5) . Other reports show that even the precipitation of mixtures of anionic 19. DelRio, J. M., Pombo, C., Prieto, G., Mosquera, V., and Sarmiento, F.,and cationic surfactants is under the control of pH (18). Also, the CMC J. Colloid Interface Sci. 172, 137 (1995).of n-alkyltrimethylammonium bromides in a pH 3.2 glycine buffer has been 20. Toullec, J., and Couderc, S., Langmuir 13, 1918 (1997).found similar to that observed at pH 10 in the same buffer and slightly

Ciro Espositolower than in the absence of glycine. This has been attributed to the additivePatrizia Colicchioglycine increasing the ionic strength of the solution (19). The last articleAngelo Facchianothat deserves attention is specifically devoted to analyzing the counterionRaffaele Ragone1

binding parameter (b) to hexadecyltrimethylammonium acetate micelles(20). This study includes a thorough examination of the dependence of

Dipartimento di Biochimica e Biofisicasurfactant aggregation on the acetate/acetic acid system. This buffer is not

Seconda Universita’ di Napolisuitable for controlling the pH in the physiological range. Nevertheless, the

via Costantinopoli 16article confirms that ionic strength effects due to weak electrolytes are not

80138 Naples, Italynegligible.

Received July 22, 1997; accepted December 5, 1997SUMMARY

1 To whom correspondence should be addressed. Fax: /39-81-5665869/The data presented here show that counterions originating from a buffersystem strongly affect micellar aggregation, as the CMC is under the control 5665863. E-mail: raragone@cds.unina.it.

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