C. B O T R ~B., DE MARTIIS, AND M. SOLINAS
3624
fied. This is true also for thin liquid films upon a solid substrate, but only a t considerably smaller radii of the films.
Acknowledgment. The author thanks Prof. Dr. A. Scheludko for his constant encouragement and helpful discussions.
On the Interaction between Macromolecules and Colloidal Electrolytes
by C. Botr6, F. De Martiis, and M. Solinas Istituto d i Chimica Farmaceutica and Centro d i Studio per la Chimica del Farmaco e dei Prodotti biologicamente attivi del C N R , Universith, Rome, Italy (Received A p r i l W 81964)
The interaction in aqueous solution between sodium lauryl sulfate either with waterinsoluble (such as polyvinyl acetate) or water-soluble (such as bovine serum albumin) macromolecules was studied. Potentiometric determinations with permselective membrane electrodes or cationic glass electrodes and specific conductivity were employed in this investigation. Several evidences clearly confirm the formation of a L*complex’’between macromolecule and colloidal electrolyte. Data are also reported about the shift observed in the c.m.c. values, as well as the binding of sodium and calcium ions in solution of these complexes. I n spite of the poor solubility of the calcium salt of a detergent in aqueous solution, it is shown that the solution of the “complex” before precipitation may sequester significant amounts of calcium. The behavior of the complex was studied both as a function of the molar detergent-polymer ratio and as a function of the concentration of the complex in solution. On the basis of these experiments, a theoretical approach is proposed to treat the complex as a polyelectrolyte.
Introduction It was shown that water-insoluble polymers can be dissolved in a solution of ionic detergents.1 The mechanism of solubilization was explained by assuming the formation of a “complex” between the macromolecule and the detergent. Similar “complexes” are also built up in the presence of surfactants and a watersoluble polymer such as polyvinylpyrrolidone.2 With molecules other than detergents, as for instance an azo dye,3 similar interactions occur. The common feature in all these complexes is, however, their polyelectrolyte character, I n the field of biologically important macromolecules, the interaction with colloidal electrolytes was also studied by many author^.^-^ The study reported below refers to the complexes resulting by an interaction between sodium lauryl sulfate (NaLS) and polyvinyl acetate (PV4c) or bovine serum albumin (BSA). T h e Journal of Phgsical Chemistry
Experimental Material. Crystallized BSA obtained from Armour La,boratories was dialyzed against water by allowing the solution to stand for 5 days a t 3’, and then the resulting solution was lyophilized. Polyvinyl acetate was a sample supplied by Polymer Consultants Ltd., England. The molecular weight, determined by light-scattering (I) I. Isemura and A. Imanishi, J . Polymer Sei., 33, 337 (1958). (2) S. Saito, J . Colloid. Sci., 15, 283 (1960). (3) H. P. Frank, S. Barkin, and F. R. Eirich, J . P h y s . Chem., 61, 1375 (1957). (4) G. Strauss and U. P. Strauss, ibid., 62, 1321 (1958). (5) M. Brauer and U. P. Strauss, ibid., 64, 228 (1960). (6) B. P. Brand and P. Johnson, Trans. Faraday SOC.,52,438 (1956). (7) D. F. Waugh, J . P h y s . Chem., 65, 1793 (1961). (8) B. S. Harrap and J. H. Schulman, Di8CU88Wn8 Faraday Soc., 13, 197 (1953). (9) I. Blei, J . Colloid. Sci., 15, 370 (1960).
3625
INTERACTIOS BETWEEN MACROMOLECULES AND COLLOIDAL ELECTROLYTES
measurements, gave a value corresponding to about 975 monomers/molecule. Sodium lauryl sulfate was preprepared according to Dreger.’O Procedure. Potentiometric determinations of the activity of K a + ions were performed with both membrane electrodes and a couple of a sodium-responsive electrode (Bec liman 39278)-liquid bridge calomel, in a wide range of polymer-detergent concentration. In this latter case, the activity of sodium ions was calculated by means of a calibration curve, which allowed the conversion of e.ni.f. values into activitiee. This was obtained by diluting a sodium chloride solution stepwise, recording the e.m.f. of the cell and plotting it vs. the logarithm of ?Sa+ ion activity a t each step. A linear plot” of the e.m.f. against log axa was obtained. The asa+values of the solution were calculated from the measured e.m.f. by interpolating the calibration curve. This was dome by assuming that Y N =: ~ ~ - ~ * ( N ~ c I in ) the calibrating solution. The sequestering power determinations were performed following the procedure already described. 12,l 3 Conductonietr ic titrations were performed in a thermostatic bath at 5!5 and 30’. In some cases, the potentiometric and thle conductometric determinations were carried out simultaneously.
Results and Discussion In previous papers, 14,15 the behavior of aqueous solutions of colloidal electrolytes was studied by means of e.m.f. measurtxients and discussed in terms of an interaction between counterions and micelles, the latter being treated as a spherical polyelectrolyte. It is assumed that the liquid junction potential remains effectively unchanged, regardless of the nature of the solution, since the concentration of XaCl in the liquid bridge is higher than the concentration of the experimen tal solution. In Fig. 1 the activity coefficient of sodium ions is reported as a function of concentration of sodium lauryl sulfate in solution (open points, plot b). The c.ni.c. value, Co, is marked by the discontinuity in the plot. The concentration of ISaLS a t this point is 8.0 X 10 -,j value is 0.56. ill, and the In the same Fig. 1 (full points, plot b), the behavior of y N a refers to a solution of NaLS with PVAc dissolved The molar Sa1,S-PVAx ratio was 2.61. As extensively discussed in a previouf, work,l4 it is assumed that above c.ni.c. any addition of detergent increases the number of micelles since both the concentration of monomere, and the number of iiiononiers per micelle are considered to be constant in solution. Furthermore, since the value of the dielectric constant of the solution containing the K aLS-PVAc complex is nearly coincident with
98-
‘a
{Na’ 7 -
6-
54-
32-
0.5
0
10
1.5
2.0
25
MN~LS.~~’
Figure 1. YN*+ us. concentration expressed as molarity of NaLS. Open points refer to NaLS solution; full points, to a solution of NaLS-PYAc complex.
the one of simple IYaLS solution, no influence on -ypJa was considered. A.s may be seen, an apparent shift toward higher conlcentrations in c.1ii.c. is observed. This shift froin Co to C can be ascribed to an amount of free monomers removed from the equilibrium nionomer micelle by an oriented absorption of the iiiononiers on the polymeric chain of the PVAc, as elsewhere discussed. The relationship which gives the yoxvalue (rexbeing the experimental value of the activity coefficient) st the concentration corresponding to the c.ni.c. of NaLS in solution of the “complex” ?SaLS-PVAc may ble written as
When the complex with a NaLS-PVAc ratio of 2.61 is present in solution, the concentration of NaLS itself required to form micelles is C = 1.0 X lo+. At t h ~ s point, the value ycx is 0.48. So, replacing the experiiiiental data in (1) 0.48
=
0.56 X 8 X loW3f 0.56a(10 X i o x 10-3
-8 X (1’)
(10) E. E. Dreger, G. I. Keim, G. A. Miles, L. Shedlovsky, and .r. Ross, Ind. Eng. Chem., 36, 610 (1944). (11) F. Ascoli, C. BotrB, and A. M. Liquori, J . Mol. Biol., 3, 202 (1961). (12) F. Ascoli and C. Bot&, Farmaco (I’svia), 17, 213 (1962). (13) C. BotrB, V. Crescenzi, and A. Mele, J . Nucl. Chem., 8 , 368 (1958). (14) C. BotrB, V. Crescenzi, and A. hfele, J . P h y s . Chem. 6 3 , 650 (1959). (15) L. Shedlovsky, C. VV. Jakob, and M. B. Epstein, ibid.,67, 2075 (1963).
Volume 68,Number 12 Decemher, 1964
C, B O T R ~F., DE ~IARYIIS, AXD XI, SOLINAS
3626
the degree of dissociation, CY, of Na+ ions in the complex KaLS-PVAc may be obtained. This value of CY is about 0.28. If a cylindrical model is assumed for the polyelectrolyte complex, then, on the basis of a relation proposed by Oosawa, the degree of dissociation, a, of counterions in solution may be evaluated
(3)
where CY = degree of dissociation, eo = charge, b = distance between neighboring charged groups, 4 = volume fraction of the polyelectrolyte, D = dielectric constant of water, and T and k have the usual meaning. I n the case within the range of complex concentrations considered, the value of 4 being small compared with unity, the relation ( 2 ) may be written as
therefore, q = 1. By assuming for our complex a = 15 H.and b = 2.5 A,, the value of CY is 0.32. At c.m.c. this value of CY is in good agreement with the value of 0.28 determined experimentally. It is to be pointed out that the hypothesis to treat the polyelectrolyte coniplex with a cylindrical model may be successful if this treatment is applied to a relatively dilute solution where inainlydetergent molecules, and not aggregates, are present inside the polyelectrolyte complex. I n fact, in a more concentrated solution, the ~ is mainly determined by contribution of the Y N value micelles, while the role played by the complex becomes negligible. This is in agreement with the experiments, and no significant differences in 7 N a values may be observed in the presence or absence of the complex when the concentration of S a L S is higher than 2.5 X M. Froin such an interaction results the polyelectrolyte character of the complex which is expressed by the typical behavior of - y N a upon dilution. Obviously, the charge density on the iiiacroion is closely dependent upon the degree of binding of the detergent molecules on the polymeric chain. When partially hydrolyzed samples of PVAc are employed, the number of active sites for the adsorption of detergent molecules on the polyineric chain is lowered. Therefore, the charge density on the iiiacroniolecular complex is also lowered, and an increase of Y N , is found. Above c.ni.c., as discussed elsewhere,14the situation is more or less the same, as in simple NaLS solutions. The Journal of Physical Chemistqi
I n both cases, in fact, the steep decrease of ?sa may be explained, considering the micelles as highly charged spherical polyelectrolytes. On the surface of the micelle, the high electrostatic field, determined by the ionic heads of the detergent toward water, affects the distribution of Ka+ ions in solution significantly. The consequent interaction between micelles and counterions leads to the lowering of free charges on the former because of an extensive binding of sodiuni counterions. Furthermore, a striking difference may be observed in the binding of divalent cations, i e . , Ca+2 ions. I n fact, when Ca+2 ions are added to a solution of detergent, a precipitation of the calcium salt takes place. On the contrary, in detergent solutions containing dissolved PVAc, after addition of Ca+2 ions, significant amounts of calcium are held in solution before the formation of a precipitate. To test the sequestering ability of this complex, the binding of Ca+2 was evaluated according to a method described elsewhere.12 To evaluate the binding of Ca+2 ions in solution of the complex, a potentiometric system was set up which employs both glass cationic electrodes and membrane electrodes. The sensitivity of cationic glass electrodes is very high toward monovalent cations while the sensitivity toward divalent cations is very low (near zero). It was observed that C a f 2 ions in the range of concentration used in sequestering power determinations do not alter the potential difference due to E a + ions alone and recorded by the couple of electrodes-cationic glass electrode-calomel electrode. I n Fig. 2 (system A), a combination of the negatively charged membrane electrode, prepared according to Yeihof,16 and the sodium electrode is reported. Yeglecting the junctions, the e.m.f. AEl,z is given by the expression
(5) and the e.m.f. AEN,is given by
--EN% = K log
aNaa+
(6)
where R, T , and F have the usual meaning, asal-and ac.,+z are Xa+ and Ca+2ion activities in reference solu~ -the ion activity measured with the S a tion, and U N ~ is electrode. Furthermore, across the membrane one has tNa+
+
tCa+2
=
1.0
(7)
and t C a i z being the transference numbers of sodium and calcium, respectively. They are functions
tSa+
(16)
R.Neihof, J . P h y s . Chem., 5 8 , 916
(1954).
INTERACTION
36:!7 -
13ETWEB:N MACROMOLECULES AND COLLOIDAL ELECTROLYTES
crlomel e I e c t r o de,
calomel
e I e e t r o d ie
H em'br a n e rWr;
ana;
resf+
rea: SY s T E
+
w 'A,
Figure 2. Apparatus for membrane electrode titration.
H e m b ra ne
rNl;
,c.;+
awl; re*:+
S Y S T E H "E.. figure 3. Reference electrode system for membrane titration.
tion is now added to the half-cell 2' of system B until AE,,,' matches AEl,2. The amount of Ca+2ions added to the half-cell 2', therefore, corresponds to the Ca.t2 ions not bound by the sequestering agent in the half-cell 2 of system A. The difference between the Ca+2 ions added to side 1' and side 2' of system B indicates the amount of sequestered Ca+2 ions. The above described steps are repeated after each addition of CaC12 to both half-cells of system A. Thus, the calcium sequestered as a function of Ca+2 ions present in a solution containing a fixed concentration of sequestering agent is obtained. In agreement with the polyelectrolyte character of the coniplex, the amount of calcium sequestered is a function of the concentration of complex in solution (Fig. 4),and of the NaLS-PVAc ratio (Fig. 5), which is in close correlation to the charge density on the polymeric chain. The strong electrostatic interaction between Ca+* ions and the macroanion leads to a release of Na+ ions and is evident from the plots of Fig. 4. In fact, the two plots differ from each other only in the total amount of complex in solution, the NaLS-PVAc ratio being the same in both cases. The curves of Fig. 4 and 5 are calculated by using eq. 5 with = 1 and ha+ = 0. On the other hand, by taking into account the sodium release by means of the preceding procedure, the plotk become more or less coincident giving a straight line with a slope of 45' (broken lines both in Fig. 4 and 5 ) .
of a N a + and %*+a so that it is impossible to determine ma+aby direct use of (5)-(7). This difficulty has been overcome by using a reference system (Fig. 3) analogous to the first one and schematized hereafter as system B and characterized by the e.m.f. values, AE'1,1 anld AENa'.
The titration is performed as follows. The solution of the complex is placed in one half-cell (side 2 of systern A) and NaCl Eiolution is placed in the other one (side 1 of system A). This latter solution is diluted or concentrated in order to have an e.m.f. equal to zero so that the activities of Na+ ion are equal in the two halfcells. The composition of the solution in side 1, l', and 2'of the two systemsA.andB are kept identical with each other throughout the titrations. After each addition of equal amounts of CaCl2 in both half-cells of the system and in half-cell 1' of system B, the potential differences AEN, andtAEN8' are read. A solution of NaCl is now added to the half-cell 2' of the system B until AENa' matches A E N (obviously ~ taking into account the necessary corrections due to small differences in the calibration curves of the two sodium electrodes). CaClz solu-
Mea+* 10 a Figure 4. Sequestering power of NaLS-PVAc. The three plots refer to the same amount of PVAc present in solution but at different XaLS-PVAc ratios. By increasing the detergent concentration, the total amount of Ca f 2 sequestered is increased.
Volume 68,Number 1% December, 1964
3628
C. B O T RF. ~ , DE MARTIIS, AND M. SOLINAS
.8
XNNa
-.
.7 .6
X104
-10
.5 .4 .3
15 .2 1
I
J
1 1
2
3
M
M ~ ~i o+3+
Figure 5 . Sequestering power of the complex toward calcium ions. The ordinate represents the amount of Ca+2 sequestered; the abscissa, the total amount of C a + 2present in solution. The two plots refer t o the same NaLS-PVAc ratio but a t different concentrations of the complex.
As it may be seen, the correction due to the sodium release is significant particularly when the concentration of ions in solution is large. However, the accuracy of released sodium ions in solution of surface active agents is significantly affected, probably by the adsorption of detergent on the surface of the glass. I n fact, the poor suitability of glass electrodes for measurhg pH in solution of detergents and similar substances was pointed out by Nebell’ who found some errors in p H measurements. Nevertheless, our aim was to study such a behavior in a semiquantitative way. The formation of a complex, with charge density strongly dependent on the charge density on the macroion, as well as on the ionic strength of the medium, was also found in solution of NaLS and BSA. In Fig. 6 (left ordinate, open points), the activity coefficient of sodium ions yNn is reported as a function of the concentration of the lauryl sulfate in solution of the NaLSBSA complex. As may be seen, a discontinuity may be observed, such a discontinuity also being evident with specific conductivity measurements of the same solution
T h e Journal of Physical Chemistry
~
a
~
~
.
i
~
2
Figure 6. y x e + (open circlets, left ordinate) and specific conductivity x (full circlets, right ordinate) us. the molarity of XaLS in a solution of the BSA-NaLS complex.
(right ordinate, full points). On the other hand, when the same experiment is performed in presence of CaC12, 5X 134, the discontinuity in the activity coefficient of sodium ions and in the specific conductivity is no longer detectable. I n agreement with other author^,^ it could be assumed that “micellar clusters” might be formed when the detergent acts on the macromolecule. Divalent cations might reduce the net charge of the complex as shown by previous plots, Such a reduction of the net charge of the complex was also claimed by some of the previously mentioned authors who ascribed to Ca+2 ions also an increase in solubilizing power of several protein detergent complexes.
Acknowledgments. The authors wish to express their best thanks to Colgate-Palmolive Co., S e w York, h-,Y., for having sponsored and financially supported this work, and to Dr. L. Shedlovsky for the useful discussion of the manuscript. The authors also wish to thank Prof. A. 31. Liquori for his extremely useful suggestions and stimulating discussions.
(17) E.Nebe, J . Electroanal. Chem., A21 (63) II/1959 52. (18) E.G.Jackson and U. P. Strauss, J . Polymer aci., 7, 473 (1951).
