D. G. HERRIES, W. BISHOP, AND F. M. RICHARDS
1842
The Partitioning of Solutes between Micellar and Aqueous Phases: Measurement by Gel Filtration and Effect on the Kinetics
of Some Bimolecular Reactions’
by D. G. Herries, W. Bishop, and F. M. Richards Departments of Biochemistry and Molecular Biology and Biophysics, Yale University, New Haven, Connecticut (Received February 3,1964)
During gel filtration on columns of Sephadex G-25, samples of sodium dodecyl sulfate at concentrations above the c.m.c. behaved qualitatively as would be expected for a reversibly polymerizing system, with the monomer units showing considerable adsorption to the gel matrix. When the eluting liquid contained sodium dodecyl sulfate (SDS) at concentrations above the c.m.c., the micelles were excluded from the interior of the gel particles. The elution behavior of additional solutes can be interpreted in terms of a partitioning between the micellar and ayueous phases; and. within a certain range, the distribution coefficients can be measured. Anisylthioethane partitioned strongly in favor of the micelles while iodine cyanide appeared to be excluded. However, the rate of the reaction between these two substances was unaffected by the detergent. The rate of the reaction between 1-fluoro-2.4-dinitrobenzene (FDXB) and glycineamide was unaffected by similar partitioning. The glycineamide cation distributes more strongly in favor of the micelle than sodium ion. However, the reactive free base form has a very low partition coefficient. The reaction of FDNB with glycylglycine was markedly reduced in the presence of SDS. The lowering can be explained if the reaction is assumed to be restricted completely to ‘the aqueous phase. Glycylglycine is apparently excluded from the micelles. The rate of the same reaction is enhanced in the presence of a cationic detergent. The ionic distribution in the double layer is clearly of great importance. The changes in absorption spectra of anisylthioethane in the presence of detergents are very similar to the difference spectra of tyrosine seen in various proteins. The chemical reactivity of the compound may or may not be changed depending upon the reagent used to study it. As in the case of many proteins, the “masking” of a functional group in the micellar systems is a relative, not an absolute, effect.
Introduction The literature reveals many instances of functional groups in proteins which appear to be “masked” or “unavailable” in the absence of denaturing conditions. Certain acid-titratable groups in hemoglobin,2 sulfhydryl groups in urease,a and methionyl and tyrosyl groups in ribonuclease4!6 are well-known examples. A commonly offered general explanation for such phenomena is the statement that the groups are embedded in a hydrocarbon environment in the interior of The
JOUTnal
of Phyaical Chemistry
the protein molecule. The spatiad separation of the functional group and the attacking reagent, or the nature of the surroundings, alters the kinetics of the (1) This work was supported by grants from the United States Publio Health Service and from the National Science Foundation. (2) J. Steinhardt and E. M.Zaiser, J . Biol. Chem., 190, 197 (1951). (3) L. Hellerman, F. P. Chinard, and V. R. Deitz, ibid., 147, 443 (1943). (4) D . Shugar, Bwchem. J . , 52, 141 (1952). (6) N. P. Neumann, S. Moore, and W. H . Stein, Biochemistry, 1, 68 (1962).
PARTITIONING OF SOLIJTES BETWEEN MICELLAR AND
AQUEOUS
1843
PHASES
was abnormally high for a "nonionic" detergent. The amberlite resins IR-120 (Hfform) and IRA-400 (OH- form) were used. The solid was recovered by lyophilization. The solidification point was 36.5' (the manufacturer's figure was 34'). Brij 35 is a polyoxyethylene(23)-lauryl ether,14 the degree of polymerization of the ethylene oxide units being an average value. The critical micelle concentration is reported as 0.014% w./v. by light scatExperimental tering14 and as 0.011% w./v.14 or 0.00.58~016 by the iodine solubilization method. The micelles appear to Materials. Anisylthioethane (ATE)6 was synthesized according to the following procedure. Anisyl be hydrated to the extent of 4-4.5 water molecules per ether oxygen. 16 Micellar molecular weights are chloride was prepared from anisyl alcohol by saturation about 49,000 as determined by light scattering, l6 of a solution in dioxane with dry hydrogen chloride. corresponding to an aggregation number of 40. The recovered product was purified by distillation, boiling point 98" a t 4-mm. pressure. Sodium ethane Cetyltrimethylammonium bromide (CTABr)6, Eastman technical grade, was extracted with diethyl ether thiolate was prepared as a white powder from ethane thiol and sodium dispersed in ether. A solution of 10.4 and then precipitated from solution in methanol by addition of ether. g. (0.066 mole) of anisyl chloride in 1.5 ml. of tetrahydrofuran was added in portions over 2 hr. to a stirred Sephadex (2-25, medium grade, was obtained from suspension of 5.9 g. (0.07 mole) of sodium ethane Pharmacia, Uppsala, Sweden. Iodoacetamide was thiolate in 100 ml. of tetrahydrofuran at 0". Stirring recrystallized from carbon tetrachloride until free of was continued a t room temperature for 3.5 hr. The iodine. All other chemicals were reagent grade comsuspension was filtered and the solvent removed by merical products and were used without further purirotary evaporation. The residue was distilled under fication. reduced pressure at 2.25 mm. The fraction boiling a t Measurement of Partition Coeficients by Gel Filtra118-120' was analyzed7 Anal. Calcd. for C1~H140S: tion. The cross-linked dextran gel Sephadex G-25 will C, 65.89; H, 7.74; S, 17.59. Found: C, 66.33; H, exclude from the interior of the gel particles a solute which is larger than about 4000-5000 in molecular 7.65; S, 17.73. Iodine cyanide was synthesized according to the weight. Micelles with molecular weights much larger procedure of Bak arid Hilleberte8 The product obthan this figure would be expected to move on a gel tained after washing with ice water and air-drying was column in the same fashion as any macromolecule. used. The iodide or iodine content was less than 0.2%. Inside the gel the detergent should exist as monomer The purest samples were obtained by washing with units at, or close to, the critical micelle concentration. petroleum ether or chloroform a t 0". The product was If the solvent for a Sephadex column consists of an stored in a refrigerator. aqueous detergent solution at a concentration well Sodium dodecyl sulfate (SDS)6 was obtained from above the c.m.c., the movement of an additional low Matheson Coleman rand Bell, Inc., and purified by recrystallization according to the method of Duynstee (6) The abbreviations used in this paper are: ATE, anisylthioethane: SDS, sodium dodecyl sulfate: FDNB, l-fluoro-2,4-dinitroand Grunwald. The large laminar crystals obtained benzene; CTABr, cetyltrimethylammonium bromide. were air-dried. (7) The microanalysis was performed by Dr. Stephen M . Nagy, The critical micelle concentration as a function of Department of Chemistry, Massachusetts Institute of Technology, Cambridge, hIass. temperature is given by Flockhartlo and as a function (8) R. Bak and A. Hillebert, O r g . Syn., 32, 29 (1952). of sodium chloride concentration by Mysels and co(9) E. F. J. Duynstee and E. Grunwald, J . Am. Chem. Soc., 81,4540, workers.11J2 The value in water a t 25' is 0.0081 M 4542 (1959). (0.234%). The micellar molecular weights are reported (10) B. D. Flockhart, J . Colloid Sci., 16, 484 (1961). as 11,500 by TartarI3 and 18,000 by Mysels and Prin(11) R. J. Williams, J. N. Phillips, and K. J. Mysels, Trans. Faraday Soc., 51, 728 (1955). cenlZ corresponding to aggregation numbers of 40 or (12) K. J. Mysels and L. H. Princen. J . Phys. Chem., 6 3 , 1696 63. (1959). Brij 35 was obtained from Atlas Chemical Indus(13) H. V. Tartar, J . Colloid Sci., 14, 115 (1959). tries, Inc., Lot No. 202. A solution of 10 g./lOO ml. (14) P. Becher and N. K. Clifton, ibid., 14, 519 (1959). was deionized by passage through columns of ion(15) S. Ross and J. P. Olivier, J . Phys. Chem., 63, I671 (1959). exchange resins since the conductance of the solution (16) P. Becher, J . Colloid Sci., 16, 49 (1961).
interaction. The experimentally observed differences in reactivity of a given group before and after denaturation can be very marked indeed. The object of this study was to see whether or not changes in rates of reaction could be observed in systems where the two reactants partition differently between an aqueous and micellar phase. Micelles were considered to be possible models for the apolar regions of a protein.
Volume 68, Nzimber 7
J d g , 1964
w.
D. G. HERRIES, BISHOP, AND F. ?vl. RICH.4RDs
1844
molecular weight solute added as a sample mill depend upon the partition coefficient of this latter material between the micellar and aqueous phases. The derivation outlined below follows closely that of Martin and Syngel7 for partition chromatography, with the inclusion of molecular sieving and micelle partitioning effects. Let h = height equivalent of one theoretical plate; A,, A,, A,, A. = cross-sectional areas of column, gel matrix, imbibed (stationary) liquid, and external V,,VI, Vo = volumes (moving) liquid, respectively; Vt, for whole column similarly defined; V, = effluent volume corresponding to maximum concentration in emerging band; p = aC, = volume fraction occupied by micelles in external liquid; d = partial specific volume of a detergent molecule in a micelle (or effective specific volume) ; K = partition coefficient of solute between micellar and aqueous phases; k = constant of proportionality between solute adsorbed per unit volume of gel matrix and equilibrium concentration of monomer solute in liquid (linear adsorption isotherm assumed) ; K D = “molecular sieving” constant, ratio of solute concentration in imbibed liquid to concentration in noiimicellar portion of external liquid; C = total concentration of detergent in eluting liquid in g./ ml.; c.m.c. = critical micelle concentration in g./ml.; C, = C - c.m.c. = concentration of micelles in starting solution in g./ml. Define
k’
=
+ V,)/V, or (&A, + A , ) / A , w = Kp + 1 - p
(kV,
I‘ = h(k’KDA1 fv
=
+ WAO)
(1)
(2)
Vi/(Ve - VO)
(3)
If m,, m,, ma,, and m, represent the fractional masses of solute initially present in the first theoretical plate adsorbed to gel matrix, in imbibed volume, in nonmicellar external volume, and in micelles, respectively, then mg
+ mi + ma, + m,
= 1
+ m,
=
Ao(1 - P)h I KpAoh - WAoh (4)
V
TFe Journal of Physical Chemistry
V
From here the development follows exactly that of Martin and Synge.I7 The band center will be a t the bottom of the column when a volume of eluent n6V = Yehas passed through. From the definition of the band position as the serial number of the appropriate theoretical plate, nWGV/V, and with substitution of the expression for V, the following equation is obtained.
Ve
~’KD
=
V, + 7Vi
Inserting the expressions for W and p and rearranging gives
Thus, the experimentally measured quantity f v should be a linear function of the micelle concentration for any given solute. Combination of slope and intercept terms will permit both K and the product k ’ k D to be determined if is known. I n the absence of micelles and adsorption effects (p = 0, IC = 0, k’ = I), eq. 6 reduces to the usual gel filtration equationLs
Ve
=
+
V O KDVi
(8)
If the solute appears only in the micellar phase ( K = a),then V , = V oand the material will appear to be completely excluded. If the solute is soluble only in the aqueous phase ( K = 0), eq. 6 becomes Ve
=
Vo
~’KD +_ _ Vi 1-P
(9)
The solute is retarded slightly on the column compared to its position in the absence of the micelles ( p = 0). The chromatography of Sephadex G-25 was carried out on columns 1.8 cm. in diameter with a bed height of about 24 cm. The columns were jacketed and operated at either 25 or 50’. The imbibed volume of the gel, Vi, was calculated from the water regain and wet density data (ref. 18, p. 28). The movement of a
Solve to get the fraction of solute in the moving liquid. mas
This solute is contained in a volume hAo. When an incremental volume of eluting liquid, 6V,is allowed to pass through the column, the fraction of the solute passing on to the next plate is
V
(17) A. J. P. Martin and R. L. M. Synge, Biochem. J . , 35, 1358 (1941). (18) P. Flodin, “Dextran Gels and Their Application in Gel Filtration,” Dissertation, Uppsala, 1962.
PARTITIONING O F SOLUTES BETWEEN h1ICELLAR AND
I
I
I
1845
AQCEOUSPHASES
R
There may be a trace of adsorption of this basic protein even in the salt solution. (In pure water it is adsorbed to the gel quite strongly.) The salt band appears just ahead of the V O Vi position. I n part b a sample of sodium dodecyl sulfate was eluted with water. The micelle peak is substantially retarded and is followed by a nearly constant plateau representing a concentration (0.24y0w./v.) close to the expected critical micelle concentration of 0.265% w./v.lQ The monomer detergent band emerges well beyond the V O Vi volume indicating marked adsorption to the gel. The retardation of the micelle peak is compatible with the theoretical treatment of Bethune and KegelesZn for countercurrent distribution of monomer-polymer equilibrium systems where the polymer partitions more favorably into the mobile phase. In part c a similar detergent sample was run on a column equilibrated with, and eluted with, detergent at the c.m.c. In this case the peak is much closer to the exclusion volume, V Oas , expected. When the column was operated in 0.2 M S a C l the micelle peak also appeared in this position, Fig. Id. At this ionic strength the c.m.c. is lowered by a factor of about 10 (0.029%) and in the figure is barely distinguishable above the background value of 1.17% NaCl. The lowered c.m.c. is equivalent to an increase in the monomer to polymer association constant; the chromatographic result is again in yualitative agreement with the treatment of Bethune and Kegeles.20 The behavior of two solutes run on a Sephadex G-25 column at various detergent concentrations is shown in Fig. 2 . Curve a1 shows the elution pattern of a sample of anisylthioethane run in 0.2 M NaC1. The large effluent volume indicates strong adsorption effects for this solute. When the solvent contained SDS at a concentration just below the c.m.c., a slight decrease in effluent volume was noted, curve a2 This effect may indicate a competition between the anisylthioethane and SDS for adsorption sites on the gel matrix. Above the c.m.c. the change in elution volume depends on the detergent concentration, curves a3 and a4. A threefold change in flow rate had no effect on the peak position. Thus, equilibration was presumably complete a t the highest rate used. The thioether partitions strongly in favor of the micellar phase. By contrast iodine cyanide is apparently excluded from the micelles as shown by the slight increase in
+
+
1' 11
vo+ vi
20
60
40
e f f l u e n t volume
-
ml
Figure 1. Elution diagrams from a column of Sephadex G-25. The gel bed was 24 cm. high and 1.8 cm. in diameter. The column was jacketed and rim a t 50". ( a ) Column equilibrated with 0.2 M NaCl. Sample of 56 mg. of ribonuclease in 0.2 M KaCl applied. Elution carried out with distilled water. ( b ) Column equilibrated with water. Sample of 110 mg. of sodium dodecyl sulfate (SDS) applied and eluted with water. (c) Column equilibrated with 0.28% w./v. SDS. Sample of 55 mg. of SDS applied and eluted with same dilute SDS solution. ( d ) Column equilibrated with 0.2 M NaCl. Sample of 91 mg. of SDS applied and eluted with same solvent. The various ordinates with different scales give the dry weights of each fraction as per cent. The abscissa is the same for all four patterns. The column parameters V Oand V,are shown by the arrows.
band of india ink or a sample of the enzyme catalase gave the excluded volume, Vn. The total bed volume was calculated from the geometry of the column. The behavior of such a column is shown in Fig. 1. Fractions of 0.5, 1.0, or 2.0 ml. were collected in weighed test tubes and the total solute determined by weight after evaporation to (dryness of the tube contents In part a the column was equilibrated with 0.2 M KaC1 and loaded with a sample of the enzyme ribonuclease in the same solvent. Subsequent elution was carried out with water. The pattern shows the protein peak appearing just behind the V o position.
(19) An increase of 12.5% in the c.m.c. for water solution of SDS between 25 and 50' was found by Flockhart.10 This increase was applied t o the value found in 0.2 M NaCl a t 25'12 to obtain an estimate for the value in the salt solution at EOo. (20) J. L. Bethune and G. Kegeles, J . Phys. Chem., 65, 433 (1961).
Volume 68, h'umbei 7
Julz/, 1964
D. G. HERRIES, W. BISHOP,A N D F. M.RICHARDS
1846
elution volume a t high detergent concentrations, curves b l and b2. Elution patterns were obtained for samples of pnitrophenol a t various concentrations of sodium dodecyl sulfate. The data have been plotted according to eq. 7 and are shown in Fig. 3. The straight line prediction from the equation is quite accurately followed and the partition coefficient is easily estimated from the slope of the line. These data and some for other compounds are summarized in Table I. a2 a1
"
"
& : a
I
- "
0 0
"
o "
Table I : Effect of Sodium Dodecyl Sulfate on the Elution Parameters of Various Solutes on Columns of Sephadex G-25 I
I
20
60
40
effluent volume
80
-
I(
ml
Figure 2. Effect of SDS on elution patterns of some low molecular weight solutes. The column is the same as that described in Fig. 1. For the curves labeled a the sample in each case was 0.34 pmole of anisylthioethane. The equilibrating and eluting solvent was 0.2 M KaCl containing in addition for a1 nothing, a2 0.02% w./v. SDS, a3 0.047, w./v. SDS, a4 1.0% w./v. SDS. In a4 the column was operated a t a flow rate of 0.25 ml./min. (solid line). The crosses refer to a duplicate run a t a flow rate of 0.8 ml./min. The effluent was analyzed by absorbance a t 276 m l . For the curve b l the sample was 9.3 pmoles of iodine cyanide and the eluting liquid 0.2 M XaC1 with 0.04% w./v. SDS, b2 5.6 pmoles of iodine cyanide and the same liquid except 1.0% w/v. SDS. The effluent was analyzed by absorbance a t 230
Parameters derived from -eq. 7-
Column volume -parameters, m1.Solutea
Iodine cyanide, Anisylthioethane
p-Kitrophenol
Sodium p-nitrophenolate 1-Fluoro-2,4dinitrobenzene Glycylglycine Glycineamide
slope = ? ( K - l ) / k ' K o
f--
.01
intercept = I / k'Ko
.02 Cm
.03
-
.C4
.05
.06
g m / ml
Figure 3. Elution of p-nitrophenol on a column of Sephadex G-25 a t 25" in the presence of SDS. The column parameters and elution volumes are given in Table I. The solvent was 0.1 M NaCl adjusted to pH 3.0 with HC1 and contained SDS a t the concentrations indicated. The data are plotted according to eq. 7 with the volume function, f v , as ordinate and the micelle concentration, C ,, as abscissa
The Journal of Physical Chemistry
'Vt
vi
r/O
Elution SDS volume, oonon., ml. g./ml. v, x lo1 k'KD
5 9 . 3 22.9 29.2 Same
75.4 27.0
Same 6 3 . 0 22.6
57.5 58.0 88 85 79 30.5 3 8 . 2 116 66 52 41 111 122 32.0 70 48
0.4 10.0 0 0.2 0.4 10.0 0.4 14.4 28.8 57.6 0.4 28.8 0.4 14.4
47 49 51 40.5 43 46.5 50
0.4 14.4 0.4 14.4 14.4 14.4 14.4
Same Same (PH 5 , 7 ) (PH 7 , 5 ) (PH 8 . 0 ) (PH 9 . 2 )
1.18
5(K -1)
b
2.12 708
2.38
94
2.20
b
1.48
62
0.76
b
1.13 1.13 1.13 1.13
40 27 12 2
a For the first two solutes the column was operated a t 50") and the solvent was 0.2 M NaCl containing the indicated concentration of SDS. For the rest of the solutes the column temperature was 25'. The solvent in each case had an ionic strength of 0.1 M made up largely of NaCl. For p-nitrophenol the pH was adjusted to 3.0, for sodium p-nitrophenolate to 11.0, for F D X B and glycylglycine to 8.0, for glycineamide as indicated. a The retardations are larger than the simple theory would predict as possible. The calculated partition coefficients would be negative. The value of K is clearly small but no numerical value can be assigned.
In the case of p-nitrophenol a separate estimate of the partition coefficient was made. The apparent ionization constant is affected by the presence of SDS
1847
PARTITIONING OF SOLTJTES BETWEEN MICELLAR AND AQTJEOUS PHASES
a t concentrations above the c.m.c. The pK’ values were obtained from spectrophotometric titrations using the absorbance at 400 mp as a measure of the concentration of the p-nitrophenolate ion. The protonated form of the phenol shows no absorption at this wave length. The ionized form does not enter the micelle and its spectrum is not appreciably affected by the presence of the detergent. The total ionic strength of the solvent was 0.1 N made up in part by phosphate buffer. The pH of the various solutions was adjusted with 1 N acid or base. The absorbance was measured on a Perkin-Elmer Model 350 recording spectrophotometer. The total concentration of p-nitrophenol was 1 X low4M . Complete ionization gave an absorbance at 400 mF of 1.8. The data are shown in Fig. 4. Let
6
5
I
I
I
7
8
9
I
Figure 4. Ionization of p-nitrophenol in the presence of sodium dodecyl sulfate. The fractional ionization, a , was calculated from the absorbance of the p-nitrophenolate ion a t 400 mp. The total p-nitrophenol concentration was 1 X M . The solvent was 0.004 Af sodium phosphate buffer, or 0.004 M glycylglycine lbuffer for the higher pH values, adjusted to the pH indicated and to a constant ionic strength of 0.1 M with TaCl. The solid lines are standard titration curves for the dissociation of a monobasic acid. The SDS concentrations in g./ml. were: a, 0; b, 0.0144; c, 0.0288; d, 0.0576.
C N p H = concentration of un-ionized p-nitrophenol in aqueous phase; CXP- = concentration of ionized p-nitrophenol in aqueous phase; C’NPHand C’NP- = equivalent concentrations in micellar phase; KNPHand KNP- = partition coefficients for the indicated forms; Q: = fraction of total p-nitrophenol in ionized form. Then PC’NPPC’NPH
+ (1 -
+ (i -
=
pK’
+ log CNPH CNP -
where pK’ refers to the ionization of p-nitrophenol in the absence of micelles.
KNp- is assumed to be zero. rearrange. ApK‘
=
log
1
Substitute for P and
+ ~ ( K N P-H1)Cm 1-
ac,
(14)
~(KNPH 1) = 94.3 is obtained directly from Table I. If 8 is assumed to be 0.9 ml./g.,21ApK’ can be calculated for various values of C,. The results are shown in Table 11. A more detailed series of measurements would be required to decide whether the small differences between the calculated and observed values are real.
Table 11: The Influence of Sodium Dodecyl Sulfate on the Ionization of p-Nitrophenol“ ApK’--
PH
__1- a
pH
P)CNP-
-
P)cNPH
+ +
(PKNP- 1 -. @)CNP(10) (PKNPH 1 -- P)CNPH
SDS conon., g./ml.
Caicd.*
Obsd.
0,0144 0.0288 0.0576
0.38 0.58 0.83
0.32 =t0.04 0 . 5 2 f 0.04 0.80 f 0 . 0 4
a
The total ionic strength was 0.1 M and the temperature 25’. See text.
* Values obtained from eq. 14. _ I
The partition coefficient for anisylthioethane between Brij 35 micelles and water at 50” was estimated from solubility measurements. Solutions of the detergent varying in concentration up to 2.6% w./v. ~ e r satue rated with the thioether. The total thioether content was estimated spectrophotometrically on aliquots diluted to a constant detergent concentration, by reference to standard solutions in the same solvent. The results are summarized in the equation y = 0.00068
+ 0.390Cm
(15)
where y is the solubility of anisylthioethane in moles per liter of solutjon, and C, is the concentratioii of Brij 35 in g./ml. (The c.m.c. is so low for this detergent that it may be neglected.) (21) P. Mukerjee, J . Phys. Chem., 66, 1733 (1962).
Volzime 68, Xtimber 7 Jirly, 1964
1848
D. G. HERRIES,W. BISHOP,AND F. M. RICHARDS
I n a unit volume of solution, let n,, be the moles of solute in the aqueous phase and n, the moles in the micellar phase. Then
Let y represent the fraction of the solute in the aqueous phase
For a saturated solution the concentration of solute in the aqueous phase is constant, ys = naq'(l - 0). The total concentration, y = nap n,, is then
+
Y
=
YdKP
+ 1 - P)
cated the assay but corrections could be applied. The iodide analysis has been described by I-Ierries and Richards23 The reaction was carried out at 50' with a substantial excess of iodine cyanide. The rates were thus pseudo-first-order in anisylthioethane. The second-order rate constants were calculated by dividing the first-order values by the initial concentration of the iodine cyanide and by applying the appropriate unit conversion .factors. The results of some typical measurements are shown in Table 111. The rate of the reaction does not appear to be significantly affected by the presence of the micelles in spite of the fact that one reactant partitions stroiidy in favor of the micelles while the other appears to be excluded. Similar results were obtained for this reaction in the presence of the nonionic detergent Brij 35.
or y =
~s
+ ys(K - 1)eCm
(18)
From eq. 15 and 18 it is seen that ( K - l)fi = 574 for anisylthioethane in Brij 35. This value is similar to that for the same thioether in SDS, 708, as determined by the gel filtration method (see Table I). In the derivations for both methods ideal behavior is assumed, that is, K is independent of solute conceiitration. Within the limits of error of the present measurements, this assumption appears to be valid. Kinetic Measurements The initial kinetic studies were made on the following system (HOCH2CHz)zS
+ ICH~COSHz+ +
(HOCW,CH2)ZSCHzCONH2
+
+ I-
(19)
Anisylthioethane, M x 103
Iodine cyanide, M x 10s
Slope of firstorder plot, min.-1 X 104
Second-order rate constant, A4 -1 see. - 1
None 0,678
18.5
0.542
14.8
-4.8 -4.4
0.361
21.1
-6.4
0.0010 0.0012 0.0012
0.05 A!! sodium dodecyl sulfate 0.542
13.7
-6.3
0.0018
0.542
19.6
-7.1
0,0014
a The conditions for the reaction were 0.001 M acetate buffer pH 5.5 at, 50". Other additions to the solvent are indicated. The reaction waa followed by the appearance of iodide ion.
A different set of experinleiits was carried out on the reaction of l-fluoro-2,4-dii~itrobeiizeiie with both glycylglyciiie aiicl glycineamide. I n each case the amine
--+
Here the reaction was followed by a chemical measuremeiit of the appearance of iodide ion. Aliquots of the reaction mixture were acidified. The reaction of Iwith excess IC?; produced iodine which was estimated colorimetrically. Oxidative side reactions compliThe Journal of Physical Chemistry
Iodine Cyanide"
0.2 M sodium chloride
The rates of 50' were estimated frbm the change in conductance as a function of time. For constant coiiceiitratioiis of the two reactants, no changes in rate were observed when the solvent was changed from very dilute acetate buffer a t pH 5.5 to an equivalent solution contaiiiiiig the nonionic detergent Brij 35. Sulfonium salt formation was studied in a second reactionz2 where the partition coefficients of the reactants were also estimatrd. C I I ~ - O ~ C H ~ - S - C ~ HICN ~
Table 111: The Reaction of Anisylthioethane with
(22) I t is assumed that the initial step is the formation of the cyanosulfonium salt as in the similar reaction with methionine peptides (B. Witkop, A d a m . Protein Chem., 16, 265 (1961)). If the initial step is in fact the formation of a thiocyanate and an alkyl iodide, subsequent hydrolysis of the halide to yield iodide ion would be a necessary second step. The interpretation of possible solvent effects would depend on which is the rate-limiting step. (23) D. G . Herrie$ and F. M . Richards, Anal. Chem. 3 6 , 1155 (1964)
PARTITIONING OF SOLUTES BETWEEN MICELLAR AND AQUEOUS PHASES
was present in a large molar excess and the reactions were followed by the a,ppearance of the yellow color of the dinitrophenyl glycyl derivative. Measurements were made a t 355 ink in a Beckman R'Iodel D B spectrophotometer using the therinostated cell compartment a t a temperature of 25'. The rates followed first-order kinetics ace urately to at least 80% reaction. The rate data for the FDNB-amine'reactions and the calculated values of y (eq. 17) for FDNB in the presence of SDS are given in Table IT. The aqueous hydrolysis of FDXB in SDS solution under the conditions used was negligible in comparison to the rate of the amine reaction and no correction was required.
1849
concerning purity of the detergent and nature of the blank reaction, no detailed kinetic studies have yet been made with this cationic detergent, nor are the relevant partition coefficients known. Spectra of Anisylthioethane. The ultraviolet absorption spectra of anisylthioethane in a nuniber of different solvents are shown in Fig. 5. The data were
A
-Table IV : Reaction of l-Fluoro-2,4-dinitrobenaenewith Glycineamide and Glycylglycine' Concentration of detergent, g./ml.
Pseudo-first-order rate --constant, min. - 1 - 7 Glycineamide Glycylglycine
Relative rate glycylglycine
Fraction of FDNB in aqueous phase, y
Sodium dodecyl sulfate
0 0,0144 0.0288 0.0576
0.0170 0.0173
0,0153
1.00
1.00
0.0089
0.59
0.0177
0.0058 0.0043
0.38 0.28
0.52 0.35 0.26
0.0169
Cetyltrimethylammonium bromideb
0.02
0.072
0,228
a Reactions were carried out in sodium phosphate buffer pH 8.00 ionic strength 0.1 .et 25". The amine concentration in each was 0.0193 M. ThLe F D S B concentration was the same in all experiments and equal to about 1 X M . Change in absorbance a t 360 rn@ was used to follow t'he reaction. y was calculated from eq. 17 witJhB(K - 1) = 62.0as given in Table I. The c.m.c. was assumed t o be 0.00147M as determined in 0.1 M NaCl (J. S.Phillips and K. J. Mysels, J . Phys. Chem., 59,325 (1955),and fl t o be 0.9 ml./g.21 * The reaction of F D X B in the presence of detergent but without added amine was not negligible. The rate was about 0.007 min.-'. The values given for the two amines have not been corrected.
Some preliminary nieasureinents in the presence of cetyltrimethylaminoniuin bromide are also listed in Table IV. Apparently the sample of detergent still contained traces of amine even after purification. The rate of color appearance with FDNB in the absence of added amine was much greater with CTABr than with SDS. From color change on acidification the product appeared to be a inixture of a dinitrophenylamine and of dinitrophenol. The appearance of a significant quantity of the latter compound indicated that the rate of hydrlolysis of FDNB was enhanced in the presence of CTABr. Because of the .uncertainties
I I I I I I 1 'ru-s_lI 260 270 2 8 0 290 300 310 wavelength - mjJ. Figure 5. Spectra of anisylthioethane in various solvents. The concentration of the thioether in each case was 7.25 X M : a, water; b, 5.11% w./v. Brij 35 aqueous solution; c, pure liquid Brij 33; d, 1 0 0 ~ o Ethyl Cellosolve; e, 100% ethanol; f, dry diethyl ether; g, n-heptane. All measurements mere made a t 2 2 f 1" except for c where the sample was warmed above the melting point. The ordinates of the curves have been displaced for clarity. The base lines for the various curves are shown by the pairs of arrows.
obtained on a Perkin-Elmer ' "Spectracord" recording spectrophotometer. The usual red shift and peak sharpening are observed in going from water to hydrocarbon solvents. In water the absorbance niaxiniuni of the solute is a t 276 nip with a molar extinction coefficient of 1150; in n-heptane the values are 279 nip and
D. G. HERRIES,W. BISHOP,AND F. $1. RICHARDS
1850
1570. The spectrum in the liquid Brij 35 (curve c) is clearly different from that in the aqueous detergent (curve b) even though 97% of the solute is associated with the micelles in the latter case. The solutions used for curves a and b in Fig. 5 provided the difference spectrum shown in Fig. 6, curve b. For curve a in the latter figure the detergent concentration was lowered by a factor of 10. Under these conditions 757& of the solute is in the micellar phase. The observed lowering of the difference spectrum by about 20% between curves b and a (Fig. 6) is compatible with this estimate. The difference spectra, relative to pure water, given with various Ethyl Cellosolve-water mixtures are shown in the upper part of Fig. 6. These solutions were picked as models for the possible environment of the chromophore in the micelle. Curve e representing a 50:50 mixture of Cellosolve and water is in fact very similar to the lower curves a or b. However, this is not a very sensitive test, as a variety of solvent mixtures could probably be designed to give about the same fit.
T I
‘I
I
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I
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I
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The difference spectra resemble those found for anomalous tyrosine residues in a number of proteins or for tyrosine itself and various derivative^.^^ !\luch of the recent work in this field has been reviewed by Wetlaufer.25 From these data alone it is not possible to specify the location of the chromophore in the micelle (ie.,whether it is in the surface region or in the interior). The elegant solvent perturbation technique of Herskovits and Laskowski26 mould be difficult to apply in this case because of the unknown effect on micellar structure.
Discussion
~
A
Lo
a, v
IT
a,
U a,
wavelength
-
mp.
Figure 6. Difference spectra for anisylthioethane. An M , was placed aqueous solution of the thioether, 7.25 X in the reference beam. The sample consisted of the same concentration of the solute in the various solvents: a, aqueous solution of Brij 35, 0.51170 w./v.; b, same as a but5.1170 w./v.; c, Ethyl Cellosolve (1 part):water ( 5 parts); the following same a s c e x c e p t : d, 1:2; e, l:l;f,2:1;g,5:1;h,pureEthylCellosolve. All measurements a t 22 f 1’. The ordinates for the curves e-h have been displaced for clarity. The base lines are shown by the pairs of arrows.
T h e Journal of Physical Chemistrv
The gel column procedure would appear to be suitable for the estimation of partition coefficients in the range of 10-1000 or perhaps even 10,000. However, it is of little use for quantitative estimates of values close to or less than unity because of the small volume fraction occupied by the micelles. For materials excluded from the micelles eq. 9 predicts a small increase in elution volume. For three of the substances in Table I such an increase was observed, but in all cases it was larger than expected. There is some uncertainty in the estimate of the effective micelle volume. Calculation on the basis of partial specific volume of the detergent may not be appropriate. In particular, with an anion such as glycylglycine, charge repulsion in the region of the double layer of a negatively charged micelle would be expected to increase the effective volume over that applicable to an uncharged species. At an ionic strength of 0.1 M such an effect could not explain the very large retardation observed for the p-nitrophenolate ion. S o explanation has yet been found for the behavior of this substance. The titration data indicate that p-nitrophenol does not ionize while associated with a micelle of SDS. Substantial changes in pK’ values are thus produced when the detergent concentration is increased. The effect is presumably a result of the combination of the apolar environment, which would discourage charge separation, and the repulsion by the negatively charged micelle of the anion resulting from the ionization. The single set of data reported is not sufficient to provide any experimental basis for estimating the relative magnitude of the two effects. In 0.1 M aqueous sodium chloride the measured pK’ for glycineamide was 7.88 .fi 0.04 at 25’. At pH 5.7 where more than 99% of the compound is in the form (24) D. B. Wetlaufer, J. T. Edsall, and B. R. Hollingworth, J. Bid. Chem., 233, 1421 (1958). (25) D. B. Wetlaufer, Advan. Protein Chem., 17, 304 (1962). (26) T. T. Herskovits and M. Laskowski, Jr., J. B i d . Chem., 237, 2481 (1962).
PARTITIONING OF SOLUTES BETWEEN MICELLAR ASD AQUEOUS PHASES
of the cation, the measured partition coefficient was 44 in the presence of SDS (Table I). The lower observed values a t more alkaline pH can be predicted from this coefficient and the pK ’ if, in addition, it is assumed that the partition coefficient for the uncharged form of the amide is very low. The free base is apparently sufficiently polar that it shows little tendency to enter the micelle. The binding a t acid pH is obviously directly related to the positive charge. I n a solution 0.1 M in sodium chloride and 0.05 M in SDS, essentially all of the detergent is present as micelles. If it is assumed that the full complement of counterions moves through the solution a t the same rate as the micelles, then one-third of all the sodium ions present in the solution would so move. From eq. 17 with y = 0.67, one obtains a value for K for the sodium ions of 38. This is a maximum value. The charge neutralization is not so effective. The estimate for the number of bound sodium ions and the derived value of K may easily be too high by a factor of 2. The discrepancy with the directly measured value of K for the glycineninide cation, 44, becomes larger. It would appear that the glycineamide cation is preferentially bound to the micelle relative to the sodium ion. The effective binding of the sodium ion could, of course, be directly measured by this technique using an appropriate isotope as a sample. Only a few kinetic studies of reactions in the presence of detergents have been found in the literature. If neither reactant enters the micelle little change in rate would be expected. Erikson and IJingafelterZ7have reported that the effect of long-chain alkylsulfonates on the reaction between thiosulfate and bromoacetate ions was predictable entirely on the basis of ionic strength. These small anions were almost certainly excluded from the negatively charged micelles. When one or both of the reactants partitions in favor of the micelles, a variety of effects have been found. Kolthoff and JohnsonZ8not,edthat the reaction between iodine and acetone to yield l-iodo-2-oxopropane, hydrogen ion, and iodide ion was unaffected by the presence of dodecylamnioniuni chloride, even though most of the iodine was solubilized by the micelles. (The partitioning of the acetone is uncertain.) There could be a cancellation of opposing effects in this system, higher effective reactant concentrations in t,he micelles and a lower intrinsic rate ctonstant in the less polar environ-. merit. I n the reaction of beiizylideneaniline with water to yield benzaldehyde and aniline the rate was retarded by the presence of cetyltrirriethylammoiiiuni bromide.29 The reaction was carried out a t pH 9 in borate buffer. The formal reactants and products are all uncharged but their distribution is unknown. Duyns-
1851
tee and Grunwald9 have examined both the kinetics and the equilibria of the reaction of triphenylmethane dyes with hydroxide ion in the presence of various detergents. The equilibria and rate effects paralleled each other and were in qualitative agreement with the rules established by Hartley30 for the interaction of ions and micelles. The complexing of porphyrins with detergents can produce very dramatic changes in porphyrin-metal ion reactions.31 The divalent, cation is repelled by micelles with positive charge and attracted by micelles with negative charge. Rate differences of several order of magnitude are produced. The principal effect appears to be through the entropy of activation. In a particularly interesting study, I i u r ~has ~ ~examined the rate of hydrolysis of monoalkyl sulfates as a function of chain length. Micelle forniation with the higher homologs had no efiect on the uncatalyzed rate. However, the proton catalyzed rate was accelerated and the hydroxide catalyzed rate retarded by micelle formation. In this system the reaction takes place a t or close to the surface of the micelles since the polar sulfate “heads” must be immersed in the aqueous phase. The acceleration of the proton catalyzed rat2 appeared to be principally the result of a lowering of the enthalpy of activation. I n the present study the rates involving neutral reactants (eq. 20 or 21) were unaffected by the presence of micelles even though in each case one species partitioned strongly in favor of the micelles and the other was apparently excluded. The products of the reactions are ions, and the transition state must involve charge separation. For such reactions the rate would be expected to decrease as the polarity of the environment is lowered. For the FDNB reactions, however, the effect may not be very great. As shomn by Bunnett and R l ~ r a t hthe ~ ~o-nitro group niay serve as “internal solvation” for the reaction. It is probable that the reactions are taking place at the niicelle surface. However, there is no obvious reason why a substance such as anisylthioethane should prefer the surface of the micelle to its interior.
(27) J. A. Erikson and E. C. Lingafelter, J . Colloid Sci., 10, 71 (1955). (28) I. M. Kolthoff and W. F. Johnson, J . Am. Chem. Soc., 73, 4563 (1951). (29) K. G. Van Senden and C. Koningsberger, Tetrahedron Letters, 1 , 7 (1960). (30) G. S. Hartley, Trans. Faraday Soc., 30, 444 (1934). (31) M. B. Lowe and J. N. Phillips, Nature, 190, 262 (1961). (32) J. L. Kurz, J . Phys. Chem., 66, 2239 (1962). (33) J. F. Bunnett and R. J. Morath, J . Am. Chem. Soc., 77, 5051 (1955).
Volume 68, Number Y
J u l y , 1964
1852
G. R. BELTON A N D R. L. R~ICCARRON
A comparison of the last two columns in Table I V shows that the reaction between glycylglycine and FDXB occurs principally, if not entirely, in the aqueous phase when the detergent is SDS. The concentration of anions, even at an ionic strength of 0.1, must be markedly reduced near the micelle surface for a radial distance of at least the length of the glycylglycine niolecule. The reverse effect is seen in the presence of the cationic detergent cetyltrimethylammonium bromide. The preliminary experiments indicate that glycylglycine reacts with FDNB coiisiderabIy inore rapidly than does glycineamide as a result of the reverse distribution in the double layer. The binding of the glycineamide cation to a micelle of SDS does not alter the kinetics of the FDNB reaction since it is only the free base forni of the amine which enters into the reaction. In the case of the cationic detergent and the glycylglycine anion, the charge interaction is with the carboxyl group and the effective c( iicentration of the amine will be increased at the micelle surface.
It is unfortunate that the low partition coefficieiits cannot be measured more accurately. The conclusions about the surface nature of the reaction depend very largely on the assumption of complete exclusion of one of the reactants. The thioether portion of anisylthioethane resembles the side chain of methionine while the benzene ring provides a chromophore whose absorbance is sensitive to its environment. The measured partition coefficient indicates that such a conipound, were it part of a polymer chain, would contribute significantly to the stabilization energy of a folded structure which would resemble a micelle. In so doing, its spectral characteristics would be changed but its cheinical reactivity would depend very much on the exact nature of the reagent used in studying it. In this sense the detergent systems have reproduced many of the characteristics frequently found in proteins. However, the micelles appear just as complicated as the proteins for which we had hoped to use them as models.
The Volatilization of Tungsten in the Presence of Water Vapor
by G. R. Belton and R. L. McCarron Department of .lfetalZurg~, University of Pennsylvania, Philadelphia, Pennsylvania (Received Februarv 6 , 1964)
h study has been made, using the transpiration technique, of the enhanced volatility of tungsten in mixtures of hydrogen and water vapor. The volatilization is shown to occur 3Hz(g). The standard free energy 4H*O(g) = WO,H,O(g) by the reaction W(s) change for the reaction between 1200 and 1500° is given by: A F o = 26,700 - 5.56T cal.
+
Introduction The enhanced volatility of tungsten oxides in the presence of mater a t 1000" arid 1 atm. total pressure was first reported by AZillner and Neugebauer.l Froin their data Grossweiner aiid Seifert2 later showed that the gaseous species W03H20 could be responsible for the enhancrd volatility. Gleinser and his co-n orkers have used a transpiration technique to study in more detail the volatility of solid W 0 3 in nitrogen-water The Journal of Physical Chemistry
+
vapor mixtures at 1100-1200 "3 and in oxygen-water vapor mixtures at 900-1100".4 AIeyer, et al.,5 have (1) T. hlillner and J. Neugebauer, Nature, 163, 601 (1949). (2) L. Grossweiner and R. L. Seifert, U. S. Atomic Energy Commission Unclassified Paper AECU-1573. (3) 0. Glemser and H. G. Voltz, Third International Congress on the Reactivity of Solids, Madrid, April, 1956: also Araturwissenschaften, 43, 33 (1956). (4) 0. Glemser and R. Haeseler, 2. anorg. allgem. Chem., 316, 163 (1962).
