Oct., 1963
HYDROPHOBIC CONTRIBUTIOX TO MICELLE FORMATIOX
activities of CaClz have been determined in the presence of NaCl and KCl in the ratio of Carl/(Kf or N a f ) of 1/100. Harned’s rule has been corroborated for systems such as BaClz-KaC1, B a C k K C l , CaClz-NaC1, CaC12-KC1 in ratios of (alkali metal cation)/(alkaline earth cation) 5 100. I n principle, one could measure pX, where X could be any ionic species. The multilayer membranes are ion-selective as well as ion-specific. Anions have no effect on the transport of cationic species in the membrane phase. However, they will affect the activities of these ions in solution. L. SHEDLOVSKY.-’rhe “glass electrode” which we used to determine pNa respoiids to various monovalent cations. However, the influence of salts of divalent cations such as Ca+z is primarily a function of ionic strength. E. D. GODDARD (Lever Brothers Company).-The fractionating data are interesting and it would be useful to have con-
2079
firmatory evidence from another source. A possibility here would be the use of ultrafiltration, although complete separation of micellar from non-micellar components, of course, could not be achieved. Changes in composition with time would probably yield the required information.
L. SHEDLOVSKY.-E.Hutchinson [Z. physik Cherr,., 21, 38 1929)] has shown that after ultrafiltration of sodium decgl or sodium dodecyl sulfate solutions alone above the c.m.c., the concentration of the filtrate remained almost constant a t the critical value, while the concentration of the solution inside the nitrocellulose membranes steadily increased throughout the course of the experiment. If i t should prove to be possible to apply Hutchinson’s method to binary mixtures of homologs of alkyl sulfates, then a direct measure of non-micellar compositions will be available; thus, referring to Fig. 3, from a solution of composition B, a filtrate of composition A would be recoverable.
THE HYDROPHOBIC CONTRIBUTION TO MICELLE FORMATIOX : THE SOLUBILITY OF ETHANE, PROPANE, BUTANE, AND PENTANE I N SODIUhI DODECYL SULFATE SOLUTION1 BY
ARNOLD W I S H N I A
Department o j Biochemistry, Dartmouth Medical School, Hanover. New Hampshire Received March 8, 1963 From the ratio of the solubility of the alkane in detergent solutions and in water a t different temperatures one can obtain AF, AH, and A S for the transfer of alkane from water to the SDS micelle. Such studies, using both manometric and radioactive tracer techniques, have been carried out. The parallelism between these data and the parapeters for transfer of these alkanes from water to nonpolar liquids strongly supports the hydrocarbonliquid model of the micellar interior. At 25’ AFT RAN^ per CHZis 0.8 kcal. ( - 1.7 kcal. per half-ethane). The data for AHTRANSare not as certain and cannot yet be used to predict the hydrophobic contribution to AH of micelle formation for longer chains, but it is clear from the large positive values of the entropies of transfer of the CZ-C~alkanes (17-18 e.u.) that the effect of the alkanes on the structure of water, as pictured in current theories, must be the key factor in micelle formation.
-
Introduction
It has long been realized that to obtain micelle formation in detergent solutions rather than phase separation the cohesive ]forces leading to association must be accompanied by repulsive forces in such a way that the ratio of repulsive to cohesive energies diverges as the association number n becomes sufficiently large. I n Debye’s theory”‘ the decrease in free energy arising from hydrocarbon interactions given by -nwa is eventually overcome by electrostatic repulsion growing with Debye also argued that the distribution of micellar sizes was sufficiently sharp to be represented by a single size His theory has been criticized as to its detailszb; the parallel plate model of micelles has also been modified or discarded. More recently, Hoeve and Benson3 proposed a statistical mechanical theory of micelle formation in which the repulsive forces appear as a “crowding” term depending on Che ratio of volume to surface, as well as, for ionic detergents, an electrostatic term. The interior of the micelle is treated as a hydrocarbon liquid; the rest of the cohesive forces is contained in the partition function of the monomer, which contains a (not explicitly evaluated) contribution from the so-called “hydrophobic interactions” between the water structure and the alkyl chains; finally, a term for the residual interfacial energy of the oily micellar surface is included. (1) This work was supgorted in part b y PHS Grant RG 8121 and NSF Grant G 13973. (2) (a) P. Debye, Ann. N . Y. Acad. Sei., 61, 576 (1949); (b) J. J. Hermans, Koninkl. Ned. Akad. Wetenschap. Proc., Ser. B, 68, 91 (1955). (3) C. A. J. Hoeve and G. C. Benson, J. Phys. Chem., 61, 1149 (1957). A discussion of several previous theories is included.
The largely entropic role of “icebergs4” in micelle formation has been emphasized in recent years by investigators who obtained relatively IOW heats of micellization ~alorimetrically~-~ from temperature dependence of critical micelle concentration ( C . ~ . C . or ) ~ ,from ~ vapor pressure measurements.10 This author’s own interest in hydrophobic interactions stems from studies of their role in stabilizing protein structures. The demonstration by Ross and Hudson11 that c.m.c. could be determined from the solubility of butadiene in micellar solutions led this author to investigate the solubility of propane and butane in protein soIutions.12 From a comparison with solutions of sodium dodecyl sulfate (SDS) the thermodynamic parameters for the transfer of an alkyl group from aqueous surroundings to a hydrocarbon cluster as well as other information about the protein could be derived. This paper, which also includes data for ethane and pentane, will be directed to the problem of micelle formation. The work reported here is consistent with a liquid hydrocarbon model for the interior of micelles, and will allow an estimation of the hydrophobic con(4) H. s. Frank and M. W. Evans, J. Chem. P h y s . , 13, 507 (1945). (5) E. Hutchinson and L. Winslow, J . Phys. Chem., 60, 122 (1956). (6) E. D. Goddard, C. A. J. Hoeve, and G. C . Benson. %bid., 61, 593 (1957). (7) P. White and G. C. Benson, ibid., 63, 599 (1960). (8) G. Stainsby and A. E. Alexander, Trans. Faraday Soc., 46, 587 (1950). (9) B. D. Flockhart, J . Colloid Sei., 16, 484 (1961). (10) D. Moule, P. White, and G. C. Benson, Can. J . Chem., 87, 2086 (1957). (11) S. Ross and J. B. Hudson, J . Colloid Sei., 12, 523 (1957). (12) A. Wishnia, Proc. Natl. Acad. Sci., 48, 2200 (1962).
Yol. 67
ARNOLD?VISHXIA
2050
first, there is no other functional group giving additional interactions; second, not only can equilibrium measurements be made, but the activity of the hydrocarbon in the gas phase in equilibrium with the aqueous solution can be varied a t will, allowing determination of AF, A H , and A S of transfer of the alkane for water to the micelle under conditions where the concentration of solute in the micelles as well as in water is quite low, so that neither c.m.c. nor micellar size is altered. Studies of the solubilization of alcohols by detergents can give good values of AH and Acp,12,14 but equilibrium with the organic phase, cannot be attained with unmodified micelles, so that a straightforward estimation of AF is excluded. Experimental
0
0I
1
3.25
I
I
3.35 lOJ/T
I
3.45
.OK-'.
Fig. 1.--The solubility of n-allranes in water: ordinate log SS, minoles of alkane1103 g. of water at 1 atm. Smallcirclesand X , t,his work; curves, ref. l i ; dashed curve, see text.
Materials.-Ethane, propane, and butane (Phillips research grade hydrocarbons), pentane (Matheson, Coleman and Bell Chromatoquality), and sodium dodecyl sulfate (SDS) (gifts of Dr. Arno Cahn of Lever Brothers and Dr. James Barnhurst of Colgate-Palmolive Company) were used without further purification. Pentane-1 ,2-H3(Yew England Nuclear), 66 millicuries/ mmole, was diluted 300-fold with cold pentane in a vacuum s> stem and treated as described below. Solubility Determinations.-The manometric apparatus has been described previously.12 The compressibilities for ethane, propane, and butane" and pentanelB were used to determine molal solubilities. Ethane solubility was determined a t 0.4-0.7 atm., pentane a t 0.15-0.25 atm.,with the others between. All the SDS experiments mere performed on 1.80 wt.7, SDS, 0.100 M KaC1 solution. The data are normalized to one atmosphere. The solubility increment, SM in mmoles/103 g. SDS/atm., is computed from Ssoln
-----
I
I
3.25
I
I
3.35
I
I
I
I
3.45
i03/T, O K - ' .
Fig. 2.-The solubility of n-alkanes in SD8 micelles a t 1 atm.: solid lines, open symbols, SM, mmoles of alkane/l03 of g. SDS at 1 atm.; dashed lines, filled symbols. Sii/Ss. 0, 0 , pentane; 0,W, butane; A, A, propane: 0, V, ethane. N.B. Data for pentane and butane to be multiplied by 100; for propane and ethane, by 10.
tributiori to the AF, A H , a i d AX of micelle formation. The advantage of using the alkane gases is twofold:
=
XM.ftI
+ Ssfs
where f~ and f s are weight fractions of micellar component and solvent, respectively. The data of Morrison and Billett'7 were used to correct XSto 0.1 M NaC1, where the experimental values were for pure water. This correction (4% for ethane, 6% for butane) increases SM (ethane) by 107, and has negligible effect on the other SM, but is also included in the computation of SM/SS. The c.m.c. changesvery little between 15 and 3509; no correction has been made for the presence of 2.37,18monomer. The manometric apparatus, which gave satisfactory results for the smaller hydrocarbons, produced anomalously high results for the solubility of pentane in water (about twice the butane solubility). Control experiments with empty cells showed that some of the pentane (about 257, a t 15" and 0.3 atm.) was adsorbed on the glass or, more likely, dissolved in the small amount of grease a t the stopcock to the cell; it could be shown that no pentane left the cell. Presumably because the surface, volume, and greasing are fairly uniform among replicates, precision is not much worse than the other experiments; moreover, since fs is close to 1, the Sx/Ss is large, the whole error is included in Ss,and b'>i is insensitive to rather large corrections in S a . However, since Sai/Ss is the desired quantity, a vessel for by tracer methods was built, consisting of a determining S.o~n/S~ four-tube manifold (8-ml. volume in each tube), with rubber stoppers from blood-bank vacutainers as sampling ports. Ha pentane was admitted to the evacuated manifold a t its vapor pressure at O", and duplicate samples of SDS and YaCl solutions introduced through the stoppers. Enough air was introduced so that no gas bubble would form in the syringes during eubsequent sampling. After 1-2 hr. of thermostated shaking 0.30-ml. (weighed) aliquots were delivered into vials containing Bray scintillation liquid.18 (Because of the high solubility of pentane in organic liquids less than 1% will go into the gas phase, and the (13) E. Hut,chinson, A. Inaba, and L. G. Bailey, Z. physik. Chem., 6 , 344 (1955). (14) E. Hutchinson and L. G.Bailey, ibid., 21, 30 (1959). (15) I. 13. Silberberg, P. K. Kuo, and J. J. McKetta, Jr., Petrol. BrLyi.., 24, CQ (1952). (16) "Physical Properties of Chemical Compounds," 11, ACS Publication, Washington, D. C.. 1969. (17) T. J. Morrison and F, Billett, J . Chen. Soc., 3819 (1852). (18) J. N. Phillips and K. J. Mysels, J . Phys. Chem., 69, 325 (1953). (19) G. A. Bray, -4naZ. Biochem., 1, 279 (1960).
HYDROPHOBIC CONTRIBUTIOK TO MICELLEFORMATION
Oct., 1963
error introduced depends only on the differences in gas space among vials.) The samples were counted in a Nuclear-Chicago Model 703 liquid scintillation counter for a t least 2 X lo4counts. If (counts/g. of soln./sec.)/(counts/g. of solvent/sec.) = R then s u / s s = ( R - J$)!fN and 8s = S M / ( ~ N / S S ) . The first experiment,s gave good replication, but with unexpectedly low values of R (1.78 ct 0.02), in a way that indicated unusually high counting rates for the NaCl solvent. On the assumption that this arose from a tritiated impurity of very high water solubility (possibly HaOH) the procedure was modified. Small quantities of pentane H3 were distilled into water, equilibrated, and the gas phase a t 30" allowed to expand into the manifold. More reasonable values of R were obtained. were obtained in The filled circles in Fig. 2, (SM/~S)PENTANE, this way; from the reasonably well established SM,the values of (SW)PEKTAXE given in Fig. 1 as crosses were computed. Because relatively few experiments were done this way, and because the impurity quei3tion may not be settled, these values should be regarded as tentative.
Results van't Hoff plots for the solubility of the alkanes in water are given in Fig. 1. The data for ethane, propane, and butane are consistently, but not greatly, higher than previously rep0rted.l' The dashed line represents a predicted curve for pentane extrapolated from the data for the lower homologs17and is probably an absolute lower limit for pentane solubility if errors have not been eliminated in the tracer study. AFTRANS (H20-+ liquid hydrocarbon) and, where available, AH are given in Table I. and TABLE I 'hERMODY?iAMI(:
P A R A M F T s R S FOR THE TRANSFER O F n-ALKANES
FROM
WATE:R TO LIQUIDALKANE' AF," kea1
a
e.u.
A H , b kcal.
Pentane --6.62 ... Butane --5.82 1.0 Propane --4.85 2.0 Ethane --3.90 ".. Mole fraction basis. Taken from ref. 20.
.. 23 23 At 25".
vaii't Hoff plots for and S M / X s are given in Fig. AH of solution of' gas in SDS is more negative by 1 kcal. than the heat of condensation of the pure hydrocarbon20 (see, however, ref. 13), but the series is otherwise normal. Attention should be directed toward the filled symbols representing XM/SS, the ratio of solubility in SDS to that in NaCl solution. These data give the therrnodyiiamic constants for the transfer of the alkanes from water to the micelle interior listed in Table 11. 2.
TABLE I1 THERMODYNAMIC PARAJIETERS FOR THE TRANSFER O F n-ALKANES FROM LVATER TO
a
sI>sMICELLES
AF," kcal.
A H , kcal.
AS," e.u.
Pentane - S .72 Butane -5.13 Propane -4.23 Ethane -3.45 Mole fraction basis.
-1.08 0.0 1.00 2.00
15.6 17.2 17.5 18.3
Discussion The first result of this study is that the average A F of transfer of a, methylene group froin water to an 51)s micelle is -0.76 kcal. Other transfers between water and an organic liquid yield: -0.91 (pure alkane, or to ail ideal solution in decane), -0.9 (to 2-propanol),12 (20) G. NBmethy and H. A. Soheraga, J . Chem. Phgs., 36, 3401 (1962).
2081
-0.81 (on a n alcohol, to the alcohol),21 -0.664.73 (on an amino acid, to ethanol).22 The AFTRANS are between 0.45 kcal. and 0.90 less negative than for transfer to ideal solutions (the pure alkane or a decane solution) (see below) ; for propane and butane, transfer to SDS is similar to transfer to 2propanol (propane, -4.2 and -4.1; butane, -5.1 and -5.0 kcal., respectively). The notion that the interior of micelles is essentially a liquid seems quite plausible. It remains to be seen whether these data can be used in a statistical mechanical theory of micelle formation. There has been a beginning in the detailed statistical mechanical treatment of water structure and the hydrophobic interaction^.^^,^^ It is still reasonable, however, to appeal to the equation3 A = -kTln (partition function), and to assert, more or less by handwaving, that certain additive terms in A give rise to identifiable, separable factors in the total partition function of the system. This may be done for AFTRANS per CH2 between water and micelle; it does not matter in the end whether the corresponding factors in partition function are included in the partial partition function for the monomer o? for the aggregates, but for calculating size distributions it would be more convenient to include them in the latter.' It should be noted at this point that the experimental AFTRANS represents a differential increment to a fully formed micelle; for building a micelle there is a primary surface effect in that only part of AFTRANSis realized: let us say the outer spherical annulus the thickness of the diameter of one methylene group gains only some fraction (half?) of AFTRANSper CH2. Another surface effect, the exclusion of the hydrocarbon tails from the region in which the polar ends and their adjacent CH2 are constrained to move, is probably already included as part of the smaller ASTRANSfor SDS relative to liquid alkanes. I do not know how far it is safe to extrapolate the alkane series, in particular, whether AHTRANS really decreases l kcal. per CH2. On the other hand, AHTRANS for a dodecyl group is not likely to be more than - 1 kcal., and could conceivably be as low as -8 kcal., although an intermediate figure is probable. With AFTRANS = - 10 kcal., AHTRANS is not negligible. A AH as well as the required repulsive forces source of is to be found in electrostatic interactions, which it is a mistake to neglect. The centers of negative charge in SDS micelles are a t most a t the surface of the sphere excluded to counterions, if not within it. The treatment of Tanford and KirkwoodZ4or Hi1126is applicable : Even a t infinite ionic strength AFel is not reduced to zero-the terms for interaction of the charges through the low dielectric constant internal medium remain. Moreover, the temperature dependence of AFel is not inconsiderable. If AH = - T 2 a(AF,l/T)/bT and AFel = constants of the geometry X ( l / D i ) , then for models appropriate to large micelles AH can easily be one to several kilocalories. It is possible that a theory of micelle formation based on the imperfect gas model3 can be constructed using Tanford-Kirkwood electrostatics (because of crowding
+
(21) K. Kinoshita, H. Ishikawa, and K. Shinoda, BUZZ. Chem. SOC.Japan, 1081 (1958). (22) C. Tanford, J . Am. Chem. Soc., 84, 4240 (1962). (23) H. S. Frank and B.S. Quist, J . Chem. P h p . 34, GO3 (1961). (24) C. Tanford and J. G. Kirkwood, J . Am. Chem. Sac., 79, 5333 (1'357). (25) T. L. Hill, zbzd., 18, 5527 (1956).
2082
RIZWANUL HAQUE AND WAHIDU. MALIK
AFel grows with a power of n greater than unity) and the hydrophobic parameters reported here (- A F ~ , grows at less than the first POTVerOf Calculations using only these terms in the Partition functions give maxima between 50 and 100 monomers per micelle. DISCUSSION E. D. GODDARD (Lever Brothers Company).-It has yet to be established whether the main contribution to the positive A S values is due to the hydrocarbon chains or to the water molecules. Very likely it is a combined effect. These high values are in keeping with theories of micelle formation recently advanced; regarding the AH values, however, I would think it somewhat hazardous to extrapolate from a butane or propane chain to a dodecane chain. It would be very useful indeed if the experiments could be repeated to include higher chain length hydrocarbons. A. Wrs”Ia.-If it is agreed that the micellar interior is not very different from a hydrocarbon liquid, in which alkanes would form nearly ideal solutions, then the large positive A S of transfer represents some interaction between the alkane and the solvent. I t is the difficulty of conceiving either (a) exceptional van der Waals interactions between HzO and CH, or CH3 groups or ( b ) eignificant restriction of the translational, vibrational, and rotational motions of the short alkanes (beyond the effect of a condensed
Vol. 67
phase) which has led current theorists of water structure (e.g., ref. 20 and 23) to attribute the greatest part of the entropy of transfer of alkanes to some sort of redistribution in the number of structural bonded and unbonded water molecules. I nould expect that such considerations still hold, in large measure, for longer alkyl chains. A linear extrapolation of the Cs-Cr data for AH to Cl9 would indeed be rash, even if the data were firmly established; as I indicated in the text, this sets an upper limit, with the true A H ~ s a x s very likely less negative. Moreover, as I remarked, the pentane data, after a good many more experiments, are still tentative, and given to too many significant figuresin Table 11: it is not excluded that AHTRAKS for pentane is closer to zero. It is not yet clear whether the uncertainty arises from some variability in residual radioactive impurity or from a real effect of pentane on the micelles, even a t the low pressures used. I have just concluded a eeries of H3 butane experiments a t 25’ between 0.1 and 1.0 atm.; the observed increase in the ratio of Henry’s law coefficients Sx/Ss of about 5% cannot arise from the type of impurity encountered with pentane, and may represent an effect of high levels of butane binding on micellar size which should be easily accessible to light-scattering measurements. Such a system, which is more flexible than studies of detergents of increasing chain length, may illuminate some problems of niicellar structure and afford ~ micelle formation. another F a y of estimating Ape, and A F E of
A SPECTROPHOTOMETRIC STUDY OF THE INTERACTION OF SURFACE-ACTIVE AGEXTS WITH DYES1& BY RIZWANUL H A Q U EAND ’ ~ WAHIDU. MALIK Department o j Chemzstry, Alagarh Muslzm Vnlzverszty, Alagarh, Indza Receked ,March 8, 1963 The interaction of the anionic surface-active agents sulfonated phenyl-, tolyl-, and xylylstearic acids with rosaniline hydrochloride and the cationic agents like dodecyl pyridinium bromide and isothiourea dodecyl ether hydrobromide with congo red, methyl orange, and alizarin sulfonic acid was studied spectrophotometrically. A definite change in the absorption maxima of a dye in the presence of one of these surface-active agents was observed. ,This change in maximum was interpreted in terms of compound formation. The effect of pH and critical miaelle concentration also was Atiidied.
Introduction agents. Later, other ~ o r k e r s ~ 5used - ~ ~this method for the determination of the c.m.c. value. The other The interaction of surface-active agents with subof the interaction are the existence of “metaaspects stances such as proteins,2-5 polymers,6 nucleic acid,’ c h r ~ m a c y ” ~and ~ . ~ ~dye-detergent ~ o m p l e x i n g ~ ~ - * ~ hydrophobic S O ~ S , ~ Jmetal ions, lo and organic dyesll which have not been fully studied and need further has been studied by a number of workers to establish work t o understand the phenomenon, especially the their properties and extend their various uses. Among mechanism of dye-detergent interaction. I n conthese the interaction of dyes and surface-act’ive agents tinuation of our earlier work on the properties of surpresent some interesting features worth considering. face-active agentsZ4v29it was thought worthwhile to Hartleyl‘ noticed that the color of the dye changes investigate the abore aspects of the problem. The with the addition of surface-active agents, and he present conimunicatioii deals with the new interactions utilized this fact, in determining the c o i i c e n t r a t i ~ n l ~ ~ ~ ~ (i) anionic soaps like sulfonated phenyl-, tolyl-, and and critical micelle ~oncentratioiil~ of surface-active xylylstearic acid with rosaniline hydrochloride and (ii) (1) (a) Presented before the 37th National Colloid Symposium of the cationic soaps like dodecyl pyridinium bromide and isoAmerican Chemical Society held at, Ottawa, June 24-26, 1963; (b) Departthiourea dodecyl ether hydrobromide with congo red, ment of Chemistry, University of British Columbia, Vancouver 8, B. C.. Canada. (2) F. W. Putnam, “Advances in Protein Chemistry.” Vol. I V , Academic Press, Inc., New York, N. Y., 1948, p. 80. (3) E. G. Cockbain, Trans. Faraday Sac., 49, 104 (1953). (4) B. S. Harrap and J. H. Schulman, Discussions Faraday Sac., 13, 177 (1953). ( 5 ) K. Aoki and J, Hori, J . A m . Chem. Soc., 81,1885 (1959). (6) (a) S . Saito, J. Colloid Sci., 16, 283 (1960); (b) Kolloid-Z., 168, 128 (1960). (7) D. Guerritore and L. Bellelli, Nature, 184, 21, 1638(1950). (8) R. H. Ottewill and -4.Watanabe, Kolloid-Z., 170, 38, 132 (1960). (9) E . Rlatijevi6 and R. H. Ottewill, J . Colloid Sci., 13, 242 (1958). (10) J. H. Schulman, Australian J. Chem., 18, 236 (1960). (11) G. 9. Hartley, Trans. Faraday Soc., 30, 44 (1934). (12) G. 6 . Hartley and D. F. Runnicles, Proc. R o y . Sac. (London), A168, 420 (1938). (13) G. S.Hartley and C. S.Samis, Trans. Faraday Soc., 34, 1288 (1938). (14) G . S. Hartley, J . Chem. Soc., 1968 (1938).
(15) M. L. Corrin and W. D. Harkins, J . A m . Chem. Soc., 69,679 (1947). (16) I. hf. Kolthoff and W. Stricks, J. Phys. Colloid Chem., 62, 915
(1948).
(17) L. Arkin and C. R. Singletcrry, J . Am. Chem. Soc., 70, 3965 (1948). (18) P. Mukerjee and K. J. Mysele, ibid., 7 7 , 2937 (1955). (19) L. Lison, Arch. Biol., 46, 599 (1935). ( 2 0 ) W. C. Holmes, Stain. Technol., 1, 116 (1926). (21) C F. Hiskey and T. A. Downey, J. P h w . Chem., 6 8 , 835 (1954). (22) JI. Hayashi, Bull. Chem. 5oc. Japan, 84, 119 (1961). (23) T. Kondo and K. Meguro, ~ V i p p o nKagaki Zasshi, 77, 1240 (1956). (24) W. U. Malik and R. Haque, Anal. Chem., 82, 1628 (1960). (25) W. U. Malik and R. Haque, Z . anal. Chem., 180,425 (1960). (26) W. U. Malik and R. Haque, Naturwiss., 49,346 (1962). (27) TI‘. E. Malik and R. Haque, Z. anal. Chem., 189, 179 (1962). (28) TV. U. Malik and R. Haque, Nature, 194, 863 (1962). (29) R. Haque and W. U. Maiik, J. PoEarog. Soc., 8 , 36 (19d2).
