2067
BINDING OF PHENOLS BY HAIR
Binding of Phenols by Hair.
I
by M. M. Breuer' Gillette Research Laboratories, Reading, Berkshire, England
(Receiued December 3, 1.963)
The binding by hair of five different phenolic compounds was studied. The results indicate that the adsorption occurs on the peptide groupings of the polypeptide chains, probably by formation of H bonds. Resorcinol was found to exhibit unique binding characteristics among the compounds studied. From the experimental data presented, conclusions can also be drawn regarding the water binding sites and the inechanisiii of hydration of hair. Changes in adsorption isotherms of resorcinol were found when the hair was "treated" with HCl. These changes point to the occurrence of transformations in the tertiayy hair structure below the p H range 2.4-2.6.
Introduction The adsorption of phenols on wool and hair has been studied by several workers. Thus, Zahn2 found that reversible changes occur in the mechanical properties of wool when it is dipped a t 25' into solutions of phenols. Chipalkatti, Giles, and Vnllance3" measured the adsorption of many compounds, among thein phenols, on wool and other textile fibers from aqueous and nonaqueous solutions. The data gathered by these authors, however, were far from being sufficient to allow the postulation of a binding mechanism for phenols on wool. Reports of other investigations carried out on this subject contain only scanty data mostly of a qualitative nature; the interpretation of these data is very difficult."b In the present study, adsorption isotherms of various phenols on hair were measured a t a number of temperatures. On the basis of the experimental results, a model is suggested for the binding mechanism off phenolic compounds. The behavior of resorcinol is shown to differ markedly from that of the other phenols studied.
Experimental lllaterials. Brown "De-Meo" Caucasian hair was used in all experiments. The hair was purified by extracting it in a Soxhlet apparatus with chloroforin and acetone consecutively €or 24 hr. each. Subsequently, it was soaked in distilled water for 6 days, the water being changed twice a day. The hair was then allowed to dry while hanging in the atmosphere. Chemicals. Where available, B.D.H. Aiialar grade
materials were used for all experiments; in all other cases B.D.H. laboratory reagent grade chemicals were used after recrystallization. Methods. The adsorption measurements were carried out in the following manner. About 0.5 g. of hair was weighed into a test tube equipped with a groundglass stopper and dried in vacuo over Pz05for 16 hr. The test tube was stoppered in a dry cabinet and weighed; then a known volume of an accurately prepared aqueous solution o€ a phenol or HCI was added. A set of such tubes was suhsequently placed in a thermostatically controlled bath and left there until equilibrium was attained, which, as a rule, required 24 hr. After the equilibrium had been completed, aliquot quantities of the solutions were withdrawn and, aft,er suitable dilut,ion, the phenol concentrations were measured. The amounts of phenols bound to the hair ( r ) were calculated from the decreases in concent,rstions and the volumes of the solutions originally added.4 The coiiceiitrations of the phenolic solutions were obtained by measuring their opt'ical densities by means (1) Unilever Research Laboratory, Isleworth, Middlesex, England. (3) W. Zahn, Z . n'aturfonrsch.. 213, 286 (1947). (3) (a) W. R. Chipalkatti, C. H. Giles, and D. G. >I. Vallanre, J. Chem. Soc., 4375 (1954); (b) a detailed hibliography is given in this reference. (4) Simultsireously with the adsorption of phenols, a hydration of the hair also takes place. The volume of the "free" phenolic solution is therefore slightly less than the volume of the original solut.ion, and the calculated amounts of the phenol adsorbed in hair a1.e thus lower than the real values. An estimat,e of this error Yhowed t,hat it must be less than 2% and no correction was applied. The mole g . - l . This was values of r were reprodurible within established by carrying out many repeated experiments under identical conditions.
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of a Hilger Uvispek spectrophotometer a t the wave length of the maximum absorbancy of the phenol in question. The values of the latter are available in the 1iterat~i-e.~Calibration curves were prepared for the individual phenols and the molar extinction coefficients calculated. Adherence to the Lambert-Beer law was found in all cases, within the concentration ranges studied ( to mole/l.), The water adsorption measurements were carried out by exposing weighed samples of hair to atmospheres of constant humidities and determining the weight increases of the hair samples. The attainment of equilibrium was ascertained by taking weighings at several time intervals. No further weight change was observed after 10 days. An Electronic Instruments Ltd. direct reading pH meter was used for the pH measurements. The reproducibility of the measurements was within 0.1 pH unit. The mechanical properties of single hair fibers were measured using an Instron extensometer. The work required for stretching to 300/, extension was determined with fibers totally immersed in distilled water.
1.5
i
0.2
0.4
0.6
/, mole 1. -1.
Figure 1. Binding isotherms of resorcinol hair: 0, 2’; 6,26”; 0 , 37”.
7’ 2.0 bi
1
Results In each experiment, r, the quantity of bound phenolic compound (measured in moles/g. of hair), was calculated and plotted against $, the equilibrium concentration (moles/l.) of the phenolic compound in solution. Two types of isotherms were obtained ; resorcinol, catechol, and pyrogallol each yielded a curve coiivex with regard to the j-axis in its entire course, whereas phenol and hydroquinone gave slightly sigmoid curves (for a typical isotherm, see Fig. 1 and 2). The difference between the two types of adsorption isotherms can be better demonstrated by plotting l l r against l/f. Resorcinol, catechol, and pyrogallol then yield straight lines (typical curves are shown in Fig. 3), whereas phenol and hydroquinone give pronounced signloids (for typical curves, see Fig, 4). All of the isotherms represent fully reversible processes a t the temperatures and coilcentrations studied, confirming Zahn’s similar previous result^.^ The mechanical properties of single fibers, although influenced by the binding of phenols, were entirely restored after washing out of the phenols. For the interpretation of these results, we now consider the relevant theoretical treatments of systems of this type. Thus, Klota6 derived equations for various types of binding equilibria of small molecules to proteins in the following manner. Postulating a model where the small molecules are bound on independent, fixed, arid equivalent sites (k.,Laiigmuiric T h e Journal of Physical Chemistry
0.2
0.4
0.6
f, mole 1. -1. Figure 2.
Binding isotherms of hydroquinone on hair.
10
20 30 l/f, 1. mole-’.
40
50
Figure 3. Reciprocal plots of resorcinol binding isotherms: 0, 2’; 6, 26”; 0 , 37”. (5) H. E. Ungnade, “Organic Electronic Spectral Data,” Interscience Publishers, Inc., New York, N. Y., 1960. (6) I. M. Klotz in “Proteinq,” Vnl. l B , H. Neurath and K. Bailey, Ed.. Academic Press, Inc., New York, N. Y., 1953, pp. 724-804.
2069
BINDING OF PHEKOLS BY HAIR
Table I : Binding Constants and Thermodynamic Quantities of Hair-Phenol Interactions T,
n X 102, mole g. -1
OK.
Compound
5.2 5.2 4.5 4.5 4.6 4.6
275 299 275 299 275 299 275 299 311 275 299 275 299 275 299
Phenol Hydroquinone Catechol Resorcinol
Pyrogallol Resorcinol and acid.treated hair Catechol and acidtreated hair
1.8 1.8 1.8 4.8
4.8 5.0 5.0 4.6 4.6
15
5.0
5
15
10
K, mole-' 1.
0.21 1.82 1.83 1.32 1.05 1.69 3.50 5.75 6.80 0.91 1.25 1.26 0.89 1.26 2.20
AF, cel. mole-'
AH,
kcal. mole-'
+858 - 358 +91 - 166 - 25 -312 -815 - 1050 - 1220 +50 - 137 - 126 84 - 126 -472
t15.85
+50.5
+3.09
+10.9
+3.21
t12.0
+2.50
+12.0
+
+2.12
+7.82
-2.43
-8.75
+3.96
a = -
Figure 4. Reciprocal plots of phenol binding isotherms: 0, 2'; 0 , 26".
1 1 r Knf
1
AFi
RT
(where AFi, R, and 7' are the free energy of interaction, the gas constant, and the absolute temperature, respectively) we obtain the adsorption equation7 exp 2 w r l n K
+;
where n and K are the number of sites and the equilibrium constant of binding of each individual site, respectively. The straight lines obtained in l / r us. l/f plots in the caiies of resorcinol, catechol, and pyrogallol, therefore, indicate that this type of binding mechanism operates with these compounds. From the slopes,and the intercepts of the lines, the n and K values for the individual phenols were obtained and are given in Table I (columns 2 and 3). The curves of Fig. 4 show that this simple model is not adequate for the interpretation of the adsorption
+14.8
data of phenol and hydroquinone, and a slightly more complicated model is used, therefore, to account for the experimental results. l n this model it is assumed that the binding occurs on fixed sites with equal intrinsic binding constants, but that the sites are not independent and that interaction occurs between two molecules bound to adjacent sites.? This interaction can have either a facilitating or hindering influence depending on the nature of the interacting forces. Thus, if electrostatic repulsion forces (for instance) are involved, the adsorption on the second site will be hindered. Should, however, van der Waals attraction forces exist, the binding next to an occupied site will be facilitated. If we introduce the quantity
t/f, 1. mole-'.
i'sotherm), he obtained the equation
AS, e.u.
2.303 wr n
== log
K
+ loa f('
(3) -
When the quantities log f ( ( n ' r ) - 1) were calculated from the phenol-hair and hydroquinone-hair adsorption data and plotted against r,'n, straight lines were obtained, suggesting that the modified model is the correct one. (For typical curves, see Fig. 5 . ) From (7) R. Fowler and E. A . Guggenheini, "Statistical Therinodynainlcs 'i Cambridge Cniversii y Pless, London, 1966, p. 429.
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0.5
-
0.0
i
1
4 v
+ ,
tL
3 -0.5
- 1.0
0.2
0.6
0.4
0.8
r/n.
Figure 5 . Phenol binding d a t a represented according t o eq. 4 : 0 , 2'; 0, 26".
+
the intercept and slope of each line, the appropriate values of K and w were evaluated. The values are given in Tables I and 11,respectively. Table I1 : Values of the Thermodynamic Quantities of Next-Neighbor Interactions
T> K.
275 299 275 299
Phenol Hydroquinone
u
-0.92 -1.49 -1.90 -0.82
AFi,
AHi,
cal. mole-'
ked. mole-'
ASi,
-505 -895 -1040 -490
+5,12
C20.4
-6.94
-21.4
e.u.
An alternative method for the evaluation of small molecule binding data has been suggested by Schumaker and COX.^ Their method of representation of the experimental data provides additional information on the hydration properties of the protein. Schuniaker and Cox pointed out that a serious error in the value of K is possible if the calculations are carried out on the tacit assumption that changes in the solute concentration of the solution do not influence the amounts of water which are bound to the protein. These workers derived the equations QI=-
-r55.51
f = ZAi
+ n 1 -1 +K55.51 Kf
The quantity 6: is known as the degree of preferential hydration. It is equal to the number of water molecules which would need to be released or bound in order The Journal of Physical Chemistru
-0.3
d
- 0.2
-0.1
- 100
(5)
and QI
to restore the same solute to solvent ratio at the binding site as the one which exists in the solution. The symbol A , represents the nuniber of sites of the ith type, capable of binding water but incapable of adsorbing solute molecules; n and K are the number of solute binding sites which can also bind solvent inolecules and the appropriate equilibrium constant, respectively. With the help of eq. 5 and 6, it is possible to check the correctness of the values calculated for K by means of eq. 1. Furthermore, one may also calculate the number of sites in hair which bind water only. Using the experiniental values of T and f and calculated vaIues of K , the values of 6: were calculated and plotted against the quantity 1 - 55,51K/(1 jK) (Fig. 6). Straight lines passing through the origin mere obtained in all cases. It is obvious therefore that the water- and phenol-binding sites in hair are the same (since A, is always positive and ZA, = 0), and no exclusively water-binding sites exist. The values of the slopes give the number of sites available for the binding of phenolic compounds. I n case of the resorcinol-virgin hair system, the value n = 1.7 X lop3mole,'g. of hair was obtained; in all other cases ( i e . , cat>echolor pyrogallol on virgin hair, catechol or resorcinol on acid-treated hair), the value n = 4.8 X mole/g. of hair resulted. These figures are in very good agreement with those obtained by extrapolation from eq. 1. It also follows
- 200
1 - K55.51, 1 K/
+
Figure 6a. Plot of binding data according t o the Schumaker-Cox equation-phenols on virgin hair : 0, 1.75", resorcinol; 8 , 26", resorcinol; c), 2', catechol; 8 , 20", catechol; 0 , 2", pyrogallol.
( 8 ) V. N. Schumrtker and D. T. Cox, J . Am. Chem. Soc., 83, 2445
(1961).
207 1
BINDING OF PHENOLS BY HAIR
3.0
-0.3
, 8 2.0 w
2 s X -0.2
b
1.0
-0.1
0.2
0.4
0.6
0.8
f, mole 1. -1.
Figure 7. Resorcinol binding isotherms a t different p H values: 0, p H 1.0; 8 , pH 2.3-2.7; 0, p H 2.7-2.9; a, pH 3.2-4.0.
1
- 50 - K55.5. 1 Kf
- 100
+
Figure 6b. Plot of binding d a t a according to t h e SchumakerCox equation-phenols om acid-treated hair: 0 , 2", catechol; 8 , 2", resorcinol; 0, 26O, resorcinol.
from the above data that the values calculated for K by means of eq. 1 are correct. The enthalpy and entropy changes AH and A S of' the various phenol bindings and next-neighbor interactions were calculated from isotherms obtained a t different temperatures (column 5 and 6, Table I and 11)using the well-known thermodynamic relations
AF AF
RT In K
(7)
AH - TAS
(8)
=
=
The heats and entropies of adsorption are positive in all cases and are of very similar magnitudes, except for the binding of phenol itself, which exhibits significantly higher AH and AX value^.^ The binding of phenols on hair did not induce any changes in the pH of solutions in equilibrium. The initial p H of the solul,ions, if not otherwise stated, was adjusted to a value between 5.50 and 5.80 and remained unchanged (within h0.1 pH unit) during the entire duration of the experiment. It can be concluded therefore that no change in the state of ionization of the protein occurs. The adsorption of resorcinol was also measured from solutions containing iincreasing concentrations of HCI (Fig. 7). No change in the shape of the isotherin occurred until a critical p H value (pH -2.4) was reached; at p H lower than 2.4, n was found to have
twice the normal value. These results suggest that a t or below this critical pH, irreversible structural changes occur in the hair. The irreversibility of the change of resorcinol binding capacity has been further demonstrated by measuring resorcinol and catechol binding on acid-treated hair. In these experiments the hair was soaked in 0.1 N HCI for 12 hr. and subsequently washed free of HC1 with distilled water until no positive reaction for C1- was observed. The isoelectric point for the acid-treated hair was found to be 3.80 in contrast to the value of $60-5.80 for the untreated hair. The value of n for resorcinol in the acidtreated hair iiicreased to 4.5 X l o + mole/g. in line with the n values observed with other phenols, The AH and A S values for resorcinol adsorption on acid-pretreated hair were entirely diff ereiit from those obtained with untreated hair. It is especially interesting to note the reversal of signs of both the enthalpy and entropy of resorcinol binding. No significant changes in the values of AH and A S were observed in the case of catechol. The effect of acid treatnient on hair was further investigated. The amount of HCl absorbed by hair was determined as a function of the pH of the solution in the absence of neutral salts (Fig. 8, curve A). The maximuin acid binding capacity was found to be 8.2 X lou4equivalentlg. of hair, in good agreement with the (9) The AH and A S values stated in Tables I and I1 might he in error up to about 20%. This, however, does not alter the main conclusions which can he drawn from itn inspection of these thermodynamic qiiantities: (a) the entropies and the enthalpies of binding are positive; (b) the values of AH and A S for phenol are higher than for the other phenols investigated; and (c, acid treatment, of hair cause3 a reversal of signs in the vnlues of both AH and AS in the case of resorcinoi, but does not influence the therrnoclynamic quantities of other phenol-binding processes.
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20
40
60
80
% humidity.
Figure 9. Water adsorption isotherms of virgin and acidtreated hair a t 25”: 0, virgin hair; 0 , acid-treated hair.
2
4
6
8
PH.
Figure 8. HCl titration curve of virgin and acidtreated hair: 0, virgin hair; 0 , acid-treated hair.
values found for wool. When, however, the back titration was carried out from low toward high pH values, a different curve was obtained (Fig. 8, curve B). Any subsequent titrations with the same hair specimen followed curve B whether carried out from high to low or low to high pH values; curve B was also obtained when the HCl adsorption of acid-treated hair was measured. The difference between curves A and B is due to the fact that curve B represents the titration curve of an increased number of carboxylic groups ( i e . , 9.5 X mole/g. of hair as against 8.2 X loF4 mole/g. of hair titrated on curve A). Exposure to low pH (less than 2.0) causes, therefore, the appearance mole/g. of hair of new acidic groups.’O of 1.30 X The water binding capacities of both native and acidtreated hair were measured (Fig. 9). The acid-treated hair was found to have lower water uptake capacity (tit a given humidity) than the native hair. The difference between the two curves amounted to a decrease in the water binding, 0.01 g. of water/g. of hair or 5 X mole of water/g. of hair. The work required to stretch a single fiber to 3Oy0 extension was also measured bcfore and after HC1 treatment ( L e , , soaking in 0.1 N HCl and subsequent washing out of the acid). It was found that on the average, a decrease of 4.201, in the required stretching work results from the acid treatment. A subsequent second acid treatment, however, restores the stretching work required to a value only 2% less than that of the The Journal of Physical Chemistry
original stretching work. The same recovery of tensile strength was also observed without HCl treatment, but only after a sufficient lapse of time (48 hr.). Untreated and acid-treated hair did not exhibit any differences in their X-ray diffraction patterns.
Discussion The number of the sites capable of binding phenolic compounds far exceeds the number of ionizing grqups in hair (about 0.8 x mole/g. of each of acidic and basic groups). It seems, therefore, that groups other than the carboxylic and basic groups are responsible for the binding of the phenolic compounds. Zahn2 suggested the possibility that the peptide linkages may act as sites for phenol adsorption. The total number of peptide groupings in hair‘is about 8.60 X lop3mole/g. of hair. l1 The fraction of peptide linkages which stay hydrogen bonded, even when brought into contact with water, has been determined in wool by ~~
~~
(10) I n order t o account for the appearance of new acid-binding groups, the following three alternative explanations can be suggested: (a) some amide groups are present in virgin hair, which are hydrolysed t o carboxylic groups; (b) some of the carboxylic groups in virgin hair are in the form of Ca or Mg salts and get freed after exposure t o low pH values: or (c) in virgin hair some of the acid groups are buried in hydrophobic regions of t,he protein and only become unmasked when the polypeptide chains unfold under the influence of an exposure to low pH medium. The third possibility seema the most plausible in view of the following considerations. The number of new arid-binding groups is the same after a relatively short exposure to acid (15 niin.) as after a long acid treatment (24 hr.). This result implies that the amide groups in hair a.re completely hydrolyzed a t room temperature within 15 min., a result, which does not seem likely. Finally, together with the appearance of new acidic groups, other properties of hair (e.g., resorcinol binding capacity, volume changes arcompanying the adsorption of various phenols-see part 111) also undergo changes, implying that some deeper structural changes also accompany the appearance of the new acidic groups. (11) P. Alexander and R . F. Hudson, “Wool,” Chapman and Hall Ltd., London, 1954.
2073
BINDING OF PHENOLS BY HAIR
Burley, Xicholls, and Speakman12 from measurements of the rates of hydrogen exchange reactions and was found to be 14% of the total amount, ie., about 7.40 X mole/g. of bonds are readily accessible. Additional support for the number ,of accessible peptide bonds is found from the analysis of water adsorption curves of hair. The number of fixed water binding sites in hair can be calculated from Chamberlain and Speakman’s13 data, using Hill’s equation, l1 giving the value 6.12 >< mole/g.-l sites. The corresponding figure for wool is 6.80 X male/g.-l. The dif-. ference is in accordance with the view that hair has higher degree of order than wool.11 The relatively close correspondence between the numbers of water- and phenol-binding sites (6 X lop9 mole/g. and about 4.5-5.0 X lop3mole/g., respectively, Table I) suggests thatt they are identical and are presumably the peptide groups. The observation that) these numbers are slightly less than the total available peptide sites suggests that not all the latter are equally accessible to water and to the phenolic compounds The difference can be easily explained on the assump-. tion that some of the peptide linkages will be hinderedl by bulky side groups and will not be readily accessible to the larger aromatic molecules. The fact that the resorcinol- and water-binding sites seem to be identical (since water could not be displaced from other types of site by the resorcinol-a conclusion which follows from Fig. 6) strongly implies that resorcinol is atta,ched by two of its phenolic hydroxyls to the polypeptide chain. It is postulated, therefore, that the two metahydroxyls of resorcinol form H bonds with two consecutive peptide groups of polypeptide chains. It can be demonstrated on molecular models that this type of binding is easily feasible for resorcinol; the angles of both bonds are about 110-120”, in agreement with the values found for similar bonds in other system^.'^ Compounds containing two phenolic hydroxyl groups in the ortho or para ]positions, on the other hand, can be fitted to two consecutive peptide groups of the p o l y peptide chains with two H bonds only if a smaller value is assumed for the C=O. . .H- bond angle. Examination of the values of enthalpies and entropies (Table I) reveals thle fact that the positive entropy changes are the driving forces in the binding of phenols to hair; the enthalpies of binding are unfavorable The source of the positive entropy changes must be sought in the release of hydration water possibly from both the polypeptide chain of hair and from the phenolic molecules. The adsorption of the subsequent inolecules of phenol and hydroquinone are facilitated by a next-neighbor
interaction process. The thermodynamic quantities of these interactions show that different mechanisms are at work in the cases of phenol and hydroquinone. I n the former case, the positive values of the interaction, entropy and enthalpy (see Table 11) point toward a mechanism in which the adsorption of a phenol molecule on a neighboring site’ to an occupied site will squeeze out the remainder of the hydration water from around the phenol group and bring about a hydrophobic-type interaction between the two benzene rings. With hydroquinone, the negative values of interaction entropies and enthalpies point rather to an interaction where the hydration shells are not disturbed. I n one of the models which can be postulated, the hydroquinone molecule can be assumed to be bound through hydrogen bonds to the peptide links while the rest of the molecule is directed from the polypeptide chain. It caii further be assumed that the neighboring molecules are interacting through their para-substituted hydroxyl groups. It is difficult to account for the nature of changes which occur in hair near the critical pH region, at -2.4, and which causes the changes in resorcinol adsorption characteristics. Xo significant differences could be seen in the X-ray diffraction patterns of the untreated and acid-pretreated hair, and therefore probably only’ the tertiary structure is involved. The same conclusion may be drawn from the fact that the adsorption characteristics of resorcinol are affected by the acid treatment, while those of catechol are not, since a more fundamental structural modification would be expected to affect the adsorption characteristics of all the phenols. The change in the signs of the entropy and enthalpy of adsorption of resorcinol on pretreated hair can be taken as an indication that the attachment of a resorcinol molecule induces a smaller loss of hydration water and consequently is more loosely attached to the polypeptide chain. I n any case, the foregoing again demonstrates the differences which exist between resorcinol and the rest of the phenols, with regard to their adsorption on hair. Our results also confirm that the great majority of peptide bonds in hair are easily accessible, and consequently only a small fraction can be regarded as bound in an organized structure. Finally, it has been shown in the present work (12) R. W. Burley, C. H. Nicholls, and J. B. Speakman, J . Textile Trans., 46, 427 (1954). (13) N. H. Chaniherlain and J. B. Speakman, 2. Elektrochem., 37, 375 (1931).
Inst.
(14) G. C. Pimeritel and A. L. MrClellan, “ T h e Hydrogen Bond,” Reinhold Publishing Corp., New York, N. Y., 1960.
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that only 4.8 X lop3equivalent of water/g. of hair is firmly bound to hair (Fig. 6) and that this hydration water is replaced by phenolic compounds in a molar proportion of 1 : l in the case of most of the phenols, and 2 : 1 in the case of resorcinol, l5 also that no preferential hydration on other sites occurs or, in other words, the rest of the water adsorbed by hair contains solutes (phenolic coliipounds) in the same ProPortioll as in the external so]ution, This water can, therefore, be regarded only as included solution.
The Binding of Phenols by Hair.
11.
Acknowledgments. The author wishes to thank Dr. A. D. Jenkins for many valuable suggestions and discussions, Mr. G. E. Jones for his skillful technical assistance, Dr. E. Graminski for permission to refer to his results, and the Management of Gillette Industries Ltd. for permission to publish this paper. (15) The displacement of water, in these ratios, was also demonstrated directly by Dr.
