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On the mechanism of collapse of monolayers of macromolecular substances: bidimensional mixtures of poly(dl -alanine) with arachidic acid
On the Mechanism of Collapse of Monolayers of Macromolecular Substances: Bidimensional Mixtures of Poly(DL-alanine) with Arachidic Acid GABRIELLA GABRIELLI AND PIERO BAGLIONI Institute o f Physical-Chemistry, The University o f Florence, Via Gino Capponi 9, 50121 Florence, Italy Received November 11, 1980; accepted January 27, 1981 The collapse process in bidimensional mixtures of poly(DL-alanine) and arachidic acid at the water-air interface was studied. Useful information can be obtained about compatibility in the collapsed phase through a study of the kinetics of this process. The process of separation is essentially a crystallization phenomenon, which comprises a nucleation phase followed by a phase in which the formed nuclei increase in size. Furthermore, it has been seen that the two components are incompatible in the collapsed phase. Observations made by means of the electron microscope confirm this incompatibility. INTRODUCTION
about the reciprocal compatibility of the components. This study is of particular interest because the separation mechanism of components of bidimensional mixtures has not yet been studied, and also because the kinetics of separation is used for the first time to obtain information about bidimensional compatibility. This latter fact is particularly interesting because in static conditions it is possible to obtain information about the compatibility of the components in the bidimensional state, specifically from the additivities of the areas, from the values of the thermodynamic functions of mixing, and from the collapse pressures as a function of the molar ratios, which is impossible in the separate bulk state.
In earlier works (1) studies made on binary bidimensional mixtures composed of a polypeptidic compound and a fatty carboxilic acid were reported. It is known that these mixtures are particularly important as simple and useful models of biological membranes (2). In a recent work (3) the behavior of bidimensional mixtures of poly(L-, D-, and DL-alanine) and arachidic acid was reported and the behaviors are incompatible with one another. This reciprocal insolubility is shown by the spreading isotherms, which show two collapse pressure, the lower of which is found at the separation of the monolayer of the polypeptidic component and is independent of the composition of the mixtures. It has also been proved that useful information can be obtained from the study of the collapse process of compounds of low (4) and high (5) molecular weight, particularly polypeptidic compounds (6). The aim of the present work is, therefore, to broaden the study of the mechanism of separation from bidimensional phases of mixtures, and, furthermore, to obtain in this way information
EXPERIMENTAL
Poly(oL-alanine), lot number AL69, was furnished by Miles Yeda Ltd., Israel; molecular weight 3900 was determined by titration of the end group in nonaqueous solvents; the arachidic acid, special reagent for gas chromatography, was furnished by Merck, Darmstadt, West Germany. 221
Journalof Colloidand InterfaceScience, Vol.83, No. 1, September1981
0021-9797/81/090221-09502.00/0 Copyright© 1981by AcademicPress, Inc. All rightsof reproductionin any formreserved.
222
GABRIELLI AND BAGLIONI TABLE I Values Selected (m z mg-*) for the Areas of Collapse
Area 1st 2nd 3rd 4th 5th
Poly(DL-alanine) 0.78 0.66 0.53 0.41
Mixture 4/1
Mixture 2/1
Mixture 1/1
Mixture 1/2
Mixture 1/4
Arachidicacid
0.58 0.51 0.45 0.38 0.32
0.52 0.46 0.41 0.35 0.30
0.48 0.44 0.39 0.34 0.30
0.44 0.39 0.34 0.30
0.34 0.30
0.34 0.30
The substrate consisted of a solution of 2.5 x 10-a N CaC12, HC1 0.01 N in bidistilled water purified of any colloidal impurities by means of activated carbon. The solutions of the mixtures were obtained by first dissolving the polyamino acid in dichloroacetic acid (6% by volume) and then adding chloroform and the arachidic acid in the desired molar ratio, calculated on the basis of the moles of monomeric unit of the polyamino acid and moles of the arachidic acid, and bringing the final solution to 100 ml. The solutions were used in the freshly prepared state only, renewed every 2 days, and stored between two successive procedures at about 4°C. The initial surface area was approximately 2.6 m 2 mg -1 while the surface pressure becomes measurable (i.e., ¢ 0) at values less than 1.8 m 2 mg -1 according to the temperature and molar ratios of the mixtures. The compressions were carried out in a discontinuous way, with interruptions every 0.05-0.04 m 2 mg-L The time interval between two successive compressions was increased with the decrease of the area (from 3 to 60 rain) so as to reach equilibrium before each new compression. The fact that the surface pressure was constant for a period of at least 5 min proved that the equilibrium had been reached for each area. The values of the areas are reported in Table I. The first area corresponds to the beginning of the collapse process, and the successive areas correspond to the following Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
stages. The corresponding collapse pressures have already been reported (3). The kinetics were studied at temperatures of 15, 20, 25, and 30°C. The apparatus used for the surface pressure measurements has been described earlier (5). The pressure measurements are approximated to +_0.01 dyn/cm. The temperature, which was checked at the beginning and at the end of each isotherm, was kept constant within _+0.05°C. The micrographs of the collapsed monolayers were obtained by transferring portions of the collapsed films on brass grids; these latter were covered with a gold layer, evaporated under vacuum, so as to avoid damaging the collapsed film with the electron beam of the microscope. The grids so obtained were examinated by means of an electron scanning microscope JSM-U3 (Japan). RESULTS AND DISCUSSION
A. Kinetics
The kinetics of collapse have been studied for monolayers of the pure components and for their mixtures by following the decrease of surface pressure as a function of time. The considerations according to which the equations describing the kinetic behavior of ordinary chemical reactions can be applied to the collapse process have already been fully illustrated and proved to be applicable in an earlier work, to which the reader is referred (6). Following the known method, the formal order of the kinetics of the collapse process
COLLAPSE OF MONOLAYERS
223
has been determined by plotting various mechanism of the collapse process, which, functions of the decrease in surface pressure as has been mentioned above, is essentially as a function of time. a process of nucleation followed by the These functions, elaborated by means of growth of the nuclei formed. a computer, are those that are usually folC. The process of separation for the mixlowed in reactions that are referred princi- tures is represented by two phases: (i) in the pally to the solid state (7). first (i.e., at greater areas) for every area a It has not been possible to represent all kinetic mechanism is involved that can be the experimental data with a single function. represented by two stages, namely, one that In Table II the kinetic laws for the pure is characterized by an equation of the P r o u t components and their mixtures are repre- Tompkins type (P-T) (8), and later one that is sented, and we shall examine them briefly. characterized by kinetics of "zero order" A. The collapse process and the laws that (Z-O). These two steps are characteristic of describe it in the case of poly(oL-alanine) the separation of the polyamino acid only, has already been fully considered (6). It has as has been shown previously (6) and this is been shown that the process of separation already an indication that there has been of the monolayer of this polyamino acid fol- separation of only one component, and that lows two kinetic laws, the first [ln (~-0 - 7r)/ therefore there is bidimensional incompati7r = In k + In t, where 7r represents the sur- bility. A confirmation of this is found in the fact face pressure (dyn cm -1) at the time t, ~r0 the surface pressure at the time t = 0, and t the that kinetics of this type, characteristic of time (sec)] can be attributed to the detach- the polypeptide component, are prevalent ment of the compound from the substrate, when the quantity of polypeptide present in and the second (Tr - 7r0 = kt corresponding the mixture is increased. In fact for the mixto a zero-order kinetics) is related to the ture containing the molar ratio of poly(oLformation of a bilayer, with activated sliding alanine) 1/4 arachidic acid, at none of the of the second layer on the first. surface areas considered is a kinetics of sepB. The collapse process of arachidic acid aration characteristic of the polypeptide has been reported in an earlier work (4) to compound observed. (ii) for smaller areas consist essentially in a process of nucleation the process of separation can be represented and growth described by a kinetic law of the by a P-T kinetic law, not followed, however, second order. In the present work the proc- by a kinetic law of Z-O; this can be attributed ess of separation of the arachidic acid is to the arachidic acid. The fact that a second kinetic process is again represented by a process of nucleation and growth according to an equation of the characteristic of the nonpolypeptidic comsecond order, but at areas that are smaller ponent and that this process takes place at than the limiting area the law of nucleation areas smaller than those corresponding to is of the Prout-Tompkins type (P-T), and the separation of the polypeptidic comcan be attributed essentially to a process of ponent is further proof of the separation, not nucleation with interference between the of a mixture, but of the individual comnuclei during the growth of these latter. This ponent, and therefore, of the incompatibilbehavior, different from the behavior previ- ity, at least in the condensed state, of the ously found, can be attributed to the differ- two components. ent experimental conditions, as, for exIn Table III the activation energies are ample, the different spreading solvent and shown for the various kinetic laws, for both pure components and their mixtures. From the different support. However, the different experimental con- the results obtained the following consideraditions do not seem to alter the general tions can be made: Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
5th
4th
3rd
2rid
1st
Area
'~ For the values of the area see Table I.
Prout-Tompkins
One law
lst: Prout-Tompkins 2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
Two laws
lst: Prout-Tompkins 2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
Two laws
lst: Prout-Tompkins 2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
Two laws
lst: Prout-Tompkins 2nd: Zero-order
lst: Prout-Tompkins
Two laws
Mixture 4/1
2nd: Zero-order
Two laws
Poly(oL-alanine)
Prout-Tompkins
One law
Prout-Tompkins
One law
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
Mixture 2/1
Prout-Tompkins
One law
Prout-Tompkins
One law
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
Mixture 1/1
Prout-Tompkins
One law
Prout-Tompkins
One law
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
Mixture 1/2
Kinetic Laws Followed by the Pure Components and Their Mixtures at the Various Areas Studied a
TABLE II
Prout-Tompkins
One law
Prout-Tompkins
One law
Prout-Tompkins
One law
Mixture 1/4
Prout-Tompkins
One law
Second order
One law
Arachidic acid
225
COLLAPSE OF MONOLAYERS T A B L E lII Activation Energies, E (kcal/mole) for the Process of Collapse of Poly(DL-alanine) and Its Mixtures with Arachidic Acid a Area
Poly(DL-alanine)
1st
First law 3.4
First law 2.6
First law 2.0
First law 3.8
First law 4.3
Second law 6.4
Second law 6.6
Second law 6.7
Second law 10.4
Second law 10.2
First law 3.6
First law 2.6
First law 1.9
First law 1.8
First law 1.0
Second law 6.4
Second law 6.6
Second law 6.7
Second law 10.4
Second law 10.2
First law 3.4
First law 2.4
First law 2.0
First law 1.7
Second law 6.4
Second law 6.6
Second law 6.7
Second law 10.4
First law 3.6
First law 2.4
Second law 6.2
Second law 8.0
2nd
3rd
4th
5th
Mixture 4/1
Not activated
Mixture 2/I
4.6
Not activated
Mixture l/1
Mixture 1/2
Mixture 1/4
Arachidic acid
8.6
Not activated
Not activated
3.6
5.8
5.5
Not activated
Not activated
~' For the values of the area see Table I.
1. All the kinetic processes regarding the mixtures are activated; for the pure components the process is activated in both steps for the poly(DL-alanine), it is activated only at the second area for arachidic acid. In the greatest area, where the nucleation process does not demonstrate regular behavior with regard to activation energies, the relation between the activation energies and the composition of the mixture is not evident; on the contrary for the successive areas, the activation energies of the first step of the process of separation of poly(DLalanine) (P-T law) are proportional to the molar ratios, i.e., they depend on the quantity of poly(De-alanine) present in the mixture, see Fig. 1. 2. During the second part of the collapse process of the poly(oL-alanine) (Z-O law), corresponding to the process of formation of a bilayer and the sliding of the second layer
over the first, the energy of activation increases with the increase of arachidic acid present in the mixture for all the areas studied, and in particular for the ratios of 1/l and 1/2 between moles of monomeric units of poly(DL-alanine) and moles of
/,
pL
z
.
oz
~
o,~ o 6
ae
AA
at.
ae
o*
a6
oe
AA
FIG. 1. Energies of activations, E (kcal mole -1) for the separation process of the mixtures. In A (2nd area) and B (3rd area) the activation energies for the first step (P-T law) of the separation process of the polyamino acid (DL) as a function of molar ratios of arachidic acid (AA) are reported: in C those for the process of separation of AA (for further explanations see text). Journal
of
Colloid
and
Interface
Science,
Vol. 83, No. I, September 1981
226
GABRIELLI AND BAGLIONI T A B L E IV
Enthalpies, AH$ (kcal/mol), Entropies, ASS (cal/mol.K), and Free Energies, A G , (kcal/mol), o f Activation for the Collapse Process of Poly(OL-alanine) and Its Mixtures with Arachidic Acid at 25°C a Area
Poly(DL-alanine) Mixture4/1
Mixture2/1
Mixture1/1
Mixture1/2
Mixture1/4
Arachidic acid
8.1 -33.9 18.1
Not activated
Not activated
3.0 -49.1 17.6
1st Ist law AH~: AS:) AG:~
2.8 -51.5 ,18.1
2.0 -54.4 18.2
1.4 -56.2 18.1
3.2 -50.0 18.1
3.9 -47.6 18.1
2nd law AH:~ ASS AG:~
7.6 -46.7 21.5
5.9 -52.8 21.6
6.0 -52.5 21.6
9.8 -39.7 21.6
9.7 -39.6 21.5
3.0 -51.1 18.2
2.0 -54.5 18.2
1.3 -56.6 18.2
1.2 -57.8 18.4
0.4 -60.3 18.4
2nd law AH~: AS:~ AG$
7.6 -46.7 21.5
5.9 -52.8 21.6
6.0 -52.5 21.6
9.8 -39.7 21.6
9.7 -39.6 21.5
3rd 1st law AH:I: AS:~ AG:~
2.8 -51.5 18.1
1.7 -55.6 18.3
1.4 -56.7 18.3
1.1 -57.8 18.3
5.2 -42.9 18.0
2nd law AH~ AS:~ AG~
7.6 -46.7 21.5
5.9 -52.8 21.6
6.0 -52.5 21.6
9.8 -39.7 21.6
3.0 -51.1 18.2
1.9 -55.3 18.0
4.0 -47.9 18.3
5.0 -44.5 18.3
5.6 -53.0 21.4
5.9 -52.8 21.6
2nd 1st law AH~ AS:~ AG:)
4th 1 st law
AH~ ASS AG:~ 2nd law AH:~ AS:~ AG:~ 5th
Not activated
Not activated
Not activated
Not activated
For the values of the area see Table I.
arachidic acid. It is reasonable to suppose that the high percentage of arachidic acid still in the monolayer strongly interferes Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
with the sliding of the second layer over the first because of the disposition it presents at the interphase, which is practically orthog-
COLLAPSEOF MONOLAYERS onal with respect to that of the poly(DLalanine), and of the high energies of interaction between the hydrophobic chains. 3. The energies of the process of separation of the arachidic acid are proportional to the quantity of arachidic acid present in the monolayer, but the values are much higher than those relative to the presence of only arachidic acid. This can be attributed to the presence of poly(DL-alanine) in the collapsed phase which can interfere with the process of nucleation of the second component (see Fig. 1). In Table IV the values of AG~, AH~, and AS~ are reported, representing, respectively, the free energy of activation, the enthalpy of activation, and the entropy of activation, determined as previously (6) by the application of the transition state theory. From the analysis of Table IV it can be deduced that: 1. The values of ASJ/for the second stage of the collapse process for poly(OL-alanine) decrease in correspondence with the mixtures that are rich in arachidic acid and confirms what was previously stated with regard to the activation energies. 2. The values of AG~ are identical for the nucleation phase of all the mixtures, and the processes of separation, both in the case of poly(oL-alanine) and in that of arachidic acid. MICROSCOPIC OBSERVATIONS In order to obtain further confirmation of the immiscibility of the poly(DL-alanine) and the arachidic acid in the condensed phase as well, micrographs were obtained with the scanning electron microscope. The micrographs were taken for the poly(oL-alanine) 2/1 arachidic acid mixture because the minimum value of AGmix determined in an earlier work (3) competes for this mixture and was attributed by us not to a reciprocal miscibility between the two components, but to interactions between the lipidic chains and the macromolecular micelles in the "bad fit" zones of the latter.
227
The study of this mixture is therefore particularly useful in confirming the insolubility between the two components, as deduced both by the classic study of bidimensional mixtures and by the kinetic process of separation of the pure components and of the mixtures from the bidimensional phase and from the formation of the tridimensional phase. In Fig. 2 the micrographs relative to the collapsed film of poly(oL-alanine) (A), arachidic acid (B), and the mixture in the above mentioned molar ratio (C) are shown. In (A) organized collapsed structures can be seen in which the hexagonal structure of the polyamino acid is evident; in (B) no organized structure can be shown; in (C) it is possible to distinguish structures of different morphology, separate one from another, not regularly alternating, and which can be attributed, by comparison with the other structures present in (A) and (B), to poly(oL-alanine) and to arachidic acid. The absence of mixed crystal and the heterogeneity of the structures in the collapsed phase constitute further proof of the incompatibility of the two components. CONCLUSIONS From the experimental results reported it is possible to draw the following main conclusions: 1. The separation of poly (oL-alanine) and arachidic acid from mixed monolayers is referred, at higher area values to a kinetic mechanism that is characteristic of the polypeptide component and at lower area values to a mechanism that is ascribable to the lipidic component. Both of the mechanisms involve a stage referring to a process of nucleation (P-T law). 2. It is possible to obtain information concerning reciprocal compatibility in bidimensional mixtures through the study of the kinetics of the separation from monolayers. In the case of the poly(DL-alanine) and arachidic acid mixtures it was proved Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
228
GABRIELLI AND BAGLIONI
FI6.2. Micrographs taken with the electron scanning microscope of the collapsed films. (A) collapsed film of poly(oL-alanine) (x 6000); (B) collapsed film of arachidic acid (x 5000); (C) collapsed film of the mixture in molar ratio 2/1 between moles of monomeric unit of poly(DL-alanine) and moles of arachidic acid.
that the two c o m p o n e n t s are incompatible in the collapsed phase as well. It seems very useful to cerrelate the kinetic results with the t h e r m o d y n a m i c data deduced from the spreading isotherms. 3. The kinetic relationships valid for one Journal of Collold and Interface Science, Vot. 83, No. 1, September 1981
of the c o m p o n e n t s by itself seems valid in the case of separation from mixtures, at least for the system studied. It seems also reasonable to assume, even if further study is necessary for a general extension, that the process of separation is essentially
COLLAPSE OF MONOLAYERS
a phenomenon of crystallization, comprising a phase of nucleation and a successive phase of growth. The mechanism of separation and the energies connected with it depend strongly on the distribution and on the interactions of the components in the monolayer. 4. Observations made with the electron scanning microscope make it possible to confirm that the structure of the poly(DLalanine) obtained after the collapse process of the monolayer is analogous to that found in the tridimensional phase, and that the poly(DL-alanine) is immiscible with arachidic acid. REFERENCES 1. Gabrielli, G., J. Colloid Interface Sci. 53, 148 Polym. Sci. 256, 1165 (1978); Gabrielli, G., and D'Aubert, C., ColloidPolym. Sci. 258, 56 (1980).
229
2. Colacicco, G., J. Colloid Interface Sci. 29, 345 (1969); Kafka, M., and Park, C. Y., J. Colloid Interface Sci. 41, 148 (1972); Gerfeld, M. L., in "Methods of Membrane Biology" (E. D. Korn, Ed.), pp. 69-103, Plenum, New York, 1974. 3. Gabrielli, G., Baglioni, P., and Fabbrini, A., Colloid Surfaces, in press. 4. Gabrielli, G., Guarini, G. G. T., and Ferroni, E., J. Colloid Interface Sci. 69, 352 (1976); Gabrielli, G., Guarini, G. G. T., and Bastianini, F., J. Colloid Interface Sci. 69, 352 (1979). 5. Gabrielli, G., and Guarini, G. G. T., J. Colloid Interface Sci. 64, 185 (1978); Baglioni, P., Gabrielli, G., and Guarini, G. G. T., J. Colloid Interface Sci., 78, 347 (1980). 6. Gabrielli, G., Baglioni, P., and Ferroni, E., J. Colloid Interface Sci., 81, 139 (1981). 7. Sestak, J., and Berggren, G., Thermochim. Acta 3, 1 (1971). 8. Prout, E. G., and Tompkins, F. C., Trans. Faraday Soc. 40, 488 (1944); 42, 468 (1946).
Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
about the reciprocal compatibility of the components. This study is of particular interest because the separation mechanism of components of bidimensional mixtures has not yet been studied, and also because the kinetics of separation is used for the first time to obtain information about bidimensional compatibility. This latter fact is particularly interesting because in static conditions it is possible to obtain information about the compatibility of the components in the bidimensional state, specifically from the additivities of the areas, from the values of the thermodynamic functions of mixing, and from the collapse pressures as a function of the molar ratios, which is impossible in the separate bulk state.
In earlier works (1) studies made on binary bidimensional mixtures composed of a polypeptidic compound and a fatty carboxilic acid were reported. It is known that these mixtures are particularly important as simple and useful models of biological membranes (2). In a recent work (3) the behavior of bidimensional mixtures of poly(L-, D-, and DL-alanine) and arachidic acid was reported and the behaviors are incompatible with one another. This reciprocal insolubility is shown by the spreading isotherms, which show two collapse pressure, the lower of which is found at the separation of the monolayer of the polypeptidic component and is independent of the composition of the mixtures. It has also been proved that useful information can be obtained from the study of the collapse process of compounds of low (4) and high (5) molecular weight, particularly polypeptidic compounds (6). The aim of the present work is, therefore, to broaden the study of the mechanism of separation from bidimensional phases of mixtures, and, furthermore, to obtain in this way information
EXPERIMENTAL
Poly(oL-alanine), lot number AL69, was furnished by Miles Yeda Ltd., Israel; molecular weight 3900 was determined by titration of the end group in nonaqueous solvents; the arachidic acid, special reagent for gas chromatography, was furnished by Merck, Darmstadt, West Germany. 221
Journalof Colloidand InterfaceScience, Vol.83, No. 1, September1981
0021-9797/81/090221-09502.00/0 Copyright© 1981by AcademicPress, Inc. All rightsof reproductionin any formreserved.
222
GABRIELLI AND BAGLIONI TABLE I Values Selected (m z mg-*) for the Areas of Collapse
Area 1st 2nd 3rd 4th 5th
Poly(DL-alanine) 0.78 0.66 0.53 0.41
Mixture 4/1
Mixture 2/1
Mixture 1/1
Mixture 1/2
Mixture 1/4
Arachidicacid
0.58 0.51 0.45 0.38 0.32
0.52 0.46 0.41 0.35 0.30
0.48 0.44 0.39 0.34 0.30
0.44 0.39 0.34 0.30
0.34 0.30
0.34 0.30
The substrate consisted of a solution of 2.5 x 10-a N CaC12, HC1 0.01 N in bidistilled water purified of any colloidal impurities by means of activated carbon. The solutions of the mixtures were obtained by first dissolving the polyamino acid in dichloroacetic acid (6% by volume) and then adding chloroform and the arachidic acid in the desired molar ratio, calculated on the basis of the moles of monomeric unit of the polyamino acid and moles of the arachidic acid, and bringing the final solution to 100 ml. The solutions were used in the freshly prepared state only, renewed every 2 days, and stored between two successive procedures at about 4°C. The initial surface area was approximately 2.6 m 2 mg -1 while the surface pressure becomes measurable (i.e., ¢ 0) at values less than 1.8 m 2 mg -1 according to the temperature and molar ratios of the mixtures. The compressions were carried out in a discontinuous way, with interruptions every 0.05-0.04 m 2 mg-L The time interval between two successive compressions was increased with the decrease of the area (from 3 to 60 rain) so as to reach equilibrium before each new compression. The fact that the surface pressure was constant for a period of at least 5 min proved that the equilibrium had been reached for each area. The values of the areas are reported in Table I. The first area corresponds to the beginning of the collapse process, and the successive areas correspond to the following Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
stages. The corresponding collapse pressures have already been reported (3). The kinetics were studied at temperatures of 15, 20, 25, and 30°C. The apparatus used for the surface pressure measurements has been described earlier (5). The pressure measurements are approximated to +_0.01 dyn/cm. The temperature, which was checked at the beginning and at the end of each isotherm, was kept constant within _+0.05°C. The micrographs of the collapsed monolayers were obtained by transferring portions of the collapsed films on brass grids; these latter were covered with a gold layer, evaporated under vacuum, so as to avoid damaging the collapsed film with the electron beam of the microscope. The grids so obtained were examinated by means of an electron scanning microscope JSM-U3 (Japan). RESULTS AND DISCUSSION
A. Kinetics
The kinetics of collapse have been studied for monolayers of the pure components and for their mixtures by following the decrease of surface pressure as a function of time. The considerations according to which the equations describing the kinetic behavior of ordinary chemical reactions can be applied to the collapse process have already been fully illustrated and proved to be applicable in an earlier work, to which the reader is referred (6). Following the known method, the formal order of the kinetics of the collapse process
COLLAPSE OF MONOLAYERS
223
has been determined by plotting various mechanism of the collapse process, which, functions of the decrease in surface pressure as has been mentioned above, is essentially as a function of time. a process of nucleation followed by the These functions, elaborated by means of growth of the nuclei formed. a computer, are those that are usually folC. The process of separation for the mixlowed in reactions that are referred princi- tures is represented by two phases: (i) in the pally to the solid state (7). first (i.e., at greater areas) for every area a It has not been possible to represent all kinetic mechanism is involved that can be the experimental data with a single function. represented by two stages, namely, one that In Table II the kinetic laws for the pure is characterized by an equation of the P r o u t components and their mixtures are repre- Tompkins type (P-T) (8), and later one that is sented, and we shall examine them briefly. characterized by kinetics of "zero order" A. The collapse process and the laws that (Z-O). These two steps are characteristic of describe it in the case of poly(oL-alanine) the separation of the polyamino acid only, has already been fully considered (6). It has as has been shown previously (6) and this is been shown that the process of separation already an indication that there has been of the monolayer of this polyamino acid fol- separation of only one component, and that lows two kinetic laws, the first [ln (~-0 - 7r)/ therefore there is bidimensional incompati7r = In k + In t, where 7r represents the sur- bility. A confirmation of this is found in the fact face pressure (dyn cm -1) at the time t, ~r0 the surface pressure at the time t = 0, and t the that kinetics of this type, characteristic of time (sec)] can be attributed to the detach- the polypeptide component, are prevalent ment of the compound from the substrate, when the quantity of polypeptide present in and the second (Tr - 7r0 = kt corresponding the mixture is increased. In fact for the mixto a zero-order kinetics) is related to the ture containing the molar ratio of poly(oLformation of a bilayer, with activated sliding alanine) 1/4 arachidic acid, at none of the of the second layer on the first. surface areas considered is a kinetics of sepB. The collapse process of arachidic acid aration characteristic of the polypeptide has been reported in an earlier work (4) to compound observed. (ii) for smaller areas consist essentially in a process of nucleation the process of separation can be represented and growth described by a kinetic law of the by a P-T kinetic law, not followed, however, second order. In the present work the proc- by a kinetic law of Z-O; this can be attributed ess of separation of the arachidic acid is to the arachidic acid. The fact that a second kinetic process is again represented by a process of nucleation and growth according to an equation of the characteristic of the nonpolypeptidic comsecond order, but at areas that are smaller ponent and that this process takes place at than the limiting area the law of nucleation areas smaller than those corresponding to is of the Prout-Tompkins type (P-T), and the separation of the polypeptidic comcan be attributed essentially to a process of ponent is further proof of the separation, not nucleation with interference between the of a mixture, but of the individual comnuclei during the growth of these latter. This ponent, and therefore, of the incompatibilbehavior, different from the behavior previ- ity, at least in the condensed state, of the ously found, can be attributed to the differ- two components. ent experimental conditions, as, for exIn Table III the activation energies are ample, the different spreading solvent and shown for the various kinetic laws, for both pure components and their mixtures. From the different support. However, the different experimental con- the results obtained the following consideraditions do not seem to alter the general tions can be made: Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
5th
4th
3rd
2rid
1st
Area
'~ For the values of the area see Table I.
Prout-Tompkins
One law
lst: Prout-Tompkins 2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
Two laws
lst: Prout-Tompkins 2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
Two laws
lst: Prout-Tompkins 2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
Two laws
lst: Prout-Tompkins 2nd: Zero-order
lst: Prout-Tompkins
Two laws
Mixture 4/1
2nd: Zero-order
Two laws
Poly(oL-alanine)
Prout-Tompkins
One law
Prout-Tompkins
One law
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
Mixture 2/1
Prout-Tompkins
One law
Prout-Tompkins
One law
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
Mixture 1/1
Prout-Tompkins
One law
Prout-Tompkins
One law
2nd: Zero-order
lst: Prout-Tompkins
Two laws
2nd: Zero-order
lst: Prout-Tompkins
Two laws
Mixture 1/2
Kinetic Laws Followed by the Pure Components and Their Mixtures at the Various Areas Studied a
TABLE II
Prout-Tompkins
One law
Prout-Tompkins
One law
Prout-Tompkins
One law
Mixture 1/4
Prout-Tompkins
One law
Second order
One law
Arachidic acid
225
COLLAPSE OF MONOLAYERS T A B L E lII Activation Energies, E (kcal/mole) for the Process of Collapse of Poly(DL-alanine) and Its Mixtures with Arachidic Acid a Area
Poly(DL-alanine)
1st
First law 3.4
First law 2.6
First law 2.0
First law 3.8
First law 4.3
Second law 6.4
Second law 6.6
Second law 6.7
Second law 10.4
Second law 10.2
First law 3.6
First law 2.6
First law 1.9
First law 1.8
First law 1.0
Second law 6.4
Second law 6.6
Second law 6.7
Second law 10.4
Second law 10.2
First law 3.4
First law 2.4
First law 2.0
First law 1.7
Second law 6.4
Second law 6.6
Second law 6.7
Second law 10.4
First law 3.6
First law 2.4
Second law 6.2
Second law 8.0
2nd
3rd
4th
5th
Mixture 4/1
Not activated
Mixture 2/I
4.6
Not activated
Mixture l/1
Mixture 1/2
Mixture 1/4
Arachidic acid
8.6
Not activated
Not activated
3.6
5.8
5.5
Not activated
Not activated
~' For the values of the area see Table I.
1. All the kinetic processes regarding the mixtures are activated; for the pure components the process is activated in both steps for the poly(DL-alanine), it is activated only at the second area for arachidic acid. In the greatest area, where the nucleation process does not demonstrate regular behavior with regard to activation energies, the relation between the activation energies and the composition of the mixture is not evident; on the contrary for the successive areas, the activation energies of the first step of the process of separation of poly(DLalanine) (P-T law) are proportional to the molar ratios, i.e., they depend on the quantity of poly(De-alanine) present in the mixture, see Fig. 1. 2. During the second part of the collapse process of the poly(oL-alanine) (Z-O law), corresponding to the process of formation of a bilayer and the sliding of the second layer
over the first, the energy of activation increases with the increase of arachidic acid present in the mixture for all the areas studied, and in particular for the ratios of 1/l and 1/2 between moles of monomeric units of poly(DL-alanine) and moles of
/,
pL
z
.
oz
~
o,~ o 6
ae
AA
at.
ae
o*
a6
oe
AA
FIG. 1. Energies of activations, E (kcal mole -1) for the separation process of the mixtures. In A (2nd area) and B (3rd area) the activation energies for the first step (P-T law) of the separation process of the polyamino acid (DL) as a function of molar ratios of arachidic acid (AA) are reported: in C those for the process of separation of AA (for further explanations see text). Journal
of
Colloid
and
Interface
Science,
Vol. 83, No. I, September 1981
226
GABRIELLI AND BAGLIONI T A B L E IV
Enthalpies, AH$ (kcal/mol), Entropies, ASS (cal/mol.K), and Free Energies, A G , (kcal/mol), o f Activation for the Collapse Process of Poly(OL-alanine) and Its Mixtures with Arachidic Acid at 25°C a Area
Poly(DL-alanine) Mixture4/1
Mixture2/1
Mixture1/1
Mixture1/2
Mixture1/4
Arachidic acid
8.1 -33.9 18.1
Not activated
Not activated
3.0 -49.1 17.6
1st Ist law AH~: AS:) AG:~
2.8 -51.5 ,18.1
2.0 -54.4 18.2
1.4 -56.2 18.1
3.2 -50.0 18.1
3.9 -47.6 18.1
2nd law AH:~ ASS AG:~
7.6 -46.7 21.5
5.9 -52.8 21.6
6.0 -52.5 21.6
9.8 -39.7 21.6
9.7 -39.6 21.5
3.0 -51.1 18.2
2.0 -54.5 18.2
1.3 -56.6 18.2
1.2 -57.8 18.4
0.4 -60.3 18.4
2nd law AH~: AS:~ AG$
7.6 -46.7 21.5
5.9 -52.8 21.6
6.0 -52.5 21.6
9.8 -39.7 21.6
9.7 -39.6 21.5
3rd 1st law AH:I: AS:~ AG:~
2.8 -51.5 18.1
1.7 -55.6 18.3
1.4 -56.7 18.3
1.1 -57.8 18.3
5.2 -42.9 18.0
2nd law AH~ AS:~ AG~
7.6 -46.7 21.5
5.9 -52.8 21.6
6.0 -52.5 21.6
9.8 -39.7 21.6
3.0 -51.1 18.2
1.9 -55.3 18.0
4.0 -47.9 18.3
5.0 -44.5 18.3
5.6 -53.0 21.4
5.9 -52.8 21.6
2nd 1st law AH~ AS:~ AG:)
4th 1 st law
AH~ ASS AG:~ 2nd law AH:~ AS:~ AG:~ 5th
Not activated
Not activated
Not activated
Not activated
For the values of the area see Table I.
arachidic acid. It is reasonable to suppose that the high percentage of arachidic acid still in the monolayer strongly interferes Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
with the sliding of the second layer over the first because of the disposition it presents at the interphase, which is practically orthog-
COLLAPSEOF MONOLAYERS onal with respect to that of the poly(DLalanine), and of the high energies of interaction between the hydrophobic chains. 3. The energies of the process of separation of the arachidic acid are proportional to the quantity of arachidic acid present in the monolayer, but the values are much higher than those relative to the presence of only arachidic acid. This can be attributed to the presence of poly(DL-alanine) in the collapsed phase which can interfere with the process of nucleation of the second component (see Fig. 1). In Table IV the values of AG~, AH~, and AS~ are reported, representing, respectively, the free energy of activation, the enthalpy of activation, and the entropy of activation, determined as previously (6) by the application of the transition state theory. From the analysis of Table IV it can be deduced that: 1. The values of ASJ/for the second stage of the collapse process for poly(OL-alanine) decrease in correspondence with the mixtures that are rich in arachidic acid and confirms what was previously stated with regard to the activation energies. 2. The values of AG~ are identical for the nucleation phase of all the mixtures, and the processes of separation, both in the case of poly(oL-alanine) and in that of arachidic acid. MICROSCOPIC OBSERVATIONS In order to obtain further confirmation of the immiscibility of the poly(DL-alanine) and the arachidic acid in the condensed phase as well, micrographs were obtained with the scanning electron microscope. The micrographs were taken for the poly(oL-alanine) 2/1 arachidic acid mixture because the minimum value of AGmix determined in an earlier work (3) competes for this mixture and was attributed by us not to a reciprocal miscibility between the two components, but to interactions between the lipidic chains and the macromolecular micelles in the "bad fit" zones of the latter.
227
The study of this mixture is therefore particularly useful in confirming the insolubility between the two components, as deduced both by the classic study of bidimensional mixtures and by the kinetic process of separation of the pure components and of the mixtures from the bidimensional phase and from the formation of the tridimensional phase. In Fig. 2 the micrographs relative to the collapsed film of poly(oL-alanine) (A), arachidic acid (B), and the mixture in the above mentioned molar ratio (C) are shown. In (A) organized collapsed structures can be seen in which the hexagonal structure of the polyamino acid is evident; in (B) no organized structure can be shown; in (C) it is possible to distinguish structures of different morphology, separate one from another, not regularly alternating, and which can be attributed, by comparison with the other structures present in (A) and (B), to poly(oL-alanine) and to arachidic acid. The absence of mixed crystal and the heterogeneity of the structures in the collapsed phase constitute further proof of the incompatibility of the two components. CONCLUSIONS From the experimental results reported it is possible to draw the following main conclusions: 1. The separation of poly (oL-alanine) and arachidic acid from mixed monolayers is referred, at higher area values to a kinetic mechanism that is characteristic of the polypeptide component and at lower area values to a mechanism that is ascribable to the lipidic component. Both of the mechanisms involve a stage referring to a process of nucleation (P-T law). 2. It is possible to obtain information concerning reciprocal compatibility in bidimensional mixtures through the study of the kinetics of the separation from monolayers. In the case of the poly(DL-alanine) and arachidic acid mixtures it was proved Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981
228
GABRIELLI AND BAGLIONI
FI6.2. Micrographs taken with the electron scanning microscope of the collapsed films. (A) collapsed film of poly(oL-alanine) (x 6000); (B) collapsed film of arachidic acid (x 5000); (C) collapsed film of the mixture in molar ratio 2/1 between moles of monomeric unit of poly(DL-alanine) and moles of arachidic acid.
that the two c o m p o n e n t s are incompatible in the collapsed phase as well. It seems very useful to cerrelate the kinetic results with the t h e r m o d y n a m i c data deduced from the spreading isotherms. 3. The kinetic relationships valid for one Journal of Collold and Interface Science, Vot. 83, No. 1, September 1981
of the c o m p o n e n t s by itself seems valid in the case of separation from mixtures, at least for the system studied. It seems also reasonable to assume, even if further study is necessary for a general extension, that the process of separation is essentially
COLLAPSE OF MONOLAYERS
a phenomenon of crystallization, comprising a phase of nucleation and a successive phase of growth. The mechanism of separation and the energies connected with it depend strongly on the distribution and on the interactions of the components in the monolayer. 4. Observations made with the electron scanning microscope make it possible to confirm that the structure of the poly(DLalanine) obtained after the collapse process of the monolayer is analogous to that found in the tridimensional phase, and that the poly(DL-alanine) is immiscible with arachidic acid. REFERENCES 1. Gabrielli, G., J. Colloid Interface Sci. 53, 148 Polym. Sci. 256, 1165 (1978); Gabrielli, G., and D'Aubert, C., ColloidPolym. Sci. 258, 56 (1980).
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2. Colacicco, G., J. Colloid Interface Sci. 29, 345 (1969); Kafka, M., and Park, C. Y., J. Colloid Interface Sci. 41, 148 (1972); Gerfeld, M. L., in "Methods of Membrane Biology" (E. D. Korn, Ed.), pp. 69-103, Plenum, New York, 1974. 3. Gabrielli, G., Baglioni, P., and Fabbrini, A., Colloid Surfaces, in press. 4. Gabrielli, G., Guarini, G. G. T., and Ferroni, E., J. Colloid Interface Sci. 69, 352 (1976); Gabrielli, G., Guarini, G. G. T., and Bastianini, F., J. Colloid Interface Sci. 69, 352 (1979). 5. Gabrielli, G., and Guarini, G. G. T., J. Colloid Interface Sci. 64, 185 (1978); Baglioni, P., Gabrielli, G., and Guarini, G. G. T., J. Colloid Interface Sci., 78, 347 (1980). 6. Gabrielli, G., Baglioni, P., and Ferroni, E., J. Colloid Interface Sci., 81, 139 (1981). 7. Sestak, J., and Berggren, G., Thermochim. Acta 3, 1 (1971). 8. Prout, E. G., and Tompkins, F. C., Trans. Faraday Soc. 40, 488 (1944); 42, 468 (1946).
Journal of Colloid and Interface Science, Vol. 83, No. 1, September 1981