Langmuir 1997, 13, 5933-5940
5933
Capillary Wave Studies of Multiblock Polypeptide Copolymers at the Air/Water Interface J. V. Gandhi and J. V. Maher* Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
K. A. Shaffer and T. M. Chapman Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received May 19, 1997. In Final Form: August 8, 1997X To observe the changes in the mechanical properties of copolymer interfacial films when controlled changes are imposed on molecular architecture, we have studied capillary waves in spread monolayers of triblock and pentablock copolymers of γ-benzyl-L-glutamate and D,L-glutamic acid having similar molecular weights and hence different block sizes. Using the mechanically generated capillary wave method and the capillary wave dispersion relation, we have extracted the values of viscoelastic parameters including surface pressure, transverse viscosity, longitudinal elasticity, and longitudinal viscosity of copolymer monolayers at the air-water interface, as a function of the interfacial number density of molecules of the triblocks and the pentablocks. The surface pressure, π, shows a clear saturation as a function of the number density for both copolymers. The saturation surface pressure of the pentablocks is not dramatically larger than that of the triblocks, in contradiction to expectations from a steric blocking argument. The longitudinal elasticity increases at a larger rate as the function of the interfacial number density for the pentablocks as compared to the triblocks. We observe negligibly small values of the transverse and the longitudinal viscosity at all number densities of the two copolymers.
I. Introduction Fluid-fluid interfaces covered with polymer films have been of great interest to researchers for a long time.1-9 Recently block copolymers have gained interest because of their increased importance in many technologically challenging areas.10-12 One interesting aspect of the behavior of block copolymers with more than two blocks is that they might localize at a fluid-fluid interface by weaving back and forth across the boundary, forming loops in both fluids.13 When the number of blocks in the copolymer is changed, the number of such potential crossing points can be changed. One possible mechanism of surfactant action is steric blocking, where surfactants reduce the effective interfacial area between the two fluids.14,15 If the contact between the surfactant and the fluid is energetically favorable, a reduction of surface X
Abstract published in Advance ACS Abstracts, October 1, 1997.
(1) May-Colaianni, S. E.; Gandhi, J. V.; Måløy, K. J.; Maher, J. V.; Kuhar, K. A.; Chapman, T. M. Macromolecules 1993, 26, 6595. (2) Gandhi, J. V.; Maher, J. V.; Shaffer, K. A.; Chapman, T. M. Langmuir 1997, 13, 1592. (3) Gaines, G. L., Jr. Insoluble monolayers at liquid-gas interface; Wiley: New York, 1966. (4) Bringuier, E.; Vilanove, R.; Gallot, Y.; Selb, J.; Rondelez, F. J. Colloid Interface Sci. 1985, 104, 95. (5) Zhu, J.; Eisenberg, A.; Lennox, R. B. J. Am. Chem. Soc. 1991, 113, 5583. (6) Li, S.; Hanley, S.; Khan, I.; Varshney, S. K.; Eisenberg, A.; Lennox, R. B. Langmuir 1993, 9, 2243. (7) Sauer, B. B.; Yu, H.; Tien, C.; Hager, D. F. Macromolecules 1987, 20, 393. (8) Lin, B.; Rice, S. A. J. Chem. Phys. 1993, 99, 8308. (9) Runge, F. E.; Kent, M. S.; Yu, H. Langmuir 1994, 10, 1962. (10) Ultracki, L. A. Polymer Alloys and Blends; Hansen Publishers: Munich, Germany, 1987. (11) Paul, D. R. In Polymer Blends; Paul, D. R., Newman, S., Eds.; Academic Press: New York, 1978; Vol. 2, Chapter 12. (12) Committee on Polymer Science and Engineering. Polymer Science and Engineering: The Shifting Research Frontiers; National Academy Press: Washington, DC, 1994. (13) Lyatskaya, Y.; Gersappe, D.; Gross, N. A.; Balazs, A. C. J. Phys. Chem. 1996, 100, 1449. (14) Leibler, L. Physica A. 1991, 172, 258. (15) Halperin, A.; Pincus, P. Macromolecules 1986, 19, 79.
S0743-7463(97)00519-2 CCC: $14.00
energy would then occur. Change in the architecture of the copolymer might result in changes in steric component of the interfacial energy reduction, resulting in interesting changes in interfacial properties. In the present experiment, we have measured changes in interfacial viscoelastic properties when the number of blocks are changed from three (triblocks) to five (pentablocks) while keeping the copolymer molecular weight almost constant. In this experiment, air-water interfaces have been decorated with triblock and pentablock copolymers of γ-benzyl-L-glutamate (Bzl-L-Glu) and D,L-glutamic acid (D,L-Glu). The Bzl-L-Glu residue is hydrophobic while D,LGlu residue is hydrophilic. The molecular weights of the two copolymers are almost the same, indicating different sizes of the blocks in each copolymer. The decision to make block polypeptide amphiphiles was based upon the perceived ease of making multiblocks by the N-carboxyanhydride approach16-18 with reasonable control of molecular weights and polydispersities. One obstacle was the insolubility of the poly(amino acids) derived from the naturally occurring hydrophobic amino acids. Our original strategy was to prepare block sequences using poly(γbenzyl glutamate) as the precursor of the glutamic acid. There was a report that poly(D,L-phenylalanine) was soluble in some organic solvents19 but the corresponding block copolymers prepared with poly(L-γ-benzyl glutamate) were insoluble in all common neutral solvents. We then decided to use poly(γ-benzyl glutamate) sequences as the hydrophobic moieties knowing that this molecule has been studied in organic solvents at great length as one of the prototypical R-helix forming polypeptides.20 The hydro(16) Kricheldorf, H. R. R-Aminoacid-N-Carboxyanhydrides and Related Heterocycles; Springer-Verlag: Berlin, 1987. (17) Katchalski, E.; Sela, M. In Advances in Protein Chemistry; Anfinsen, C. B., Jr., Anson, M. C., Bailey, K., Edsall, J. T., Eds.; Academic Press: New York, 1958; Vol. 13. (18) Szwarc, M. Carbonions, living polymers, and electron transfer processes; Interscience Publishers: New York, 1968, Chapter 10, pp 558-619. (19) Kania, C. M.; Nabizadeh, H.; McPhillimy, D. G.; Patsiga, R. A. J. Appl. Polym. Sci. 1982, 27, 139-148.
© 1997 American Chemical Society
5934 Langmuir, Vol. 13, No. 22, 1997 Scheme 1
philic monomer was to be an orthogonally protected glutamic acid NCA which could be deprotected in the presence of a benzyl ester; we found the p-methoxybenzyl ester to serve this function well. Initiation of NCA polymerization with a diamine gives a homopolypeptide with two growing ends; addition of the comonomer gives a triblock which then leads to a pentablock polymer. We have used the mechanically generated capillary wave (MGCW) method to measure the capillary wave dispersion relation for the film covered air-water interface and thus to determine the interfacial viscoelastic moduli as a function of polymer interfacial number density for both the triblock and the pentablock copolymers. In the following section, we will describe the synthesis of the copolymers, the experimental method and the sample preparation. Section III describes the data analysis involving the extraction of the viscoelastic parameters from the measured data and the dispersion relation. Finally, in section IV, we discuss the copolymer synthesis and characterization and present the results of our measurements of the viscoelastic parameters and compare them in the light of the differences between the very different monomer ordering of the two block copolymers. The observed surface tension reduction of the pentablocks is not dramatically larger than that of the triblocks, in contradiction to expectations from argument that the pentablocks may offer about twice the steric blocking as compared to triblocks at the air-water interface. II. Experimental Section L-glutamic acid, γ-benzyl-L-glutamic acid, 1,6-hexanediamine, phenylhydrazine, N-phthaloyl-D,L-glutamic acid, and N-phthaloyl-D,L-glutamic anhydride were purchased from Aldrich. All were used as received except for 1,6-hexanediamine which was recrystallized from toluene or sublimed. Phosgene was purchased from Fluka as a 1.93 M solution in toluene. Pyridine and DMF obtained from Aldrich (HPLC grade) were used without further purification. Water used throughout the experiment was doubly distilled from a basic permanganate solution. γ-(p-Methoxybenzyl)glutamic acid was prepared by the method of Feijen, et al. (Scheme 1)21 Dimethylformamide was distilled from P2O5 before use. 1H-NMR spectra were recorded at 300 MHz.
(20) Blout, E. R.; Karlson, R. H. J. Am. Chem. Soc. 1958, 80, 1259. (21) Feijen, J.; Sederel, W. L.; de Groot, K.; de Visser, A. C.; Bantjes, A. Makromol. Chem. 1974, 175, 3193-3206.
Gandhi et al. Amino acid N-carboxyanhydrides were prepared by the method of Fuller, et al.22 except that the phosgene solution in toluene was used. In a typical synthesis, 5 g of the amino acid is suspended in 50 mL of dry THF. A 2-fold molar excess of the phosgene solution is added and the suspension heated to 65 °C until the amino acid dissolves, typically 30-60 min. The solution is heated for an extra hour, cooled, and purged with nitrogen until phosgene is not detected by indicating strips.23 The solution is transferred via cannula to a 5-fold excess of stirring hexanes and the solution is stored at -20 °C for 1-2 d. D,L-Phenylalanine NCA. A white powdery solid was obtained: 47%, mp 127.0-127.2 °C (lit. 132-133 °C14). HRMS (EI): exact mass calculated for C10H9O3N, 191.0582; found, 191.0581. IR (KBr): 3397, 1840, 1775 cm-1. 1H NMR (CDCl3): δ 7.30 (d, 2 H, Ar), 6.89 (d, 2 H, Ar), 6.37 (s, 1 H, NH), 5.07 (s, 2 H, OCH2), 4.36 (t, H, CH), 3.81 (s, 3 H, OCH3), 2.56 (t, 2 H, γ-CH2), 2.04-2.36 (m, 2 H, β-CH2). γ-Benzyl L-glutamate NCA, 4b. A white powdery solid was obtained: 72%, mp 92.5-93.4 °C (lit. 96-97 °C24 ). HRMS (EI): exact mass calculated for C13HO5N, 263.0794; found, 263.0797. IR (KBr): 3335, 1858, 1786, 1721, cm-1. γ-o-Nitrobenzyl L-glutamate NCA, 4c. A pale yellow crystalline solid was obtained: 82%, mp 93.5-94.5 °C (lit. 9193 °C25 ). IR (KBr): 3256, 1860, 1786, 1711, 1530, 1345 cm-1. γ-p-Methoxybenzyl L-glutamate NCA, 4a (Scheme 1). A white powdery solid was obtained: 41%, mp 72.1-72.8 °C. IR (KBr): 3276, 1869, 1786, 1700, 1254 cm-1. HRMS (EI): exact mass calculated for C14H15NO6, 293.0899; found, 293.0894. 1H NMR (CDCl3): δ 7.27 (d, 2H, ArH), 6.90 (d, 2H, ArH), 6.61 (bs, 1H, NH), 5.07 (s, 2H, OCH ), 4.37 (t, 1 H, CH), 3.81 (s, 3 H, 2 OCH3), 2.57 (t, 2H, CH2C(O)), 2.28 (m, 1H, CH2CH), 2.11 (m, 1H, CH2CH). Relative stabilities of γ-p-Methoxybenzyl Glutamate and γ-Benzyl Glutamate to 10% TFA in CH2Cl2. γ-p-Methoxybenzyl N-phthaloyl-D,L-glutamate and γ-benzyl N-phthaloyl-D,Lglutamate were dissolved in 10% TFA/CH2Cl2 and the solutions monitored by TLC (ethanol/30% ammonium hydroxide 7:3, silica gel plates). After 35 min, the mixture starting with the p-methoxybenzyl ester showed only N-phthaloyl-D,L-glutamic acid. The reaction also developed a fuchsia color. After 3 days, the benzyl ester mixture showed no change. A diblock polypeptide, poly(γ-(p-methoxybenzyl) D,L-glutamate-b-γ-benzyl D,Lglutamate) was dissolved in the acid mixture and the product precipitated into anhydrous ether after 80 min. 1H-NMR showed the complete loss of the p-methoxybenzyl aromatic protons while the benzyl esters were retained. Finally, a sample of poly(γbenzyl L-glutamate) was subject to the TFA solution for 48 h without a detectable reaction taking place. Block Polypeptide Synthesis. The block polypeptides were made by sequential additions of γ-(p-methoxybenzyl) glutamate N-carboxyanhydride (pmB-D,L-Glu-NCA), 4a, and γ-benzyl glutamate NCA, 4b, to 1,6-hexanediamine (Scheme 2). Reactions were carried out in a foil wrapped round bottom flask equipped with stirring bar, septum adapter, and a drying tube. Typically 1 g of amino acid NCA was dissolved in 10 mL of dry, distilled DMF and an aliquot of initiator solution (∼1 M hexanediamine in DMF) was added via syringe. After IR showed disappearance of the carbonyl peak of the NCA, a small aliquot was removed and a solution of the next amino acid NCA was added. This sequence was continued until the desired block polymer was prepared. Precipitation into a 10-fold excess of absolute ethanol gave a precipitate which was recovered by centrifugation, washing with ethanol, and drying in vacuo overnight. When desired, the products were freeze dried from 1,4-dioxane. The details are presented in Scheme 2 and Table 1. The triblock copolymer studied was made by reaction of the activated γ-pmB-D,L-Glu derivative (4a) to give the poly (amino acid) (5a) terminated with two amino groups (albeit with hexamethylene link in the center). Reaction with the activated γ-Bzl-L-Glu (4b) derivative gave the triblock copolymer (6bab) (22) Fuller, W. D.; Verlander, M. S.; Goodman, M. Tetrahedron Lett. 1976, 15, 1869-1871. (23) Budavari, S., Ed. The Merck Index, 11th ed.; Merck and Co.: Rahway, NJ, 1989; p 1165. (24) Daly, W. H.; Poche, D. Tetrahedron Lett. 1988, 29, 5859-5862. (25) Ledger, R.; Stewart, F. H. C. Aust. J. Chem. 1966, 19, 17291734.
Multiblock Polypeptide Copolymers
Langmuir, Vol. 13, No. 22, 1997 5935 Scheme 2
Table 1. Synthesis of Block Copolymers of Bzl-L-glu and onB-L-Glu or pmB-D,L-Glua -NH2 DMF polymer initiator (mmol) (mL) 5a 6bab 5b 6aba 7babab
1,6-Hexb 5a 1,6-Hexb 5b 6aba
0.20 0.18 0.36 0.18 0.11
14 14.4 10 6.5 5.2
NCA Ester pmB,4a Bzl,4b Bzl,4b pmB,4a Bzl,4b
NCA Timec yield (mmol) (h) DPd (%) 4.74 4.33 3.8 3.8 2.3
41 40 28 40 90
24 24 11 21 21
54 53 25 85
a The structures of these polymers are summarized in Table 2. The method used to synthesize the block copolymers was that NCA’s were added sequentially to the polymerization. b 1,6-Hexanediamine. c Time elapsed before the polymerization of the NCA was complete (by IR), d ) days. d Theoretical degree of polymerization (DP) of the block(s) synthesized, based on the mole ratio of NCA (monomer) to -NH2 (initiator).
(refer to Table 2). Reaction of diaminohexane with γ-Bzl-L-GluNCA followed by γ-pmB-D,L-Glu-NCA, then in turn with γ-BzlL-Glu-NCA gave the pentablock copolymer (7babab) (refer to Table 2). Both polymers terminated with γ-Bzl-L-Glu-NCA sequence. The average lengths of the individual blocks was determined by the mole ratios of the monomer units to the amine initiators and the product ratios were verified by NMR. On treatment with 10% trifluoroacetic acid in CH2Cl2, the pmethoxybenzyl glutamates were cleaved giving block copolymers (8,9) with poly(γ-Bzl-L-Glu) and glutamic acid sequences. The structures of these copolymers are shown in Scheme 3 and Table 3. Column 9 of Table 4 shows the molecular weight of the deprotected species that we used in this experiment as calculated from column 2 of the same table. As Tables 2 and 3 show, the number of hydrophilic (D,L-Glu) and hydrophobic (Bzl-L-Glu) monomers in the triblock copolymer are equal, 48 in each case, while the pentablock copolymer has 42 D,L-Glu and 64 Bzl-L-Glu monomers. The primary difference in the block sizes of the two copolymers is the difference in the size of the hydrophilic blocks which is 48 monomers long in the triblock copolymer while only 21 monomers long in the pentablock copolymer. Methods. We have used the mechanically generated capillary wave (MGCW) technique to measure the capillary wave disper-
sion relations of the block copolymer films at an A/W interface. The details of the MGCW experimental setup and measurement method has been described elsewhere.2 Using this technique, we have measured the wavenumber q and the damping constant β of the capillary (or transverse) waves as a function of the frequency of excitation, f, in the range 500-2900 Hz. Sample Preparation. The triblock (8) and pentablock (9) copolymers were dissolved in the common solvent pyridine for the MGCW method. An optical quality Pyrex glass cell26 was used as the sample cell. All the glassware was treated with Chromerge (mixture of chromic and sulfuric acid) and rinsed thoroughly with double-distilled water. A sample of required polymer interfacial concentration was prepared by depositing drops of the stock solution on the surface of water. Care was taken not to add all the drops at one point on the interface, because it is known that the system may become entangled into a longlived metastable state if all the drops are added to a concentrated region on the interface.4 Pyridine is less dense than water and also spreads easily on the surface of water. We usually waited in excess of 24 h, during which time the pyridine would evaporate and equilibrium would be achieved. At equilibrium, the copolymer film on the A/W interface showed no spatial inhomogeneities (except at very low polymer concentrations) and showed no evidence of change in the capillary wave properties over several days after coming to apparent equilibrium. At very low concentrations of the copolymers, we found that the copolymer monolayer was not uniform even after the sample was allowed to come to equilibrium for tens of hours. The capillary wave spectra in such cases showed that there was significant inhomogeneity on the interface. The capillary waves had a nonuniform damping and/or nonuniform wavelength. We have also encountered such nonuniform monolayers with our studies of diblock copolymers at an A/W interface.2 One plausible explanation is that the polymer monolayer may be existing in a liquidgas coexistence phase.27 In such an inhomogeneous monolayer, the damping and the wavelength of the capillary wave would depend on whether the wave is in the gas phase dominant area or the liquid phase dominant area. The regions of spatial inhomogeneities are all at such low polymer concentrations as (26) G. Finkenbeiner Co., Waltham, MA 01002. (27) Miyano, K.; Tamada, K. Langmuir 1992, 8, 160.
5936 Langmuir, Vol. 13, No. 22, 1997
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Table 2. Structures of the Block Copolymers of Bzl-L-Glu and pmB-D,L-Glu Synthesized polymer
structurea
6bab 6aba 7babab
(Bzl-L-Glu)24(pmB-D,L-Glu)24NH(CH2)6HN(pmB-D,L-Glu)24(Bzl-L-Glu)24 (pmB-D,L-Glu)21(Bzl-L-Glu)11NH(CH2)6HN(Bzl-L-Glu)11(pmB-D,L-Glu)21 [(Bzl-L-Glu)21(pmB-D,L-Glu)21(Bzl-L-Glu)11NH(CH2)3]2
a The degree of polymerization (DP) indicated for each block is the theoretical value based on the NCA (monomer) to -NH (initiator) 2 mole ratio used in the synthesis.
Scheme 3
Table 3. Structures of the Amphiphilic Block Copolymers Synthesized polymer
structure
8 9
poly(Bzl-L-Glu)24poly(D,L-Glu)48poly(Bzl-L-Glu)24 poly(Bzl-L-Glu)21poly(D,L-Glu)21poly(Bzl-L-Glu)22poly(D,L-Glu)21poly(Bzl-L-Glu)22
Table 4. Characterization of Block Copolymers of pmB-D,L-Glu and Bzl-L-Glu mole % Mna sample (calcd) 5b 6bab 5b 6aba 7babab
12100 22500 4700 15100 24300
GPCb Mn 3200 3500 g 4600 6000
pmBGlu Glu % Mnh PDI (calcd)c (obsd)d (obsd)e Yieldf (calcd) 1.4 2.4 g 2.2 2.7
50 67 40
55 68 42
55 65 43
68 48 81
16700 19300
a Based on the mole ratio of NCA (monomer) to -NH (initiator) 2 used in the synthesis. b Versus a polystyrene calibration curve. c From the monomer feed ratio. d From the integrations (1H NMR of the copolymer in CDCl3) of the two aromatic H of pmB-D,L-Glu and the benzylic protons of Bzl-L-Glu and pmB-D,L-Glu. e From the integrations (1H NMR of the deprotected copolymer in TFA-d) of the aromatic H of Bzl-L-Glu and the methine H of Bzl-L-Glu and D,L-Glu. f Percent yield of the deprotected polymer. g Insoluble in THF. h Molecular weight of the deprotected block copolymer used in the experiment.
to be of rather little importance for the discussions below. The data shown in the next sections are all taken when the monolayer was uniform and the capillary wave spectra were also uniform. For the polymer number densities investigated, light scattering and visual inspection showed no evidence of polymeric structures in the bulk water phase. This suggests that most, if not all, of the copolymer resides on the interface, allowing a plausible discussion of the interfacial energetics in terms of measured viscoelastic moduli and the global copolymer concentration of the sample.
III. Data Analysis Mechanically Generated Capillary Wave Technique. As described in detail in ref 2, each measured capillary wave profile obtained using the MGCW technique was fitted, using a least-squares method, to the form
I(x) ) ae-βx cos(qx + φ) The least-squares fits for q and β typically gave very small uncertainties, but by repeating measurements, we found that q and β are reproducible for any one sample to 0.25% and 5% respectively. (The uncertainty in β is larger because fewer characteristics damping lengths, 1/β, are observable than oscillation lengths, 2π/q.) A possible source of systematic error in the damping coefficient is evaporation of water during a single scan (lowering the interface and changing the point of reflection). This would cause β to be underestimated for scans away from the wire and overestimated for scans toward the wire. We reversed the direction of scan for each new frequency scan, and when the whole dispersion relation was fitted, this systematic error was minimized. Figure 1 shows some of the measured values of q and β as a function of the interfacial number density of the triblock copolymers on the A/W interface. The figure shows an interesting trend in q and β. At very low nominal polymer number density, only β shows an increase in its value as polymer density is increased. Then, as still more polymer is added, q also increases. With further increase in the polymer number
Multiblock Polypeptide Copolymers
Figure 1. Wavenumber, q, and spatial damping constant, β, as a function of the polymer number density at two values of frequency for the triblock copolymer at the A/W interface. The error bars for q in each case are smaller than the data symbol shown. Typical uncertainties for β are approximately 5%.
density, while q continues to increase (now with a smaller slope), β (after going through a maximum) decreases before coming to a plateau value. Finally q also comes to a plateau value as more polymer is added to the system. As will be discussed below, the maximum value in β (as observed in our measurements) occurs when the transverse and the longitudinal modes of oscillations at the interface are in resonance with each other. Dispersion Relation. The dispersion relation and the calculation of the viscoelastic parameters are discussed elsewhere.2 The capillary wave dispersion relation for a thin viscoelastic interface between two simple fluids has been reported by Lucassen-Reynders and Lucassen.28 The dispersion relation is the relation between the complex wave number q* ) q - iβ and the angular frequency ω ) 2πf in terms of the viscoelastic parameters which involve the complex surface tension (σ* ) σ + Iωµ) associated with the transverse motion of the surface and the complex dilational elastic modulus (* ) + Iωκ) associated with the longitudinal motion and the material parameters of the two fluids involved. To determine viscoelastic parameters for the interface, it is necessary to insert our measured q(f) and β (f) into the dispersion relation D(q*, ω, σ*, *) and adjust σ* and * to satisfy D ) 0. To accomplish this, we minimize an effective χ2 using the Marquardt-Levenberg algorithm. At an air-liquid interface, the density and viscosity contrast is sufficiently large that there is significant coupling between the transverse and longitudinal waves. This coupling between the two modes is exhibited (in the present case where the apparatus drives transverse modes) mostly in the damping of the transverse modes. As discussed in ref 2, the measured transverse capillary wave parameters, viz. the wavenumber, q, and the (28) Lucassen-Reynders, E. H.; Lucassen, J. Adv. Colloid Interface Sci. 1969, 2, 347.
Langmuir, Vol. 13, No. 22, 1997 5937
damping constant, β, vary in a nonmonotonic way as a function of the longitudinal elasticity . In particular, β goes through a maximum (and q goes through a minimum) at a rather small value of (as compared to the surface tension of σ). This maximum in β occurs when resonance between the transverse and longitudinal modes occurs. The most prominent effect of the longitudinal viscosity κ is to flatten this maximum in β (minimum in q). At larger values of and κ, both q and β tend to become insensitive to the longitudinal moduli. This means that the calculations of such large values of the longitudinal moduli from the measurements of the capillary wave parameters tend to be imprecise. This point will be noted again in the next section while discussing the large uncertainties shown in our determination of for the two block copolymers in the ranges of polymer density where becomes large. As noted above, the dependence of β and q on the longitudinal elasticity is nonmonotonic, making it difficult to deduce four unknown viscoelastic parameters from the measurements of two parameters q and β. As discussed in ref 2, we typically found two sets of solutions (for the viscoelastic parameters) which satisfy the measured dispersion relation. An independent measurement of surface tension of the film covered interface was needed to unambiguously determine the viscoelastic moduli. We used the Wilhelmy plate method to measure the surface tension of the A/W interface covered with copolymers. New samples were prepared at copolymer number densities similar to the number densities used in the MGCW measurements. The surface tension was measured using a platinum plate with a Surface Tensiometer model Sigma 70 (KSV instruments). Our surface tension measurements clearly chose one solution over the other. It should be noted that the surface tension measurements were not performed on the same samples that were used for MGCW experiment. Subsequently, the measurements were not utilized to reduce the number of unknown parameters by one but were used only to choose the correct solution of the dispersion relation from the two solutions obtained from the analysis of our MGCW data. Figure 2 compares the measured values of the wavenumber q and β as a function with a curve calculated using the dispersion relation and the best-fit parameters, σ, µ, , and κ, at 810 × 1012 molecules/cm2 of the triblock copolymer. IV. Results and Discussion Synthesis of block copolymers of alternating glutamic acid and benzyl glutamate sequences required a poly(glutamic acid) precursor that was orthogonally protected. Block copolymer 6bcb containing poly(γ-benzyl glutamate) and poly(γ-o-nitrobenzyl glutamate) was prepared from the corresponding NCA’s 4b and 4c; although the latter could be deprotected photolytically,29 the samples were slightly discolored and the 1H-NMR spectra contained weak, but undefined resonances. Dialysis failed to effect purification and the polymer was not studied further.30 We then determined to our satisfaction that p-methoxybenzyl esters could be hydrolyzed by mild acid treatment without affecting the benzyl glutamate side chains (see Experimental Section) and proceeded from there. The fully protected triblock 6bab and pentablock 7babab were prepared and deprotected with mild acid. A problem arose in the verification of the sequence molecular weights. Although the additions of new monomers led to products with the correct stoichiometry as determined by NMR (columns 5 and 6 of Table 4), and (29) Patchornik, A.; Amit, B.; Woodward, R. B. J. Am. Chem. Soc. 1970, 92, 6333-6335. (30) Kuhar, K. A. Ph.D. Thesis, University of Pittsburgh, 1994.
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Table 5. Interfacial Polymer Number Density, Maximum Surface Pressure, Saturation Monomer Densities, Surface Compressibility and Energy Reduction per Molecule If All the Molecules Joined the Interface at Saturation for the Triblock and Pentablock Copolymer at the Air-Water Interface polymer
Ns (no./cm2)
triblock pentablock
808 × 1020 × 1012 1012
πs (dyn/cm)
(N′s)a (no./cm2)
(N′sb (no./cm2)
κ′ × 103 (cm/dyn)
(∆E)c (kbT/mon)
13 ( 1 17 ( 1
3.9 × 6.5 × 1016
3.9 × 4.3 × 1016
44 ( 5 54 ( 5
8.0 × 10-3 9.5 × 10-3
1016
1016
a Monomer number density of Bzl-L-Glu monomers. b Monomer number density of D,L-Glu monomers. c Energy reduction per D,L-Glu monomer in kbT/monomer.
Figure 2. Measured values (solid circles) and best-fit line for the wave number and damping constant for 810 × 1012 molecules/cm2 of the triblock copolymer as a function of frequency. Measurment uncertainties are indicated wherever they exceed the size of the data symbol.
GPC showed single and not bimodal distribution curves (columns 3 and 4 of Table 4), the molecular weight determinations were not conclusive. GPC indicated lower molecular weights than were calculated (columns 2 and 3 of Table 4), but these are based upon polystyrene as a standard and so are not precise though the numbers should be relative. Indeed, molecular weights increase as new blocks are added. A standard curve for standardized commercial poly(L-γ-benzyl glutamate) samples taken in N-methylpyrrolidone was prepared, but the results were not supportive. The problem is that the standardized samples were enantiopure (consisting of only the L type of optically active isomer) but the block polymers contain racemic (consisting of both the L and the D type of optically active isomers) poly(glutamic acid) sequences. Thus the standards are helical and the copolymers are partly helical and partly random coil in shape. We cannot fit these to the universal calibration as the Mark-Houwink constants would be some average based on the two conformations. Intrinsic viscosities are also model dependent and although the Mark-Houwink constants are known for poly(L-γ-benzyl glutamate), the sequences contain partially racemic monomers which would affect the calculated molecular weights as in GPC. Attempts to obtain molecular weights by MALDI mass spectrometry were unsuccessful and electrospray mass spectrometry, although very promising for the pentablock 7babab, gave inconsistent results. Nevertheless, we believe that the
Figure 3. Surface pressure, π, as a function of the interfacial polymer number density (in molecules/cm2) for the triblock and pentablock copolymers. Ns indicates the number density of molecules at which the saturation of the surface pressure occurs. The lines are a guide to the eye.
samples are useable based on the combined NMR/GPC results above. We now present the results of our capillary wave studies of the triblock and pentablock copolymers at an A/W interface and compare the results in the light of the differences in the structures of the two copolymers. The surface tension is presented as surface pressure, π, which is reduction of surface tension due to the presence of the polymer film on the A/W interface. The concentration of the polymer is presented as a nominal areal number density of the polymer (in the units of number of molecules/ cm2) calculated on the assumption that all copolymer resides on the interface. As was discussed above, light scattering from the bulk water is very compatible with this assumption. Figure 3 shows the best fit values of surface pressure, π, as functions of polymer interfacial number density of triblock and pentablock copolymer respectively. Both surface pressure curves show qualitatively the same behavior, with surface pressure increasing gradually (above a threshold) with the interfacial polymer number density until a “saturation” value is reached, beyond which point the surface pressure does not change appreciably. As can be seen from the first three columns of Table 5, both the polymer number density at which saturation is reached, Ns, and the saturation surface pressure, πs are only marginally different for the
Multiblock Polypeptide Copolymers
Langmuir, Vol. 13, No. 22, 1997 5939
Table 6. Properties of the Molecules in an Assumed Homogeneous Monolayer at the Air-Water Interface at Saturation for the Triblock and Pentablock Copolymers (Refer to Text for Details) polymer
A1 (Å2/molecule)
A2a (Å2/molecule)
overlapb (A2/A1)
A3c (Å2/molecule)
overlapd (A3/A1)
triblock pentablock
12 10
982 1335
82 134
2640 2270
220 227
a Area of hydrophobic block assuming collapsed spherical shape. b Number of overlapping hydrophobic molecules. c Area of hydrophilic block assuming flexible chain. d Number of overlapping hydrophilic molecules.
Figure 4. Expected configurations of the triblock and pentablock copolymers at the A/W interface.
two copolymers. At saturation, the pentablock copolymers have only slightly larger surface tension reduction (24%) as compared to the triblock copolymers (18%). Let us analyze this result in the light of Figure 4. As shown in the figure, the pentablock may weave across the interface about twice the number of times the triblock does. Since steric blocking depends on the number of junctions of the blocks of the copolymer at the A/W interface,14,15 we expect the steric component of the surface tension reduction to be about twice for the pentablocks as compared to the triblocks. When the molecular weight of the copolymers is kept approximately the same, other factors affecting the surface tension have been kept the same, e.g. solvation or ionization energy of each block in its preferred solvent. As noted in section II, the number of hydrophilic blocks in the two copolymers are approximately the same. This might result in about equal surface energy reduction by ionization of the hydrophilic chains in the bulk water below the interface. The above considerations indicate that if the steric blocking were the dominant mechanism of surface energy reduction, the surface tension reduction of the pentablocks would be about twice that of the triblocks. Such a large difference in the surface pressures of the two copolymers is not observed in our measurements, suggesting that the very different configurations of these copolymers at the A/W interface due to their very different molecular architecture may not be very important in reducing the interfacial energy. There is some amount of theoretical work on block copolymers at interfaces, considering the effects of molecular architecture as well as solvent qualities of fluids forming the interface on the properties of the interface.31-35 Simulations have recently become available which consider effects of the molecular architecture on the properties of interfaces.13,36-41 But unfortunately, these simulations (31) Munch, M. R.; Gast, A. P. Macromolecules 1988, 21, 1366. (32) Marques, C.; Joanny, J. F.; Liebler, L. Macromolecules 1988, 21, 1051. (33) Milner, S. T.; Wang, Z. G.; Witten, T. A. Macromolecules 1989, 22, 1454. (34) Marques, C. M.; Joanny, J. F. Macromolecules 1989, 22, 1454. (35) Rossi, G.; Cates, M. E. Macromolecules 1988, 21, 1372. (36) Balazs, A. C.; Siemasko, C. P.; Lantman, C. W. J. Chem. Phys. 1991, 94, 1653. (37) Balazs, A. C.; Zhou, Z. X.; Yeung, C. Langmuir 1992, 8, 2295. (38) Li, W. X.; Yeung, C.; Jasnow, D.; Balazs, A. C. Macromolecules 1992, 25, 3685. (39) Yeung, C.; Balazs, A. C.; Jasnow, D. Macromolecules 1992, 25, 1357. (40) Gersappe, D.; Harm, P. K.; Irvine, D.; Balazs, A. C.; Macromolecules 1994, 27, 720.
do not predict any quantitative effects of changing block numbers and block size (while keeping the molecular weight constant) of a linear block copolymer on the interfacial properties of the copolymer monolayers. Columns 4 and 5 of Table 5 show the number density of hydrophobic and hydrophilic monomers, respectively, at saturation for each of the copolymers. It is interesting to note that the saturation number density of hydrophilic monomers is about the same for both copolymers. Column 6 in Table 5 lists the values of the surface layer compressibility (κ′ ) (1/A)(∂A/∂π), where A ) 1/N for each of the copolymer films, calculated at the point where the slope of π vs number density curve is the maximum. These values are small and similar for both copolymers. The energy reduction at saturation per hydrophilic monomer has been calculated in column 7 and similar values of energy reduction are observed for both block copolymers (both being about 1% of kT). The easiest way to explain the saturation of π is to assume that the polymer molecules added beyond number density Ns do not join the interfacial monolayer, because it may not be energetically favorable to add another molecule to an already crowded interface, but that they either go into the bulk water phase, possibly forming micelles, or form multilayer structures at the A/W interface. It should be noted that our qualitative light scattering studies by visual inspection showed very little polymeric scattering from the bulk water phase, even at number densities above Ns. This suggests a very small concentration of micelles and may support formation of a multilayer structure at the interface. As noted above, absence of light scattering at number densities at and below Ns also permits a discussion of the copolymer conformations at the A/W interface assuming that the interface is formed at the true interfacial number density at and below Ns. Table 6 lists the area available at saturation per molecule, A1, for triblock and pentablock copolymers in column 2. The hydrophobic Bzl-L-Glu block is expected to form a tightly collapsed structure.42 Assuming spherical shape for such collapsed structure, the area of projection, A2, of such coils would be as shown in column 3. These areas are much larger than the area available to each molecule at saturation, suggesting that the hydrophobic block would be very stretched and the extent of such stretching has been calculated as a number of overlapping molecules as shown in column 4. It is known that the hydrophilic poly(L-glutamic acid) forms helical configurations in water in acidic conditions when it is not completely ionized;43 however, at neutral pH values such as obtained in this experiment, it is completely ionized and has a random coiled structure.43,44 Assuming a flexible chain, the area of projection, A3, of such coils will be as shown (41) Lyatskaya, Y. and Gersappe, D. and Balazs, A. C. Macromolecules 1995, 28, 6278. (42) Doty, P.; Bradbury, J. H.; Holtzer, A. M. J. Am. Chem. Soc. 1956, 78, 947. (43) Doty, P.; Wada, A.; Yang, J. S.; Blout, E. R. J. Polym. Sci. 1957, 23, 851. (44) Tanford, C. Physical Chemistry of Macromolecules; John Wiley and Sons, Inc.: New York, 1961.
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Gandhi et al.
larger triblocks. The figure shows that, after reaches a large value (g50), the uncertainties in our determination of become very large. As has been noted in the last section, this is due to the fact that, at large enough values of , the observable capillary wave properties become very insensitive to changes in . Thus these uncertainties are not the uncertainties of measurements but are simply imprecision in the relation between the measured transverse wave properties and the longitudinal moduli. Longitudinal viscosity, κ, is consistent with zero for all the number densities of triblocks and almost all the number densities of the pentablocks. Nonzero values of κ have been conjectured to be due to the diffusional exchange of molecules between the bulk and the interface.28 Hence the absence of κ may indicate that the monolayers formed by both copolymers are insoluble monolayers and the exchange of molecules between the bulk and the monolayer during expansion or contraction of the interface is negligible. V. Summary
Figure 5. Best-fit longitudinal elasticity, , as a function of the interfacial polymer number density (in molecules/cm2) for the triblock and pentablock copolymers.
in column 5. This area indicates interpenetration of these coils and the overlap number of the coils is calculated in the last column. The above estimates show that the interface may be very crowded at saturation. Additional molecules added to the saturated interface may find it energetically favorable to form micelles in the bulk or form multilayer structures at the interface. We find the values of the transverse viscosity, µ, to be consistent with zero with uncertainty of the order 10-4 (dyn s)/cm for all our measurements. We now turn our attention to the longitudinal parameters. As discussed in the last section, the density and viscosity contrast between air and water makes it possible to determine the longitudinal elasticity, , and the longitudinal viscosity κ, for the copolymer monolayers at the A/W interface even though our apparatus primarily drives transverse waves. Figure 5 shows the best fit values of , as functions of interfacial number density of triblock and pentablock copolymers respectively. As the figure shows, increases monotonically with the nominal number density of molecules until it reaches a large value (g50), indicating formation of very stiff monolayers for both copolymers. The rate of increase in as a function of the polymer number density is higher for the pentablock copolymers as compared to the triblock copolymers, as can be seen from the figure. The smaller blocks of the pentablock copolymers may be playing a role in forming a more efficiently packed monolayer as compared to the
Using the mechanically generated capillary wave method, we have compared the surfactant properties of triblock and pentablock copolymers of hydrophobic γ-benzyl L-glutamate and hydrophilic D,L-glutamic acid at the air-water interface. In particular, we measured the surface pressure, π, surface transverse viscosity, µ, surface longitudinal elasticity, , and surface longitudinal viscosity, κ, as a function of the number density of molecules for the triblock and pentablock copolymers. After a certain number density of molecules, Ns, both copolymers show a saturation of the surface pressure. The saturation of the surface pressure may be attributed to crowding of the interface making it energetically favorable for subsequently added molecules to select a different configuration such as a multilayer or a micelle. The saturation surface pressure of the pentablocks is not dramatically different from that of the triblocks, suggesting that steric blocking is not the dominant mechanism of surface energy reduction. Within the accuracy of our measurement method, we found the values of transverse viscosity, µ, to be consistent with zero. The rate of increase of the longitudinal elasticity, , as a function of the polymer number density is higher for the pentablock copolymers as compared to the triblock copolymers. This might indicate a more efficient and hence denser packing of the smaller pentablock molecules. A large increase in the values of at higher number densities indicates formation of a stiff monolayer at the air-water interface by both polymers. For all the number densities of the triblock copolymer, the longitudinal elasticity, κ, is negligible, possibly indicating an insoluble monolayer. The pentablock copolymer also shows a similar trend in κ for almost all the number densities investigated. Acknowledgment. We acknowledge helpful discussions with A. C. Balazs. We also acknowledge Alan Esker and Hyuk Yu for their very helpful advice on performing the Wilhelmy plate measurements. This work was supported by DOE Grant No. DE-FG02-84ER45131. LA970519H
