Two-Dimensionally Well-Ordered Multilayer Structures in Thin Films of

Apr 10, 2008 - Department of Microbiology, Dongguk UniVersity College of Medicine, Gyeongju 780-714, Republic of Korea. ReceiVed: NoVember 24, 2007; ...
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5338

J. Phys. Chem. B 2008, 112, 5338-5349

Two-Dimensionally Well-Ordered Multilayer Structures in Thin Films of a Brush Polypeptide Jinhwan Yoon,†,| Seung Woo Lee,‡,| Seungchel Choi,† Kyuyoung Heo,† Kyeong Sik Jin,† Sangwoo Jin,† Gahee Kim,† Jehan Kim,† Kwang-Woo Kim,† Heesoo Kim,§ and Moonhor Ree*,† Department of Chemistry, National Research Laboratory for Polymer Synthesis and Physics, Pohang Accelerator Laboratory, Center for Integrated Molecular Systems, and BK school of Molecular Science, Pohang UniVersity of Science and Technology (Postech), Pohang 790-784, Republic of Korea, School of Chemical Engineering and Technology, Yeungnam UniVersity, Gyeongsan 712-749, Republic of Korea, and Department of Microbiology, Dongguk UniVersity College of Medicine, Gyeongju 780-714, Republic of Korea ReceiVed: NoVember 24, 2007; In Final Form: February 16, 2008

In this study, we report the first production of two-dimensionally well-ordered molecular multilayers (i.e., with a well-defined molecular lamellar structure) based on the antiparallel β-sheet chain conformation in thin films of a brush polypeptide, poly(S-n-hexadecyl-DL-homocysteine) (PHHC), through the use of a simple spin-coating process and the quantitative structural and property analysis of the thin films using a grazing incidence X-ray scattering technique combined with Fourier transform infrared spectroscopy and differential scanning calorimetry. These analyses provide detailed information about the structure and molecular conformation of the self-assembled lamellae in the PHHC thin film, which is not easily obtained using conventional techniques. Moreover, we used the in situ measurements carried out at various temperatures and the data analyses to establish mechanisms for the evolution of the self-assembled lamellar structures in the film and for their melting. In addition, we propose molecular structure models of the PHHC polymer molecules in the thin film at various temperatures.

Introduction Polypeptides with well-ordered structures are of significant interest because of their self-assembly into a stable, ordered conformation. Much research effort has gone into producing ordered peptides and characterizing their structures.1-3 For example, a series of R-helical poly(L-glutamates) with n-alkyl side chains of various lengths was synthesized, and their structures and thermal properties were investigated.2 The thermal properties and structural changes of poly(R-n-alkyl β-L-aspartate)s with n-alkyl side groups were studied.3 Most research into such brush polypeptides has focused on their bulk structures and properties. However, well-ordered brush polypeptides in thin films have also attracted attention as a novel class of materials because of their potential applications in various fields such as microelectronic, optical, and optoelectronic devices.4 The structures of polypeptide thin films can be significantly different from those of the bulk because their molecular configuration and structural order can be affected by their interfaces with substrates and air or vacuum. Thus, it is important to investigate the structure and properties of brush polypeptide thin films. X-ray reflectivity (XR) has been widely used to examine the structure and orientation of polymer thin films.5 However, although XR analysis can be used to determine the film thickness and roughness of the surface and the interface as well as the electron density profile within the film, it cannot provide * To whom correspondence should be addressed. E-mail: ree@ postech.edu, Tel: +82-54-279-2120, Fax: +82-54-279-3399. † Pohang University of Science and Technology. ‡ Yeungnam University. § Dongguk University College of Medicine. | J. Yoon and S. W. Lee contributed equally to this work.

information about the structure in the film plane or the overall film structure. Conventional transmission X-ray and neutron scattering (TXS and TNS) have also been considered for this purpose, but are not applicable because of their low sensitivity and resolution, which are particularly problematic for low scattering volumes and when the substrate is much thicker than the film.6-9 Therefore, a nondestructive method is required for the quantitative analysis of thin films of polymers such as brush polypeptides. It is also important to characterize the dependence of structure on temperature, because brush polypeptides undergo thermal transitions. Grazing incidence X-ray scattering (GIXS) has recently emerged as a very powerful technique for characterizing the surface and internal structures of thin films because of several important advantages: a highly intense scattering pattern with high statistical significance is always obtained, even for thin films, because the X-ray beam path length through the film plane is sufficiently large; there is no unfavorable scattering from the substrate on which the film is deposited; and sample preparation is easy.7-11 However, the use of the GIXS technique is only possible in conjunction with the development of a scattering theory for the system of interest and intensive data analysis because grazing incidence scattering is complicated by reflection and refraction effects8-10 that are not found in conventional TXS and TNS. Because of these complexities of GIXS data analysis, the full use of GIXS in the characterization of the structures of thin films and nanostructure systems has been limited. In the present paper, we report the first production of twodimensionally well-ordered molecular multilayers based on the antiparallel β-sheet chain conformation in thin films of a brush polypeptide, poly(S-n-hexadecyl-DL-homocysteine) (PHHC) (Figure 1), which was carried out with simple spin-coating on

10.1021/jp711149k CCC: $40.75 © 2008 American Chemical Society Published on Web 04/10/2008

2D Well-Ordered Multilayer Thin Films

Figure 1. Chemical structure of the poly(S-n-hexadecyl-DL-homocysteine) (PHHC), a brush polymer used in our study.

substrates, and the quantitative structural analysis of the thin films during heating and subsequent cooling by using a GIXS technique with synchrotron radiation sources. In addition, differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy analysis were conducted on the brush polypeptide thin films, and we found that the thermal heating of the thin films induces an order-to-disorder phase transition in the n-alkyl bristles and also induces a change in the chain conformation of the brush polypeptide molecules from an antiparallel β-sheet structure to an R-helical structure; the reverse chain conformation change and bristles’ phase transition processes were found to occur when cooled from the melt. In fact, a molecular multilayer stack (i.e., lamellar stack structure) is one of the most common morphological structures observed in films and bulk samples of polymers. However, no quantitative 2D GIXS analysis, or even any TXS and TNS analyses, of such lamellar stack structures in polymer thin films and bulk samples has previously been carried out. Only limited qualitative scattering analysis has been performed with scattering profiles extracted along the direction of the lamellar stacking.6,12 In the present study, 2D GIXS measurements and quantitative data analysis using a derived GIXS formula were used to provide for the first time the comprehensive structure and orientation details of lamellar stacks formed in thin films, in this case of a brush PHHC polymer. Experimental Section Brush Polypeptide Synthesis and Thin Film Preparation. triphosgene, anhydrous dimethyl formamide (DMF), anhydrous ethyl acetate (EA), 1-bromohexadecane, and chlorobenzene were purchased from Aldrich Company and used as received. S-n-Hexadecyl-DL-homocysteine (HHC) was synthesized as follows. DL-Homocysteine (7.8 g, 0.058 mol) in 50 mL of 2 N NaOH and 60 mL of ethanol were stirred at room temperature for 30 min. 1-Bromohexadecane (17.7 g, 0.058 mol) was then added into the reaction mixture with stirring for 6 h. The reaction mixture was poured into cold water, and the pH was adjusted to 7, giving S-n-hexadecyl-DL-homocysteine with 72.0% yield. S-n-Hexadecyl-DL-homocysteine N-carboxylic anhydride (HHCNCA) was synthesized from HHC, according to a method reported in the literature.13 HHC (2.00 g, 5.57 mmol) was dissolved in 150 mL ethyl acetate and then refluxed for 30 min. Triphosgene (0.550 g, 1.86 mmol) was added immediately to this solution, and then refluxing of the reaction mixture was continued for an additional 4 h. The resulting pale yellow solution was chilled to 0 °C, and then washed with cold deionized water, followed by washing twice with cold 0.5% sodium bicarbonate solution. The organic layer was dried with MgSO4 and concentrated, and then an equal volume of hexane was added to induce crystallization. The target product was collected as white crystals in a yield of 78.5%. 1H NMR (δ, CDCl3): 4.80-3.70 (m, 1H, -CH-NH-), 2.51 (m, 4H, -CH2DL-Homocysteine,

J. Phys. Chem. B, Vol. 112, No. 17, 2008 5339 S-), 2.12 (m, 2H, S-CH2-CH2-CH), 1.55 (m, 2H, S-CH2-CH2CH2-), 1.25 (m, 26H, CH2-CH2-CH3), 0.88 (t, 3H, CH2-CH3). Poly(S-n-hexadecyl-DL-homocysteine) (PHHC) was synthesized using the ring-opening polymerization of HHCNCA as follows. HHCNCA (1.84 g) was dissolved in 10 mL of anhydrous DMF under a nitrogen atmosphere, then triethylamine (0.005 mL, 0.01 equiv mol with respect to the used HHCNCA) was added as an initiator, followed by stirring at room temperature for 1 day. This polymerization was aimed to have a PHHC product with a degree of polymerization of 100 via controlling the molar ratio of the initiator to the monomer. The product was precipitated in methyl alcohol, filtered, and then dried under vacuum at 40 °C. The product was obtained as a white powder in 90.5% yield. The inherent viscosity in chlorobenzene solution was found to be 0.45 dL/g. 1H NMR (δ, CDCl3/CF3COOD ) 5/1 in volume): 5.20-4.30 (m, 1H, -CH-NH-), 2.71-2,48 (m, 4H, -CH2-S-), 2.40-1.92 (m, 2H, S-CH2-CH2-CH), 1.56 (m, 2H, S-CH2-CH2-CH2-), 1.27 (m, 26H, CH2-CH2-CH3), 0.89 (t, 3H, CH2-CH3). PHHC thin films were prepared as follows. The dried PHHC product was dissolved in chlorobenzene, producing a 1.5 wt % solution. This solution was filtered through PTFE membranes of pore size 0.2 µm, and then spincoated onto precleaned silicon substrates at 2000 rpm for 60 s, followed by drying at 40 °C for 1 day under vacuum. The resulting PHHC films were measured to have a thickness of 50-100 nm, using a spectroscopic ellipsometer (model M2000, J. A. Woollam Inc.) and an R-stepper (model Tektak3, Veeco Company). Measurements. DSC measurements were carried out at a rate of 10.0 °C/min over the temperature of -75 to 180 °C under a nitrogen atmosphere using a Seiko differential scanning calorimeter. FTIR spectroscopic measurements were performed during heating and subsequent cooling at a rate of 10.0 °C/min over 25-180 °C by using an ATI Mattson spectrometer (model Research Series) equipped with a hot stage under a nitrogen atmosphere and a programmable temperature controller system. Samples were installed perpendicular to the incident beam direction. IR spectra were recorded at 4 cm-1 resolution with a liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector under vacuum. GIXS measurements were carried out at the 4C1 and 4C2 beamlines14 at the Pohang Accelerator Laboratory.15 The samples were measured at a sample-to-detector distance of 146 mm, using an X-ray radiation source with a wavelength λ of 0.154 nm, and imaged using a two-dimensional charge-coupled detector (2D CCD: Roper Scientific, Trenton, NJ), as shown in Figure 2. Samples were mounted on the heating/cooling stage of a homemade z-axis goniometer equipped with a vacuum chamber. The incidence angle Ri of the X-ray beam was set at 0.20°, which is between the critical angles of the films and the silicon substrate (Rc,f and Rc,s). The GIXS measurements were conducted on the film samples during heating and subsequent cooling over the temperature range 25-180 °C. Here, the heating and subsequent cooling runs were carried out according to a programmed multistep protocol [for example, a four-step (25.0, 75.0, 100.0, 120.0, and 180.0 °C) heating run and a fourstep (180.0, 120.0, 70.0, 45.0, and 25.0 °C) cooling run] where a ramping or cooling rate between the steps was 10.0 °C and a holding time at each step was 60 s. Scattering data collections were typically performed for 60 s at the programmed steps. Scattering angles were corrected according to the positions of the X-ray beams reflected from the silicon substrate interface

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Yoon et al.

Figure 2. Geometry of GIXS: Ri is the incident angle at which the X-ray beam impinges on the film surface; Rf and 2θf are the exit angles of the X-ray beam with respect to the film surface and to the plane of incidence, respectively, and qx, qy, and qz are the components of the scattering vector q.

with changing incidence angle Ri, and with respect to a precalibrated silver behenate (TCI, Japan) powder. Aluminum foil pieces were employed as a semitransparent beam stop, because the intensity of the specular reflection from the substrate is much stronger than the intensity of GIXS near the critical angle. GIXS Theory. The intensity of GIXS (IGIXS) from a thin film can be expressed by the scattering formula derived recently8,9

IGIXS(Rf, 2θf) =

1 1 - e-2Im(qz)d ‚ × 16π2 2Im(qz)

[

|TiTf|2I1(q|, Re(q1,z)) + |TiRf|2I1(q|, Re(q2,z)) + |TfRi|2I1(q|, Re(q3,z)) + |RiRf|2I1(q|,Re(q4,z))

]

(1)

where Rf is the angle between the scattered beam and the film surface (i.e., out-of-plane exit angle), 2θf is the angle between the scattered beam and the plane of incidence (i.e., in-plane exit angle), Im(qz) ) |Im(kz,f)| + |Im(kz,i)|, Re(x) is the real part of x, d is the film thickness, Ri and Ti are the reflected and transmitted amplitudes of the incoming X-ray beam, respectively, and Rf and Tf are the reflected and transmitted amplitudes of the outgoing X-ray beam, respectively. In addition, q| ) xqx2+qy2, q1,z ) kz,f - kz,i, q2,z ) -kz,f - kz,i, q3,z ) kz,f + kz,i, and q4,z ) -kz,f + kz,i; here, kz,i is the z-component of the wave vector of the incoming

X-ray beam, which is given by kz,i ) ko xn2R-cos2Ri, and kz,f is the z-component of the wave vector of the outgoing X-ray

beam, which is given by kz,f ) koxn2R-cos2Rf, where ko ) 2π/ λ, λ is the wavelength of the X-ray beam, nR is the refractive index of the film given by nR ) 1 - δ + iξ with dispersion δ and absorption ξ, and Ri is the out-of-plane grazing incident angle of the incoming X-ray beam. qx, qy, and qz are the components of the scattering vector q. I1 is the scattering intensity of the structure in the film, which can be calculated kinematically. In eq 1, I1 is the scattered intensity from the structure (i.e., scatterers and their ordering) in the film, and thus can be expressed by the following equation:8,16

I1(q) ) P(q)‚S(q)

(2)

Figure 3. Schematic representation of a paracrystalline lattice composed of lamellar-ordered molecules: a is the primitive lattice vector, which is defined by its components a1, a2, and a3, as shown in this figure; n is the vector of the lamellar orientation axis, which is parallel to a3; d3 is the long period between lamellae; h is the mean thickness of one layer in a lamella; dr is the lateral distance within a lamella; β is the angle between a1 and a3; φ is the polar angle between the n vector and the out-of-plane direction of the film; and ∆a is the lattice positional fluctuation parameter. The subscript r denotes the direction transverse to a3. As an example, the molecules shown here in an extended chain in the lamellar structure assembly are PHHC molecules, which were investigated in this study.

where P(q) is the form factor of the scatterers that describes the shape, size, and orientations of the scatterers, and S(q) is the structure factor that provides information on the positions of the scatterers such as the crystal lattice parameters, orientation, dimension, and symmetry in an ordered structure and the interdistance of the scatterers in the case of an isotropic colloidlike system. To quantitatively analyze lamellar structures formed in PHHC thin films of our study, we have considered possible models including Hosemann paracrystal model,17 Cialle model,18 and modified Cialle model.19 As a result, we have found that the paracrystal model is most suitable for the lamellar structures formed in PHHC thin films of our study (Figure 3). For an ordered layer lattice with dimensions of L1, L2, and L3, the form factor P(q) can be expressed as follows:20

P(q) ) exp

{4π1 [L

2

1

(qx sin β + qz cos β)2 + L2qy2 +

}

L3qz2] sin2 β (3) where qx, qy, and qz are the components of the scattering vector q, and β is the angle between the a1 and a3 components of the lattice vector a (see Figure 3). In this study, the lattice dimension is assumed to be identical to the interlattice distance: namely, L1 ) L2 ) dr and L3 ) h, where dr is the lateral interdistance of the lattices of a lamella and h is the thickness of one layer in the lamella defined by a long period d3 (Figure 3). For a paracrystalline system, as proposed by Hosemann and Bagchi17 and refined by Blundell,21 the structure factor S(q)

2D Well-Ordered Multilayer Thin Films

J. Phys. Chem. B, Vol. 112, No. 17, 2008 5341

(the so-called interference function or lattice factor) can be determined from the Fourier transform of a complete set of lattice points. In the paracrystal model with distortion of the second kind, its lattice points are no longer fixed at certain positions but instead described by a positional distribution function. In the simple case, where the autocorrelation function of a crystal lattice is given by the convolution product of the lattice point distributions along the three vector component axes, and the distribution function is Gaussian, S(q) can be expressed by the following equation:22 3

S(q) ) [〈|f|2〉 - |〈f〉2|] + |〈f〉2|[

Zk(q)] ∏ k)1

(4)

where

f)

sin(qzh/2) exp(-σh2qz2/2) qzh/2

(5)

In the above equation, h and σh are the thickness of one layer in a lamella, as defined above, and its deviation, respectively. The kth lattice factor Zk(q) is given by

Zk(q) )

1 - |Fk(q)|2 1 + |Fk(q)|2 - 2|Fk(q)| cos(q‚ak)

(6)

where

[( [( [(

)] )] )]

qc ) R‚c* ≡ (qc,x, qc,y, qc,z)

(10)

where R is a 3 × 3 matrix for deciding the preferred orientation of the structure in the film; and qc,x, qc,y, and qc,z are the x-, y-, and z-components of the peak position vector qc, respectively. Using eq 10, every peak position can be determined. Due to the cylindrical symmetry, the Debye-Scherrer ring composed of the in-plane randomly oriented lattice point c* cuts an Ewald sphere at two points in the upper half of the sphere: q| ) qc,| ≡ (xq2c,x+q2c,y with qz ) qc,z. In this respect, diffraction patterns with cylindrical symmetry can be easily calculated in q-space. It is then convenient to determine the preferred orientation of known structures and to analyze the anisotropic X-ray scattering patterns. However, since the q-space in the GIXS patterns is distorted by refraction and reflection effects, the relation between the detector plane (expressed as the Cartesian coordinate defined by two perpendicular axes, i.e., in-plane exit angle 2θf and out-of-plane exit angle Rf, and the reciprocal lattice points is needed. The two wave vectors kz,i and kz,f are corrected for refraction as kz,i ) ko

qr2dr2grr2 + qz2d32gr32 F1(q) ) exp 2

(7a)

qr2dr2grr2 + qz2d32gr32 F2(q) ) exp 2

(7b)

qr2dr2g3r2 + qz2d32g332 F3(q) ) exp 2

anisotropic displacement is assumed and the domain orientation is accounted for numerically. For the lamellar structure with a given orientation, its fundamental vectors can be rotated and transformed using a rotation matrix. When the structure within a thin film is randomly oriented in-plane, but uniaxially oriented out-of-plane, the peak position vector qc of a certain reciprocal lattice point c* in the sample reciprocal lattice is given by

(7c)

and

q‚a1 ) (qx + qz cot β)dr

(8a)

q‚a2 ) qydr

(8b)

q‚a3 ) qzd3

(8c)

where ak is the fundamental vector of the kth axis and qr )

x

q2x +q2y .

grr, grz, gzr, and gzz are the components of the paracrystal distortion factor, which are defined as

grr ) ∆arr/ar

(9a)

gr3 ) ∆ar3/ar

(9b)

g3r ) ∆a3r/ar

(9c)

g33 ) ∆a33/a3

(9d)

where ∆ak is the displacement of the vector ak shown in Figure 3; for example, ∆a3r represents the displacement of the fundamental vector a3 in the direction of r; here, r denotes the direction transverse (normal) to a3. In the present study,

xnR2-cos2Ri

and kz,f ) koxn2R-cos2Rf, respectively. Therefore, the two sets of diffractions that result from the incoming and outgoing X-ray beams, and denoted by q1 and q3, respectively, are given at the exit angles by the following expression:

Rf ) arccos

[x ( nR2 -

)]

qc,z ( n 2 - cos2 Ri ko x R

2

(11)

where qc,z/ko > xn2R-cos2Ri. In eq 11, the positive sign denotes diffractions produced by the outgoing X-ray beam, while the negative sign denotes diffractions produced by the incoming X-ray beam. The in-plane incidence angle 2θi is usually zero, so the in-plane exit angle 2θf can be expressed by the following equation:

[

cos2 Ri + cos2 Rf -

2θf ) arccos

( )

2 cos Ri cos Rf

qc,| ko

2

]

(12)

Therefore, diffraction spots observed at the detector plane in GIXS measurements can be directly compared to those derived using eqs 10-12 from an appropriate model, and thus analyzed in terms of the model. As can be seen in Figure 3, the distribution of the orientation vector n with respect to the film plane is given by a function D(φ), where φ is the polar angle between the n vector and the out-of-plane direction of the film; for example, φ is zero when the n vector in the film is perfectly oriented normal to the film plane. To calculate the 2D GIXS patterns, D(φ) should be represented by an actual numerical function. In relation to the distribution of the lattice orientation, D(φ) can be generally considered as a Gaussian distribution

5342 J. Phys. Chem. B, Vol. 112, No. 17, 2008

D(φ) )

1

x2πσφ

[

exp -

(φ - φ j )2 2σφ2

]

Yoon et al.

(13)

where φ j and σφ are the mean angle and standard deviation of φ from φ j, respectively. The observed scattering intensity IGIXS,φ(q) is obtained by averaging IGIXS(q) over possible orientations of the lattice

IGIXS,φ(q) )

∫-ππ IGIXS(q)D(φ) dφ

(14)

The second-order orientation factor Os can be defined as the following equation23

Os )

(3 cos2 φ - 1) dφ 2

∫D(φ)

(15)

when D(φ) is strongly peaked around φ ) 0 (i.e., vertical alignment), cos φ ) 1, and Os ) 1. On the other hand, if the orientation is entirely random, 〈cos2 φ〉 ) 1/3 and Os ) 0. Thus, Os is a measure of the orientation of lamella. Results and Discussion FTIR and DSC Analysis. Figure 4 shows FTIR spectra of a PHHC thin film, measured during heating and cooling at a rate of 10.0 °C/min. These vibrational bands can be assigned with the aid of results previously reported in the literature.24-26 As can be seen in Figure 4a, the spectrum for the PHHC film at 25 °C contains vibrational peaks at 1457 and 1471 cm-1, which are characteristic of methylene units in gauche- and transconformational structures, respectively.24 The trans-conformational band is stronger in intensity than the gauche-conformational band. These results indicate that, in the PHHC film, the n-hexadecyl groups of the bristles consist mostly of transconformational methylene units and fewer gauche-conformational methylene units. During the heating run, the intensity of the trans-conformational band at 1471 cm-1 decreases with increasing temperature and, in contrast, that of the gaucheconformational band at 1457 cm-1 increases (Figure 4a). This conformation change occurs mainly over the temperature range 45-75 °C. During the subsequent cooling run, the reverse of this change in the conformation of the n-hexadecyl groups occurs. In addition to the vibrational peaks characteristic of the methylene units in the bristles, the spectra of the PHHC film contain vibrational peaks at 1654 and 1676 cm-1 with intensities that depend on the temperature (Figure 4b), which are characteristic of peptide carbonyl groups in an R-helix and an antiparallel β-sheet conformational structure, respectively.25,26 The vibrational band at 1676 cm-1 is very strong at 25 °C, indicating that the PHHC polymer chains are mainly present in the film in an antiparallel β-sheet conformation. During the heating run, the intensity of the antiparallel β-sheet conformational structure peak at 1676 cm-1 decreases with increasing temperature, and in contrast, that of the R-helix conformational band at 1654 cm-1 increases (Figure 4b). This conformation change occurs mainly over the temperature range 75-115 °C. During the subsequent cooling run, the change in the conformation of the peptide backbone occurs in reverse; however, this reverse conformational change takes place below 75 °C. Figure 5 shows DSC thermograms of the PHHC polymer obtained during heating and cooling at a rate of 10.0 °C/min. During the heating run, the brush polymer undergoes three phase transitions. One phase transition appears at 24.6 °C. This

Figure 4. FTIR spectra of a PHHC polymer film measured in transmission mode during heating and subsequent cooling. The measurements were performed during heating and subsequent cooling at a rate of 10.0 °C/min under a nitrogen atmosphere.

Figure 5. DSC thermograms of PHHC polymer. The measurements were performed during heating and subsequent cooling at a rate of 10.0 °C/min under a nitrogen atmosphere.

transition is very weak and broad, and determined to reveal only a small heat capacity change ∆Cp (0.183 J/°C‚g). Thus, the phase transition is thus a glass transition. The second phase transition occurs over the temperature region 50-73 °C; the peak maximum temperature and heat of fusion ∆Hf of this transition were determined to be 61.1 °C and 8.2 J/g, respectively. Taking into account the above IR spectroscopy results, we conclude that this phase transition is associated with the change in the conformation of the methylene units in the bristles’ n-hexadecyl groups from the trans-conformational structure to the gaucheconformational structure, i.e., the phase transition at 50-73 °C corresponds to the melting transition of the crystalline phase of the n-hexadecyl groups in the brush polymer. Further, note that, when the polymer film is in a crystalline state below 50 °C, the trans-conformational methylene units of the n-hexadecyl groups participate in a crystalline phase in the polymer film; the less abundant gauche-conformational methylene units of the nhexadecyl groups are present in a less ordered phase in the film. The third phase transition takes place over the temperature region 80-112 °C; the peak maximum temperature of this transition is 102.7 °C and ∆Hf ) 21.8 J/g. Taking into account the above IR spectroscopy results, we conclude that this phase transition is associated with the change in the structure of the

2D Well-Ordered Multilayer Thin Films peptide backbone from the antiparallel β-sheet conformational structure to the R-helix conformational structure. During the cooling run, the polymer also undergoes three transitions (Figure 5). Two phase transitions occur in the temperature region 73-33 °C, and a glass transition is discernible around 23.4 °C (∆Cp ) 0.194 J/°C‚g). These phase transitions overlap heavily, which is very different from the transitions observed during the heating run. For the less intense phase transition peak in the high-temperature region, the peak maximum temperature is 61.1 °C and ∆Hf ) 7.4 J/g. The determined values are very similar to those (61.1 °C and 8.2 J/g respectively) obtained for the melting transition of the crystalline phase of the n-hexadecyl groups in the brush polymer during the heating run. However, this exothermic transition occurs at temperatures lower than those at which the change in the structure of the peptide backbone from the antiparallel β-sheet conformational structure to the R-helix conformational structure takes place on the heating run. In general, linear polymers and bristles require a certain degree of supercooling to crystallize from the melt state.27 Taking into consideration the results as well as the fact, the exothermic phase transition is attributed to the change in the structure of the peptide backbone to the antiparallel β-sheet conformational structure from the R-helix conformational structure, rather than the crystallization of the n-hexadecyl groups in the brush polymer. For the highly intense phase transition peak, the peak maximum temperature is 54.7 °C and ∆Hf ) 23.1 J/g. This phase transition occurs at temperatures lower than those at which the two endothermic phase transitions take place on the heating run. The determined ∆Hf value is slightly larger than that (21.8 J/g) obtained for the endothermic phase transition in the temperature region 80-112 °C during the heating run, which is associated to the change in the structure of the peptide backbone from the antiparallel β-sheet conformational structure to the R-helix conformational structure. However, the exothermic transition is relatively sharp, which is quite different in shape from that observed from the crystallization of conventional polymers but rather similar to that observed for the crystallization of n-alkyl side chains in a relatively short length.27 Taking these facts into account, the strong exothermic phase transition is attributed to the changes in the molecular ordering that accompanies the crystallization of the n-hexadecyl bristles in the brush polymer in addition to the continuation of the ordering of the antiparallel β-sheet conformational peptide backbones transformed from the R-helix conformational peptide backbones in the melt state, which has already been started at a higher temperature, around 73 °C. As discussed above, during the heating run, two different kinds of molecular conformational change occur in the brush PHHC polymer, one in the bristles’ n-hexadecyl groups and the other in the peptide backbone, which are directly associated with the respective order-disorder phase transitions; during the subsequent cooling run, the two conformational changes occur in reverse, and are directly associated with the respective disorder-order phase transitions. During the heating run, the crystals consisting of the trans-conformational methylene units in the n-hexadecyl groups of the bristles of the brush PHHC polymer melt first in the range 50-73 °C, which is then followed by disordering of the antiparallel β-sheet conformational peptide chain backbone assemblies over the range 80112 °C. During the cooling run from the melt, inversely the ordered assembling of the antiparallel β-sheet conformational peptide chain backbones occurs first from the R-helix conformational peptide backbones in a disorder state (i.e., melt state),

J. Phys. Chem. B, Vol. 112, No. 17, 2008 5343 then followed by the crystallization of the trans-conformational methylene units in the bristles’ n-hexadecyl groups from the melt. Note here that the ordered assembly of the antiparallel β-sheet conformational peptide chain backbones takes place prior to the crystallization of the n-hexadecyl groups in the bristles and overall the ∆Hf value for the formation of the peptide backbone assemblies is much larger than that for the crystal formation in the bristles. These results might result from the strong hydrogen (H) bonding that arises between the peptide units in the polymer backbones; this H-bonding was detected in the measured IR spectra as a vibration peak at 3300 cm-1 (data not shown), which corresponds to hydrogen-bonded N-H stretching.25 Taking these results into account, we conclude that the formation of the ordered assembly of the peptide backbones with antiparallel β-sheet conformations can proceed very favorably in an accelerated mode due to the strong H-bonding between the peptide units of the polymer backbones. Further, the formation of ordered assemblies in the antiparallel β-sheet conformational peptide backbones plays a critical role in inducing the crystal formation in the bristles in the melt. Static GIXS Analysis. Figure 6a shows the two-dimensional (2D) GIXS pattern measured at 25 °C of a PHHC thin film deposited on a silicon substrate in the coordinates of the detector plane, the out-of-plane scattering angle Rf and the in-plane scattering angle 2θf. As can be seen in the GIXS pattern, several strong specular reflections appear with regular spacing along the Rf direction at 2θf ) 0°, and in addition, one broad, weak peak appears along the 2θf direction at Rf ) 0°, with peak maximum at 2θf ) 22°. The appearance of these scattering peaks suggest that a multilayer structure (i.e., a lamellar structure) is present along a direction normal to the film plane and that lateral packing in the layers (i.e., lamellae) is relatively poor. Further, note that the 2D GIXS pattern contains two additional scattering features (Figure 6a). The reflection peaks along the Rf direction at 2θf ) 0° are not sharp spots but have an arc shape, and the broad, weak scattering peak 2θf ) 22° at Rf ) 0° is also not a sharp spot but has an arc shape. The observation of such arc-shaped scattering peaks suggests the presence of a distribution in the orientation vector n of the lamellae (Figure 3), which is due to undulation of the lamellar surfaces and/or a distribution in the orientation of the lamellar stacks. Further, a weak, circular scattering ring is discernible on the two reflection arcs in the low Rf region at 2θf ) 0° as well as on the very weak, broad scattering arc at 2θf ) 22° at Rf ) 0°. Such circular scattering is not observed for the higher-order reflection arcs along the Rf direction at 2θf ) 0° (Figure 6a). The observation of such weak, circular scattering rings indicates that less ordered lamellar domains with random orientation are present in the film as a minor morphological component, in addition to the well-oriented, highly ordered lamellar domains. Taking into account the qualitative GIXS, IR spectroscopy, and DSC results discussed in the previous section, the out-ofplane and in-plane scattering profiles in the 2D GIXS pattern in Figure 6a were extracted along the Rf direction at 2θf ) 0° and along the 2θf direction at Rf ) 0.15°, respectively, and are plotted in Figure 6b and c. As can be seen in Figure 6b, the out-of-plane profile clearly contains eight diffraction spots. The two diffraction peaks in the low qz region partially overlap with a scattering angle difference of 0.40°, which exactly matches twice the incidence angle of the X-ray beam, indicating that the relatively intense scattering peak (i.e., the first diffraction peak) at qz ) 1.25 nm-1 (Rf ) 1.76°) arises from scattering from the transmitted X-ray beam, while the slightly less intense scattering peak (i.e., the second diffraction peak) at qz ) 1.57

5344 J. Phys. Chem. B, Vol. 112, No. 17, 2008

Figure 6. (a) 2D GIXS patterns measured at Ri ) 0.20° for a PHHC thin film of 62.0 nm thickness deposited on a silicon substrate; the measurement was conducted at 25 °C. (b) Out-of-plane scattering profile extracted from the GIXS pattern in (a) along the Rf direction at 2θf ) 0°. (c) In-plane scattering profile extracted from the GIXS pattern in (a) along the 2θf direction at Rf ) 0.15°. (d) 2D GIXS pattern simulated for the PHHC film at 25 °C using the GIXS formula with the structural parameters (listed in Table 1) obtained by analyzing the GIXS data in (a); in this simulation, film thickness ) 62.0 nm, electron density of film ) 216 nm-3, electron density of substrate ) 699 nm-3.

Yoon et al. nm-1 (Rf ) 2.16°) arises from scattering from the X-ray beam reflected at the silicon surface.8 For the second to eighth diffraction peaks, their relative scattering vector lengths from the specular reflection position are 1, 2, 3, 4, 5, 6, and 7, indicating that these scattering peaks arise due to scattering from the reflected X-ray beam. Further, the appearance of these highorder diffraction peaks strongly indicates that a very well ordered periodic lamellar structure is present in the film, in which the lamellae are stacked together normal to the substrate. The inplane scattering profile is quite different from the out-of-plane profile: specifically, the in-plane profile contains only one broad peak at qxy ) 15.2 nm-1 (Figure 6c). Both the out-of-plane and in-plane scattering profiles can be satisfactorily fitted with the GIXS formula derived in the GIXS Theory part of the Experimental Section, as can be seen in Figure 6b,c. The determined structural parameters, including the lattice positional distortions, the orientation, and its distribution, are summarized in Table 1. The determined mean long period ()d3) of the lamellar stacks is 4.30 nm and the mean lateral distance ()dr) of the bristle paracrystals within the lamellae is 0.41 nm. The determined d3 value is larger than the length (2.38 nm) of a bristle in the fully extended conformation, which was estimated from a molecular simulation with the Cerius2 software package (Accelrys, USA), but nearly twice the length of a fully extended bristle. In addition, it was found with the GIXS analysis that the lamellae consist of two layers with different electron densities, and that the thickness ()h) of one layer is 2.02 nm. The other layer thickness ()d3 - h) was found to be 2.28 nm from the determined d3 and h values. These results indicate that a wellordered lamellar structure is formed in the polymer film and that the individual lamellae consist of brush PHHC polymer chains with polymer backbones that are fully extended in the lamellar plane and packed together laterally, and with bristles that are aligned vertically with respect to the lamellar plane and packed together laterally. Here, the question arises as to what causes the formation of the two layers of different electron densities in such well-ordered lamellae. Further, the determined h value can in fact be assigned to either the more ordered layer or to the less ordered layer of the lamellar stacks formed in the polymer film, according to Babinet’s reciprocity.6 Thus, another question arises as to how to assign the determined h value. To find answers to these two questions, we need to consider the molecular conformation and crystallization characteristics of the bristles in the brush PHHC polymer film in detail, and then to characterize the phases (the more and less ordered phases) of the two layers. A molecular simulation of the bristles in the brush PHHC polymer was conducted with the Cerius2 software package, and it was found that as mentioned earlier the length of a bristle in a fully extended conformation is 2.38 nm, where the distance from the backbone carbon to the sulfur atom in the bristle is 0.46 nm and the length of the n-hexadecyl group in the fully extended trans-conformation is 1.92 nm. Thus, 80.7% of the total length of the bristle is that of the n-hexadecyl group. As discussed in the previous section, the IR spectroscopy and DSC results show that in the PHHC film at 25.0 °C a major fraction of the bristles’ methylene units have a trans-conformation in the crystalline layers of the lamellae; only a minor fraction of the bristles’ methylene units are present in the gaucheconformation in the less ordered layers of the lamellae. The peptide backbone of the PHHC polymer is relatively rigid because of the planar peptide units and their characteristic H-bonding formation. In this peptide, the inner part of the bristle

2D Well-Ordered Multilayer Thin Films

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TABLE 1: Structural Parameters of the PHHC Thin Film at Various Temperatures, Which Were Obtained by GIXS Measurements and Data Analysis temp (°C)

d3 (nm)

g33

g3r

dr (nm)

gr3

grr

h (nm)

σh

ti (nm)

β (deg)

φ (deg)

σj (deg)

Os

25 75 100 120 c45

4.30 3.98 3.80 4.30

0.058 0.120 0.150 0.080

0.060 0.090 0.110 0.072

0.41 0.43 0.44 0.41

0.25 0.28 0.32 0.25

0.16 0.18 0.21 0.17

2.02 1.98 1.94 2.00

0.10 0.13 0.14 0.11

0.26 0.33 0.36 0.29

90 90 90 90

0 0 0 0

6.7 6.9 7.2 5.8

0.979 0.978 0.976 0.984

is fixed by its position on the rigid peptide backbone and, as a result, has limited mobility and cannot participate in the crystallization. Only the outer part of the bristle is sufficiently far from the rigid peptide backbone to take part in the crystallization. Thus, the formation of lamellae consisting of two layers (a crystalline layer and a less ordered layer) in the PHHC film is due to the partial crystallization characteristic of the bristles, which arises from the inhomogeneous conformation and differential segment mobility along the bristle skeleton. The crystalline layers in the lamellae consist of the outer parts of the n-hexadecyl groups, while the less ordered layers consist of the inner parts of the bristles. From the IR spectroscopy data and the simulated bristle length, we conclude that the crystalline layer has a thickness slightly larger than that of the less ordered layer. Therefore, the determined h value (2.02 nm) is assigned to the thickness of the less ordered layers in the lamellae. The other layer thickness (2.28 nm) is assigned to the thickness of the crystalline layers in the lamellae. This crystalline layer thickness corresponds to the length of a bilayer of the ten outer methylene units of the n-hexadecyl group in a bristle. Further, these results show that the bristle paracrystals of one lamella do not undergo interdigitation with those of the adjacent lamellae in the stack. The standard deviation σh of the thickness h of the less ordered layers in the lamellae was determined to be 0.21 nm. σh is a parameter that characterizes the interface between the less ordered layer and the crystalline layer within a lamella. The characteristic interfacial thickness ti was thus estimated to be 0.26 nm by using the relation, ti ) (2π)1/2σh.28 The positional disorders (g33 and g3r) of the bristle paracrystals in the lamellae along the direction normal to the film plane were determined to be 0.058 and 0.060, respectively. Moreover, the mean orientation angle φ j with respect to the out-of-plane projection is 0.0° and its deviation σφ is 6.7°, so the secondorder orientation factor Os ) 0.979; it was also found that β ) 90.0°. These results indicate that the PHHC film has a lamellar stack structure with individual lamellae that consist of the polymer backbone chains linked with two sets of bristle paracrystals oriented almost perfectly normal to the film plane. The results also indicate that there is almost no undulation at the lamellar surfaces. In contrast, the positional disorders (gr3 and grr) of the bristle paracrystals of the lamellae in the film plane were found to be 0.250 and 0.160, respectively. These relatively large gr3 and grr values indicate that the bristle paracrystals of the lamellae in the film are poorly packed in the film plane. By using the determined structural parameters shown in Table 1, we used the GIXS formula to calculate the 2D GIXS pattern, which is displayed in Figure 6d. The simulated GIXS pattern is in good agreement with the experimentally measured scattering pattern shown in Figure 6a. Moreover, on the basis of the determined structural parameters including the orientation factor and positional disorders, we propose a molecular structural model for the lamellar stacks formed in a PHHC film supported with a silicon substrate, as shown in Figure 7. Here, the lamella is an assembly of PHHC

chains in an antiparallel β-sheet conformation, in which the bristles are uniaxially aligned perpendicular to the film plane but are laterally packed in an irregular manner rather than in a regular manner in the film plane. Each lamella consists of two layers, namely, a crystalline layer and a less ordered layer. The crystalline layer is composed of a bilayer of ten outer methylene units of the bristles, with no interdigitation. In Situ GIXS Analysis. To elucidate the temperature dependence of the structure of the PHHC film, 2D GIXS measurements were performed during heating and subsequent cooling runs. During the heating run, the scattering pattern measured at 25 °C was found to retain its shape and intensity up to below 50 °C, the onset temperature of the bristle crystals’ melting; the scattering pattern at 25 °C was described in the previous section. However, during the heating of the film through the melting transition (50-73 °C) of the bristle crystals, the scattering pattern was found to become weak in intensity. A 2D GIXS pattern measured at 75 °C, just above the melting transition of the bristle crystals, is shown in Figure 8a, and the out-of-plane and in-plane scattering profiles extracted along the Rf direction at 2θf ) 0° and along the 2θf direction at Rf ) 0.15°, respectively, are displayed in Figure 9. As can be seen in Figures 8a and 9a, the diffraction peaks along the Rf (i.e., qz) direction at 2θf ) 0° shift slightly to the high qz region and some high-order peaks disappear, compared to those in the scattering pattern measured at 25 °C. In contrast, the one broad peak in the in-plane scattering profile shifts slightly to the low qxy region and weakens in intensity (Figure 9b). As shown in Figure 9, the out-of-plane and in-plane scattering profiles can be satisfactorily fitted with the derived GIXS formula, indicating that a lamellar structure is still present in the film at 75 °C, at which temperature the bristle crystals are completely melted. However, the melting of the bristle crystals influences the structural parameters of the lamellar structure in the film, as can be seen in Table 1; the sublayer thicknesses in the lamellae as well as the long period of the lamellae are reduced, whereas the lateral interdistance of the bristles is enlarged. The interfacial characteristic parameters (σh and ti) are also increased. Furthermore, all the positional disorders (g33, g3r, gr3, and grr) of the bristles in the lamellae are increased. However, the secondorder orientation factor Os of the lamellae does not alter. With further heating up to 112 °C, at which temperature the polymer backbone chains transform from the antiparallel β-sheet structure to the R-helix structure, the 2D GIXS pattern was found to change more drastically. A representative scattering pattern measured at 100 °C is shown in Figures 8a and 9. Only the first-, second-, and third-order diffraction peaks along the Rf direction at 2θf ) 0° are still discernible, but are very much weakened and shifted to the high qz region, whereas the other high-order diffraction peaks have disappeared completely (Figure 9a). In contrast, the one broad peak in the in-plane scattering profile is slightly shifted to the low qxy region and has weakened in intensity (Figure 9b). This scattering pattern can also be satisfactorily fitted with the derived GIXS formula (Figure 9). This result indicates that a lamellar structure is still present to a certain extent in the film even at 100 °C, at which

5346 J. Phys. Chem. B, Vol. 112, No. 17, 2008

Figure 7. Molecular model of a lamellar structure formed in the PHHC thin film on a substrate: Each lamella is composed of two sets of bristle paracrystals linked to the top side and bottom side of a peptide backbone chain, where the individual bristle paracrystals have a fully extended molecular conformation oriented uniaxially normal to the film plane, but laterally packed in an irregular manner. Each lamella consists of two layers, namely, a crystalline layer and a less ordered layer; the crystalline layer is composed of a bilayer formed from the outer ten methylene units of the bristles with no interdigitation. (a) x-z view of the molecular model; (b) y-z view of the model; (c) x-y view of the model.

Yoon et al.

Figure 8. 2D GIXS patterns of a PHHC thin film deposited on a silicon substrate measured with various temperatures at Ri ) 0.20° during (a) heating and (b) subsequent cooling. The inset in each 2D GIXS pattern is an enlargement of the scattering part in the low scattering angle region.

2D Well-Ordered Multilayer Thin Films

Figure 9. (a) Out-of-plane scattering profiles extracted from the GIXS patterns with various temperatures in (a) along the Rf direction at 2θf ) 0°. (b) In-plane scattering profiles extracted from the GIXS patterns with various temperatures in (a) along the 2θf direction at Rf ) 0.15°.

temperature a majority of the conformational changes of the polymer backbone chains have occurred. The analysis results are summarized in Table 1. The polymer backbone chains’ conformational changes further influence the structural parameters of the lamellar structure in the film (Table 1). However, the second-order orientation factor Os of the lamellae is unaltered. On the other hand, note that a new scattering peak is present at q ) 2.09 nm-1 and is of ring-type rather than arc- or spot-type (Figures 8a and 9). This scattering ring was determined to have a d-spacing of 3.01 nm, which is smaller than the long period d3 (3.80 nm) of the lamellar structure detected at 100 °C (Table 1). These scattering characteristics suggest that the scattering ring at q ) 2.09 nm-1 originates from the interdistance of the polymer chains in an amorphous state (i.e., a molten phase). Thus, the observation of the scattering ring indicates that the polymer molecules in the film at 100 °C are present in a molten state to some extent. The scattering data indicate that both lamellar structure phases and amorphous phases are present to certain extents in the film at 100 °C. Above 112 °C, the peptide backbone chains are present predominantly with an R-helix conformation in the molten state, and the GIXS pattern of the film contains only two scattering rings but no scattering peaks along the Rf (i.e., qz) direction at 2θf ) 0°. A representative scattering pattern measured at 120 °C is shown in Figures 8b and 9. This scattering pattern could not be fitted with the derived GIXS formula. This result confirms that the lamellar structure in the film is completely destroyed at 120 °C. As can be seen in the figures, one scattering ring is present in the low scattering region, which corresponds to that which is present at q ) 2.09 nm-1 during the conformational transformation of the polypeptide backbone in the range 80112 °C, and there is another in the high scattering region (around q ) 14.3 nm-1), which corresponds to the lateral interdistance of the bristles. As mentioned above, the scattering ring in the low scattering angle region originates from the mean interdistance of the polymer chains in the molten state. This scattering ring has a d-spacing of 3.01 nm. This d-spacing value is smaller than the long periods d3 (3.98 and 3.80 nm) of the lamellar structure measured at 75 and 100 °C, at which temperature the bristle crystals are completely melted; note that the d3 value

J. Phys. Chem. B, Vol. 112, No. 17, 2008 5347

Figure 10. Molecular model (y-z view) of a close-packed structure of the R-helically conformed polypeptides bearing completely disordered bristles in the melt state of a PHHC film.

corresponds to the interdistance of the brush polymer chains in a lamellar structure. In general, R-helical polypeptides are known to assemble in a hexagonally close-packed structure with a lattice size corresponding to the diameter of the R-helical polypeptide chain.29 As described in the FTIR and DSC Analysis section, PHHC polymer chains have an R-helical conformation in the film above 112 °C, and further might have sufficient mobility to assemble in a hexagonally close packed structure. Taking these facts into account, one expects that above 112 °C the polypeptide molecules would favorably assemble in the PHHC film in a hexagonally close-packed structure. However, such a hexagonally close-packed structure could not be detected in the GIXS analysis of the PHHC film. Thus, the question arises as to what prevents the formation of a hexagonally close-packed structure in the PHHC film. To find the answer to this question, we carried out a molecular simulation of the PHHC molecule with the Cerius2 software package that took into account the IR, DSC, and GIXS analysis data. It was found that an R-helical PHHC molecule with bristles in a completely disordered conformation has a diameter of around 3.00 nm. This calculated diameter of the R-helical PHHC molecule is comparable to the interdistance of the polymer chains determined from the GIXS analysis. This result indicates that above 112 °C the disordered conformational bristles of a PHHC chain in the film are fully interdigitated with those of the adjacent polymer chains. Such interdigitation in the bristles in the disordered conformation may limit the mobility of the R-helical PHHC chains, restricting polymer chain packing such as in a hexagonally close packed structure. Taking into account the results of the GIXS analysis, and the IR spectroscopy and DSC results, we propose the structural model for the PHHC molecules in the film at 120 °C shown in Figure 10, which was constructed by molecular simulation with the aid of the Cerius2 software. As can be seen in this figure, pseudo-hexagonal polymer chain packing is discernible but limited to very short range. During the cooling run, the scattering pattern measured at 120 °C was found to retain its shape and intensity with decreases in temperature down to 75 °C (data not shown). However, there were some variations in the scattering pattern when the film

5348 J. Phys. Chem. B, Vol. 112, No. 17, 2008 temperature was decreased below 73 °C, which is the onset temperature of the ordered assembling of the antiparallel β-sheet conformational peptide chain backbones. A representative scattering pattern is shown in Figures 8b and 9, which was measured at 70 °C. The scattering ring corresponding to the interdistance of the polymer chains is shifted slightly to the low scattering angle region (Figure 9a,b), whereas the scattering ring corresponding to the lateral interdistance of the bristles is shifted slightly to the high scattering angle region (Figure 9b). In addition, a new scattering peak is present near qz ) 3.70 nm-1 (Figure 9a). These changes in the scattering pattern might be due to the ordered assembling of the antiparallel β-sheet conformational peptide chain backbones in the film. When the film was cooled further, the scattering pattern began to resemble that measured at 25 °C before the heating run. A representative scattering pattern is shown in Figures 8b and 9, which was measured at 45 °C. As can be seen in these figures, several strong specular reflections are present at regular spacing along the Rf direction at 2θf ) 0°, and the scattering peak corresponding to the lateral interdistance of the bristles is present as an anisotropic ring with maximum intensity at 2θf ) 21.5° (dr ) 0.41 nm) at Rf ) 0°, rather than as an isotropic ring. The presence of these scattering peaks confirms that a lamellar structure consisting of crystalline and less-ordered layers develops in the film during the cooling process after heating to 180 °C. As shown in Figure 9, the out-of-plane and in-plane scattering profiles can be satisfactorily fitted with the derived GIXS formula, confirming again that a lamellar structure develops in the film during the cooling process. The scattering analysis results are summarized in Table 1. The determined structural parameters and the second-order orientation factor are nearly the same as those determined at 25 °C before the heating run. However, the out-of-plane positional disorders (g33 and g3r) of the bristles in the lamellae are slightly larger than those determined at 25 °C before the heating run. Conclusions The well-defined brush polypeptide bearing one ethylenylthion-hexadecyl bristle per repeat unit, poly(S-n-hexadecyl-DLhomocysteine) (PHHC), was synthesized and used to produce nanoscale thin films supported with silicon substrates with conventional spin-coating and subsequent drying processes. The PHHC thin films were characterized in detail for various temperatures by carrying out IR spectroscopy, DSC, and GIXS analysis. The PHHC polymer molecules in the thin film were found to form well-defined self-assembled nanostructures. In addition, a GIXS formula was derived for lamellar structures in thin films supported on a substrate. Using the derived formula, quantitative GIXS analysis was successfully carried out for the well-defined self-assembled nanostructures in the PHHC thin films supported on silicon substrates. The combination of the GIXS analysis with the IR spectroscopy and DSC results provided significant information about the molecular conformation and structures of the self-assembled lamellae formed in the PHHC thin films, which could not be easily obtained with conventional techniques. Moreover, the in situ measurements and data analyses during the heating and subsequent cooling of the PHHC film were used to establish a mechanism for the evolution of the self-assembled lamellar structures in the film and their melting. Temperature-dependent molecular structure models were established for the PHHC polymer in the film. Above 112 °C, the PHHC molecules in the thin film were found to be present in a melt state composed of R-helically conformed polymer backbones and completely disordered

Yoon et al. bristles. In the melt state, the bristles of each polymer chain were found to be fully interdigitated with those of adjacent polymer chains, which restrict the hexagonal close packing of the polymer chains. During the cooling run from the melt, the R-helix conformational peptide backbones in the disordered state start to transform to the fully extended antiparallel β-sheet conformational peptide chain backbones below 73 °C, and this transformation process is accompanied by ordering of the β-sheet conformational peptide chain backbones with the aid of Hbonding between the peptide backbones. This self-assembling of the β-sheet conformational peptide chain backbones was found to induce the favorable transformation of the bristle to a fully extended conformation (mainly the trans-conformation) from the disordered conformation (mainly the gauche-conformation) within individual polymer chains, leading to lateral packing of the bristles. After cooling to room temperature, the film was determined to have a lamellar structure consisting of PHHC polymer molecules with polymer backbones that are fully extended along the lamellar plane and packed together laterally and with bristles that are aligned vertically with respect to the lamellar plane and packed together laterally. The lamellae are stacked along a direction perpendicular to the film plane. The individual lamellae are composed of two layers, a crystalline layer and a less-ordered layer. The crystalline layer is composed of a bilayer of the ten outer methylene units of the bristles, with no interdigitation. Thus, the formation of a well-defined lamellar structure in the PHHC thin film is a result of the cooperation of the crystallization of the bristles in the fully extended trans-conformation and the lateral ordering of the peptide backbones in the fully extended antiparallel β-sheet conformation, with the aid of strong H-bonding between the neighboring peptide units. During the heating run, the well-defined lamellar structure in the film was found to undergo a two-step melting process as follows. The lamellar structure was maintained up to 50 °C, and the crystalline layer then underwent melting over the temperature range 50-73 °C, keeping the framework of the lamellar structure; the peak maximum temperature of the melting of the bristles’ crystals was found to be 61.1 °C, which is equivalent to that of the bristles’ crystallization. The melting of the crystalline layer was found to cause some alterations in the lamellar structure. The retention of the framework of the lamellar structure is attributed to the thermally stable lateral packing of the fully extended polypeptide backbones due to their strong H-bonding interactions. In the temperature range 80112 °C, the film was found to undergo another order-disorder transition, and the lamellar structure is completely destroyed above 112 °C; the peak maximum temperature of the melting of the laterally packed polypeptide backbones was found to be 102.7 °C, which is much higher than that of the peptide backbones’ ordering as well as that of the bristles’ crystallization. This order-disorder phase transition is attributed to the thermally induced weakening of the H-bonding interactions and the resulting change in conformation from the fully extended antiparallel β-sheet structure to the R-helical structure. Acknowledgment. This study was supported by the Korea Science & Engineering Foundation (National Research Lab Program, and Center for Integrated Molecular Systems) and by the Ministry of Education (BK21 Program). Synchrotron GIXS measurements at the Pohang Accelerator Laboratory were supported by the Ministry of Science & Technology and the POSCO company.

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