Besides being the main types of carbohydrate in food, sugars are a representative protectant in biopharmaceutical formulations. To identify the protection mechanism, researchers have extensively investigated the bulk physicochemical properties of sugars. However, whereas the glass transition of sugar has been widely studied and debated, the physicochemical properties of sugar molecules in confined circumstances such as nanometer thick films remain largely unknown. In this chapter, we introduce an experimental procedure for analyzing the glass transition of sugars in ultrathin films. The analysis is based on X-ray reflectivity (XRR) analysis, which has been often applied in glass transition studies of polymer films, but never in sugar media.
- ultrathin film
- X-ray reflectivity
Amorphous sugars form a glassy matrix under far from equilibrium conditions (i.e., supercooling or supersaturation) [1–3]. For instance, sugar glass is obtained by cooling the sugar melt to temperatures far below the melting point (
The specific heat and coefficient of expansion of a sugar melt abruptly change across the glass transition . In the glassy state, both values are much closer to the crystalline values than the liquid-state values. The glass transitions of bulk sugar-water mixtures at various concentrations are commonly studied by differential scanning calorimetry (DSC), which detects the glass transition as a change in heat capacity. However, DSC thermograms often contain a problematic overshoot peak caused by a macroscopic dynamic process accompanying structural relaxation at the glass transition, which largely depends on the thermal history of the sample . The temperature dependence of the dynamical behavior of amorphous sugar has been widely studied from a thermodynamic perspective .
On the other hand, the preparation dependence of the physical properties of amorphous sugar has also received much attention [13–15]. Surana et al. reported that together with aging, the preparation methods (i.e., freeze-drying, spray-drying, dehydration, and melt quenching) largely affect the glass transition temperature (
Specular X-ray reflectivity (XRR) is a unique and powerful analytical method (Figure 2) that is frequently applied to condensed soft-matter films, including glass transition studies of ultrathin polymer films [16–20], but which has not been applied in amorphous sugar studies. XRR can evaluate the layered structure of a material, such as the film thickness, electron density, surface roughness, and interfacial width [21, 22]. When the incident X-ray angle in the 2
The preparation of sugar film has been reported in several articles. Tre and Suc films have been prepared by drop casting  or spin coating [25–27] aqueous solutions of the sugar in atmospheric air or by vacuum deposition under reduced pressure . In those reports, the films were thicker than 100 nm. On the other hand, Zhao et al. prepared glucose nano-films with thicknesses ranging from 20 to 60 nm . They studied the adhesion and detachment behaviors of glassy, viscoelastic, and Newtonian liquid states of glucose and characterized the effect of the sugar glassy state on the surface deformations and flows. However, to our knowledge, Tre and Suc nano-films with thicknesses below 100 nm have not been fabricated, and detailed studies of their glass transitions and other properties have not been investigated. We prepared flat nano-films of Tre or Suc sugars by spin coating the aqueous sugar solutions onto silicon wafers and analyzed them by XRR. Large, fairly flat films are required, as the XRR method has a very small angle of incidence. The effects of the vacuum operation and temperature on the spin-coated films were investigated, and the usefulness of XRR and some novel phenomenon related to the features of sugar glass are disclosed.
Tre dihydrate, Suc, and ultrapure water were purchased from Wako Pure Chemical Co. Ltd. Si(100) substrate (thickness
2.2. Preparation of sugar nano-films
Tre and Suc were dissolved in ultrapure water by heating. Sugar nano-films were then prepared by spin coating the aqueous solutions on the Si(100) substrates at 4000 rpm for 45 s.
2.3. X-ray analyses on sugar nano-films
X-ray analyses of the sugar nano-films were performed in a multipurpose X-ray diffractometer (SmartLab, Rigaku Corp., Japan) equipped with a temperature control unit. Prior to measurements, the surface temperature calibration of the Si(100) substrate was checked by a conventional digital multimeter with a thermocouple wire. Specular XRR and X-ray diffraction (XRD) profiles were obtained by 2
3. Results and discussion
3.1. Estimation of film thickness of spun-films
Figure 3 shows the XRR profile of the Si(100) substrate washed with ethanol. The X-ray reflection intensity was smoothly attenuated and no oscillation fringes were discernible at X-ray incidence angles above
Figure 4 shows an XRR profile series of the spin-coated sugar films. The film thicknesses were calculated by fitting the XRR profiles. Along the series, the film thickness ranged from several to several dozens of nm, and clear oscillation fringes (Kiessig fringes) were observed in
3.2. Variation of film thickness of spun-films under reduced pressure
Water traces in the sugar matrix must be considered in studies of amorphous sugar. To confirm the existence of water, we analyzed the thicknesses of the Suc and Tre films in air and vacuum at room temperature and compared the pre-vacuum XRR profiles with those of the post-vacuum and subsequent vacuum release operations. Figure 6 shows the XRR profiles analyzed (1) before and (2, 3) after the vacuum, together with the profiles (4) after vacuum release. From these results, we recognized that the vacuum operation increased Δ
3.3. Variation of film thickness with temperature
3.3.1. Temperature-dependent behaviors of sugar nano-films
We next investigated the thickness of sugar nano-films fabricated at different temperatures. Experiments were conducted on Suc and Tre films with initial thicknesses of
The thermal expansion behavior of the Tre film depended on its thermal history (conditions are described in the caption of Figure 9). Similarly to Figure 8, the film thickness both decreased and increased during heating process (1 in Figure 9), and apparently decreased during isothermal annealing at 150°C (2 in Figure 9a). However, during subsequent cooling from 160° to 30°C, no NTE phenomenon was observed (3 in Figure 9a). In Figure 9b (Case II) the slopes of the normalized thickness versus temperature lines differ between above and below
3.3.2. Observation of reproducibility of NTE during heating and cooling cycle
NTE has received much attention as a tuning phenomenon in the overall thermal expansion of materials . It has also been observed in ultrathin polymer films [16, 17, 20]. Mukherjee et al. reported NTE below the
In Figures 7–9, an apparent hysteresis appears between the thicknesses during the first heating and first cooling and during the first and second heating processes. During the first heating process, the thickness (and its product with the electron density) monotonically decreases with increasing temperature up to 90°C and 120°C for Suc and Tre film, respectively. This effect might be attributable to the evaporation of trace amounts of water during the heating. The filling of the voids left by the departing water molecules might shrink the sugar matrix. Above 90°C for Suc film and 120°C for Tre film, the thickness and electron density product became invariant, suggesting that the water had wholly evaporated. Thus, NTE was observed during the first heating process, but apparently the first cooling process was governed by different mechanisms.
3.3.3. Sublimation problem
The dominant factor governing the inclusion or exclusion of NTE is unclear, but a likely scenario is rearrangement of the sugar matrix in the melt state, as the isothermal conditions are different in Cases I and II. In fact, the largely reduced thickness of the Tre film after isothermal annealing at 160°C was unexpected (see process 2 in Figure 8b). The product of thickness and electron density in Tre film exhibited a similar trend (2 in Figure 10b). Figure 11 plots the time-dependent thickness of the Tre film under isothermal annealing at 170°C. Prior to annealing, the film was gradually heated to 150°C. During the heating process, the decrease and subsequent increase in thickness mimicked that of stage (1) in Figure 8 (Figure 11b). In addition, the thickness began decreasing at 160°C. Subsequently, under isothermal annealing at 170°C, the film thickness continuously decreased, reaching 0 after
In this chapter, we demonstrated the temperature-dependent thickness behaviors of sugar nano-films formed on Si(100) substrates of area (2 × 2) cm2. Homogeneous flat films suitable for precise XRR analysis were fabricated by the conventional spin-coating method. The complex behaviors of the shrinking and expanding sugar nano-films during heating and cooling were successfully observed and are schematically summarized in Figure 12.
Once the aqueous sugar solutions have been spin-coated on the Si substrates, residual water remains in the nano-films (1 in Figure 12). Such water can be mostly or partly removed by evaporation under reduced pressure even at room temperature (1–2 in Figure 12). Evaporation manifests as decreased film thickness. As the
As shown in this chapter, the XRR methodology provides profound insights into the adsorption and desorption properties of amorphous sugars, the rearrangement of sugar molecules at the sugar-air interface and the glass transition. To acquire these insights, we observed how the film thickness depends on water content and temperature. By understanding the sugar-vacuum or sugar-solid interface, we might also capture the structural changes of sugar matrixes under freezing, freeze-drying, and spray-drying operations. In all of these processes, molecular transfer such as water adsorption and desorption starts at the interface.
This work was financially supported by the Amano Institute of Technology, AIST, Japan, by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities (S1201027) 2012–2016, and by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research (C) 24560033).
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