Sucrose and Trehalose - American Chemical Society

Article pubs.acs.org/molecularpharmaceutics

Dielectric Studies on Molecular Dynamics of Two Important Disaccharides: Sucrose and Trehalose K. Kaminski,† K. Adrjanowicz,*,† D. Zakowiecki,‡ E. Kaminska,§ P. Wlodarczyk,† M. Paluch,† J. Pilch,∥ and M. Tarnacka† †

Institute of Physics, University of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland Preformulation Department R&D, Pharmaceutical Works Polpharma SA, Pelplinska 19, 83-200 Starogard Gdanski, Poland § Department of Pharmacognosy and Phytochemistry, Medical University of Silesia, ul. Jagiellonska 4, 41-200 Sosnowiec, Poland ∥ Department of Biological Sciences Academy of Physical Education, Raciborska 1, 40-074 Katowice, Poland ‡

ABSTRACT: Broadband dielectric measurements were carried out in the supercooled as well as in the glassy state of two very important disaccharides: trehalose and sucrose. Multiple relaxation processes were observed. Above the glass transition temperatures of examined disaccharides structural relaxation of cooperative origin was detected, where in the glassy state more local motions (secondary modes) appeared. Our data were discussed in light of the findings reported by other groups. We pointed out that sample preparation might impact mobility and, thus, dielectric loss spectra in a significant way. Consequently, it may lead to misinterpretation of the dielectric relaxation processes. Moreover, impact of physical aging and pressure on dynamics of two secondary relaxation processes observed in the glassy state of trehalose and sucrose has been investigated. Additionally, we have demonstrated that, in contrast to the calorimetric measurements (DSC), activation energies of the β- and γ-relaxation processes observed in the glassy state of sucrose and trehalose do not change as a result of physical aging. Finally, we found out that the βrelaxation process slows down as pressure increases. We interpreted this fact in view of increasing rigidity of the structures of disaccharides. KEYWORDS: glass transition, disaccharides, molecular dynamics, dielectric spectroscopy, dc conductivity, primary and secondary relaxations

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INTRODUCTION Saccharides are a group of compounds exhibiting a lot of physicochemical properties, which make them so essential and certainly very attractive materials for different types of studies. They play a key role in the most important chemical reactions, which determine appropriate functioning of the living organisms. Recently, the great role of carbohydrates was emphasized in the process of exchanging information between the cells. This finding is a starting point for researchers to design a new category of anticancer drugs. Saccharides are also very often used as standard excipients improving tableting properties of poorly compactable drugs. More so, current research has demonstrated new potential applications of the carbohydrates. In the literature one can find a lot of examples clearly demonstrating that preparation of a solid dispersion with the use of saccharides or polyols improves the dissolution rate of poorly water-soluble drugs.1,2 It should be also mentioned that saccharides are glass formers of great hydrogen bonding abilities. For that reason they can be used as stabilizers of the amorphous form of many active pharmaceutical ingredients.3,4 In addition saccharides have extremely slow crystallization kinetics below the glass transition temperature, © 2012 American Chemical Society

and it can be considered that they will not crystallize in practical experimental time frames.5 The other, very promising application of sugars is using them as lyoprotectant.6−9 It is well-known that there are a lot of protein pharmaceuticals which are prepared by freeze-drying. However, in many cases this process breaks the native structure of protein and leads to its destabilization. A very simple solution to this problem is addition of the sucrose or trehalose which minimize unwanted processes occurring during freezedrying. Due to great ability of saccharides to form H bonds with target molecules as well as their weak tendency to recrystallize, aggregation and destabilization of proteins can be completely avoided.10 Since amorphous saccharides are very often used in protein and drug formulations, it was necessary to get more detailed information about their molecular dynamics. For this purpose many measurements with use of the very powerful spectroscopies such as dielectric,11−18 NMR,19−24 DSC,25−29 and Received: Revised: Accepted: Published: 1559

September 1, 2011 February 14, 2012 May 3, 2012 May 3, 2012 dx.doi.org/10.1021/mp2004498 | Mol. Pharmaceutics 2012, 9, 1559−1569

Molecular Pharmaceutics

Article

Figure 1. Chromatograms measured for the crystalline (black line) and glassy (blue line) sucrose (a) and trehalose (b).

theoretical computations30−32 were applied. In this paper we present broadband dielectric relaxation data measured for two disaccharides, i.e., sucrose and trehalose. Until now almost all studies on molecular dynamics of disaccharides in their glassy state have shown that there are two secondary relaxation processes differing significantly in activation energies and preexponential factors.18,33−37 Moreover, addition of water to the sample influences dynamics of the observed relaxation processes in a completely different way.38 It means that faster relaxation process slows down while the slower one becomes faster with increasing content of water. These experimental observations have clearly indicated that both relaxation modes have completely different molecular origins. One can add that many different, sometimes contradictory, interpretations were proposed for the observed secondary modes. In our previous work39 the molecular mechanism of the slow (β) process has been finally clarified. Based on comparison between dielectric measurements and theoretical computations we demonstrated that the β-process is connected to the twisting motions of the glycosidic linkage, while the most probable molecular mechanism underlying the faster relaxation process (γ) is

related to the motions of the exocyclic hydroxymethylene groups.40 Recently Dranca et al. have investigated molecular dynamics of trehalose and sucrose with use of the standard DSC technique.41 They found two secondary relaxation processes in the former, while only one was detected in the latter carbohydrate. They also demonstrated that activation energies of the secondary modes of both saccharides change with the time of physical aging. Authors interpreted these data in the view of different molecular packing of the sucrose and trehalose in the amorphous state. In this paper we present dielectric studies on molecular dynamics of trehalose and sucrose in a wide range of temperatures and frequencies. We discuss our data in the context of recently published papers by Bhardwaj et al.42 and Kwon et al.43 We have shown that they mistakenly assigned the slowest relaxation process visible in the supercooled region (surely connected to the dc conductivity) as a structural one. Moreover, dynamics of the secondary relaxation processes in the gassy state of both disaccharides have been carefully investigated. We have also checked what is the impact of 1560

dx.doi.org/10.1021/mp2004498 | Mol. Pharmaceutics 2012, 9, 1559−1569


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physical aging and pressure on the dynamics of the β- and γrelaxation processes. It is worth noting that the pressure range applied in our experiment coincides well with that applied commonly during tableting of pharmaceuticals. It should be also stressed that these types of studies are rarely performed, although drugs and excipients are very often exposed to the high pressures (even up to p = 400 MPa), during formulation stages.

and trehalose by quench cooling of the melt seems to be a very risky method. In this context one can add that the rate of chemical degradation is strongly coupled to the time of keeping saccharide at the melting temperature.44 In order to test the purity of glassy sucrose and trehalose we have carried out additional measurements with the use of high performance liquid chromatography (HPLC), which is supposed to be the best analytical method intended to such purposes. In Figure 1a,b, we compared chromatograms obtained for the crystalline and the glassy sucrose and trehalose, respectively. It can be seen that in the case of glassy samples additional peaks appear on HPLC chromatograms. However, integration of the main peaks connected to the elution of respectively sucrose and trehalose indicated that purity of the glassy disaccharides was greater than 98%. Moreover, we identified glucose, fructose and rafinose to be the main products of caramelization of sucrose and trehalose. In Tables 1 and 2 comparison of the impurity profiles of the crystalline

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EXPERIMENTAL SECTION Materials. Sucrose and dihydrate trehalose of purity greater than 99% were supplied by Sigma Aldrich. In order to vitrify both carbohydrates, samples were heated up to the melting temperatures and then were quickly cooled down. In the case of trehalose, we applied a procedure described earlier by De Gusseme et al.33 One can add that trehalose was kept for 0.5 min at melting temperature (T = 478 K) to be sure that all of the water had evaporated and the sample was completely molten. In the case of sucrose the sample was heated up to T = 454 K, and then it was kept for 0.5 min at this temperature. After melting, saccharides were supercooled very quickly to room temperature, and dielectric measurements were carried out starting from the low to the high temperatures. In the case of aging measurements, molten samples were cooled to room temperature with the speed 20 K/min. Methods. Dielectric Spectroscopy. Isobaric dielectric measurements at ambient pressure were carried out using a Novo-Control GMBH Alpha dielectric spectrometer (10−2− 107 Hz), and in the case of sucrose also the high frequency setup hp 9531 [106−109 Hz] was used. The samples were placed between two stainless steel flat electrodes of the capacitor with gap 0.1 mm. The temperature was controlled by the Novo-Control Quattro system, with use of a nitrogen-gas cryostat. Temperature stability of the samples was better than 0.1 K. In the case of high pressure measurements the capacitor filled with the test material was placed in a high pressure chamber and compressed using the silicone fluid via a piston in contact with a hydraulic press. The sample capacitor was sealed and mounted inside a Teflon capsule to separate it from the silicon oil. Pressure was measured by a Nova Swiss tensometric meter resolution of 0.1 MPa. Temperature was controlled within 0.1 K by means of liquid flow from a thermostatic bath. High Performance Liquid Chromatography (HPLC): Purity Test. Identification and quantitation of sucrose, trehalose and products of their degradation were carried out by HPLC (high performance liquid chromatography) using a refractive index (RI) detector. The HPLC system consisted of a Waters Alliance 2695 separation module with integrated solvent and sample management functions, and equipped with Waters 2414 refractive index detector (Waters Corp., Millford, MA, US). The chromatography column used was an Alltech 700 CH carbohydrate, 300 × 6.5 mm i.d. (Alltech Associates Inc., Deerfied, IL, US). An isocratic separation of carbohydrates was carried out at a flow rate of 0.5 mL/min, using high purity MilliQ water (Millipore, Bedford, MA, USA) as mobile phase and keeping the column temperature at 85 °C.

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Table 1. Comparison of Impurity Profiles of Crystalline and Glassy Sucrose amount of analyte [%] ± SDa

a

analyte identification

crystalline

glassy

unidentified impurity 1 (imp. 1) sucrose glucose unidentified impurity 2 (imp. 2) fructose total impurities

0.081 ± 0.007 99.797 ± 0.006 0.050 ± 0.004

0.743 ± 0.032 98.031 ± 0.080 0.790 ± 0.043 0.301 ± 0.026 0.135 ± 0.012 1.969 ± 0.080

0.072 ± 0.006 0.203 ± 0.006

Mean of three repeated analyses ± standard deviation (SD).

Table 2. Comparison of Impurity Profiles of Crystalline and Glassy Trehalose amount of analyte [%] ± SDa analyte identification unidentified impurity 1 (imp. 1) trehalose glucose total impurities a

crystalline 0.207 ± 0.019 99.793 ± 0.019 0.207 ± 0.019

glassy 1.099 ± 0.010 98.644 ± 0.021 0.257 ± 0.017 1.356 ± 0.021

Mean of three repeated analyses ± standard deviation (SD).

and glassy samples is presented. Moreover, we also carried out additional measurements for the sample recovered after dielectric measurements, and we found that purity changed only a little bit. Thus, we could certify that purity of the quenched samples and purity of samples recovered after dielectric measurements were almost the same. Molecular Dynamics above the Glass Transition Temperature. Dielectric loss spectra of trehalose and sucrose are shown in Figure 2 and Figure 3, respectively. For both samples multiple relaxation processes were detected. It is visible that above the glass transitions dc conductivity comes into the experimental window as a first in both studied samples. According to the Nernst equation this process is related to the concentration and mobility of the ions which can be always found in samples. Interestingly, in the case of saccharides dc conductivity is much greater than in the standard van der Waals liquids such as propylene carbonate or phthalates. Greater dc conductivity of disaccharides is direct implication of their associative character. In this context one can recall that carbohydrates form networks (clusters) connected via the

RESULTS AND DISCUSSION

Before results of dielectric studies will be presented we would like to clarify one issue. It is commonly known that saccharides, especially disaccharides, undergo the caramelization process at high temperatures. Thus, preparation of the amorphous sucrose 1561



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Figure 3. Dielectric loss spectra of sucrose measured above (a) and below (b) its glass transition temperature.

Figure 2. Dielectric loss spectra of trehalose measured above (a) and below (b) its glass transition temperature.

recovered from the freeze-drying or from the heating in a microwave oven. In this context one can recall a paper by Anopchenko et al.,46 who measured freeze-dried trehalose and glycerol mixtures. They found that in the freeze-dried material there are additional relaxation processes in the glass as well as in the supercooled state. They concluded that freeze-drying yields materials of much more complicated structure than that obtained by simple quench cooling of the melt. Moreover, it was claimed that further studies will be necessary to understand what structural changes are occurring in the freeze-dried materials. Thus, it appears that the method of amorphization may significantly influence dielectric response of the samples. Consequently, interpretation of the loss spectra obtained on such materials seems to be a hard task. One can also direct readers to the paper by Ermolina et al.38 They carried out broadband dielectric measurements on freeze-dried amorphous lactose. It was clearly shown that two relaxation modes can be observed above the glass transition temperature in this disaccharide. However, Ermolina et al. found that the slow relaxation process is connected to the self-diffusion of charge carriers over the surfaces of crystallites within an otherwise amorphous matrix. On the other hand, the faster relaxation process was certified to be the structural relaxation. Hence, based on papers mentioned above one can conclude that loss spectra of the freeze-dried materials can be quite different from those obtained for the molten samples. In fact at

hydrogen bonds.45 Consequently, new channels for the additional proton conductivity are formed. The next process observed in the loss spectra above the Tg’s of trehalose and sucrose is structural relaxation originating from the cooperative motions of the molecules. In a common view the α-process is connected to liquid structure reorganization and is responsible for the glass transition phenomenon. It is worth mentioning that in the case of disaccharides structural relaxation can be observed only at high frequency range of the loss spectra. As temperature decreases this relaxation process moves under the dc conductivity. Hence, in both sucrose and trehalose structural relaxation cannot be visible in the vicinity of Tg as a well-separated peak. Herein, one can recall a paper by Kwon et al.43 Contrary to our data, they showed that in the supercooled state of disaccharides (trehalose and maltose) there are in fact two relaxation processes. Moreover, a similar situation was also reported in ref 42, where it was demonstrated that the slower relaxation process can be visualized after subtraction of dc conductivity from the loss spectra, while the faster one can be observed as a prominent shoulder on the high frequency tail of the structural relaxation process. One can add that authors identified the slow mode as the structural process, while the faster one was assigned as the β-relaxation not seen by the others. It is also worth mentioning that loss spectra presented in refs 42 and 43 were obtained for the material 1562



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first sight it can be seen that loss spectra reported in refs 42 and 43 differ significantly from those reported herein. To find the reason for the discrepancy between dielectric data measured for the molten and freeze-dried samples following procedure was applied. First, data presented in Figures 3 and 4 in ref 42 have been digitalized and plotted in the same graph (see our Figure 4). It

trehalose and maltose.49 Moreover, one should remember that there were carried out systematic studies on saccharides by some of us which showed that with increasing molecular weight of the saccharides stretch exponent lowers (D-ribose (βKWW = 0.55), glucose (βKWW = 0.52), sucrose (βKWW = 0.38)). Moreover, a similar trend was found to occur in the case of polyalcohols.50 Thus, one can be sure that the structural relaxation process of trehalose must be much broader than the Debye like relaxation. Hence, based on the above arguments one can claim that the slow relaxation process visible in the loss spectra presented in refs 42 and 43 is surely connected to the dc conductivity process. Molecular Dynamics below the Glass Transition Temperature. In the glassy state two secondary relaxation processes were detected. The faster one is labeled by us as a γprocess and appears in the experimental window as a wellseparated loss peak. It can be seen that this process is visible at temperatures much higher than the glass transition temperature. Its characteristic feature is huge dielectric strength, which decreases with lowering temperature, and asymmetric shape. It is also seen that above the glass transition temperature (Figure 3) the structural and the γ-relaxation processes tend to merge into one relaxation peak at higher temperatures. A very similar scenario is often observed for other glass forming liquids. It was shown that merging of secondary and structural relaxation processes occurs at a temperature at which change of dynamics of the latter mode is observed. It is usually located at TB ≈ 1.2Tg. Interestingly, it was found that both modes merge when the time of the α-process is equal to τα ≈ 10−7 s. That is why this characteristic time of structural process is often called magic relaxation time. Since α- and γ-processes start to separate at τα = 10−7 s,51 it is a clear indication that the molecular origin of both processes is completely different. Briefly, structural relaxation is connected to the cooperative motions of molecules (rotation and translation) while the γ-process takes its source from the conformational changes or motions of some parts of the molecule. Finally, close to the glass transition temperature we can also find another secondary relaxation which appears in the frequency region located between structural and γ-relaxation processes. Since this mode is slower than the γ-process we labeled it as the β-process. Dielectric loss spectra presented in Figures 2 and 3 revealed that the γ-relaxation becomes more separated and visible with lowering temperature. It is also worth adding that the amplitude of this process increases with lowering temperature. In Figure 5 two representative loss spectra measured at T = 288 K and T = 287 K for the trehalose and sucrose are presented. Both spectra were scaled to have the same amplitude as the γ-relaxation process. It is well visible that two secondary relaxation modes can be found in the glassy state of both disaccharides investigated herein. At the sight, it can be seen that the faster and slower secondary relaxation processes differ in amplitude. One should also note that the difference in the amplitude between β- and γ-modes is much greater in trehalose than in sucrose. The other very interesting observation is that the relaxation times of both secondary modes in the latter system are longer than in the former. It is also clearly visible that at room temperature the β-relaxation process of sucrose is almost ten times slower than that of trehalose. Hence, we can state that the dynamics of the glassy state of both saccharides are not identical, although differences are not significant.

Figure 4. Data digitalized from Figures 3 and 4 from the paper by Bhardwaj.42

can be seen that the imaginary and the real components of the complex permittivity cross at some frequency fc which coincides perfectly with the maximum of loss of the relaxation process, labeled by Bhardwaj et al.42 as the structural one. One can add that a similar situation was observed in the case of data presented by Kwon et al.47 Accordingly to the relation48 presented below it is seen that dielectric dispersion is equal to the loss at the conductivity frequency ωσ ω ε i(ωσ ) = ε″(ωσ ) ⇒ ωσ = dc εsε0

τσ =

εsε0 σdc

where εs and ε0 are static and vacuum permittivity while τσ characterizes time scale with which the electric field decays to zero at constant charge conditions. Based on the above we can be sure that the slowest mode observed by Bhardwaj et al. is not a structural relaxation and it must be connected to dc conductivity. To justify our conclusions we also fitted loss peak visible in Figure 4 to the one sided Fourier transform KWW function with the (1 − n) = βKWW = 0.92. ϕ(t ) = exp[−(t /τα)1 − n ]

One can add that the βKWW varies within the range (0,1). At this place it is important to remember that for βKWW = 1 we have exponential response function (Debye relaxation) which is characteristic for dc conductivity. It also should be noted that De Gusseme found βKWW = 0.3 for the structural relaxation process of trehalose based on TMDSC measurements33 while Kaminski et al. obtained from BDS studies stretching exponent equal to 0.38 for primary mode of sucrose being homologue of 1563



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Marquardt (L−M) algorithm which adjusts the parameter values in the iterative procedure. ⎛ D T ⎞ τα = τVFT exp⎜ T 0 ⎟ ⎝ T − T0 ⎠

(1)

In eq 1 D is a measure of the non-Arrhenius behavior of the structural relaxation times, τVFT is pre-exponential factor and T0 is a Vogel−Fulcher−Tammann temperature. One can add that in the literature there is discussion about the physical meaning of T0. However, one needs to remember that very recently a paper by the Dyre group was published in Nature Physics54 which criticized using the VFT equation for description of relaxation times below the glass transition temperature. Thus, in light of this paper interpretation of T0 (VFT parameter) seems to be very speculative and controversial. One can add that VFT was used only to parametrize temperature dependence of the structural relaxation times. All fitting parameters for samples studied herein are collected in Table 3. Using VFT fits it was possible to calculate glass transition temperatures which are equal to Tg = 373 K and Tg = 340.5 K for trehalose and sucrose, respectively. The Tg was defined as the temperature at which structural relaxation time was equal to 100 s. Estimated values of glass transition temperatures agree quite well with those reported in the literature for sucrose while there is quite significant difference in Tg estimated from our dielectric data and a calorimetric one reported in the literature for trehalose.4,14,25,33,55 To explain this we need to recall that it can be due to measurement technique. It is worth mentioning that in dielectric measurements it is arbitrarily accepted that Tg is equal to the temperature at which structural relaxation time is equal to 100 s. Moreover, as it is commonly known Tg depends on the speed of cooling of the sample. During DSC scan the sample is usually cooled with the cooling rate 10 or 20 K/min while in the case of dielectric measurements the cooling rate is very small (k → 0). Finally, one should note that in the case of trehalose there was a huge contribution of dc conductivity to the loss spectra. Thus, precise estimation of the structural relaxation times of trehalose could be done only at temperatures far above the Tg. Consequently, there is quite large uncertainty in estimation of the glass transition temperature for trehalose based on dielectric data. The temperature dependence of the secondary relaxation times was described by the Arrhenius equation:

Figure 5. Dielectric loss spectra of sucrose (open circles) and trehalose (open squares) measured at indicated temperatures.

To get more detailed information about dynamics of the structural as well as secondary relaxation processes of sucrose and trehalose, α, γ and β loss peaks were analyzed with the use of the Havriliak−Negami52 and Cole−Cole53 functions, respectively. Next, the relaxation times of all observed modes were plotted versus temperature as shown in Figure 6.

⎛E ⎞ τγ = τ0 exp⎜ a ⎟ ⎝ RT ⎠

Figure 6. Relaxation map of sucrose and trehalose. Solid lines are the best VFT and Arrhenius fits.

(2)

It can be seen that the activation energies and relaxation times of the γ-process in sucrose (Eγ = 52 kJ/mol) and trehalose (Eγ = 51 kJ/mol) are almost the same. On the other hand the activation barriers calculated for the β-processes were found to be Eβ = 98 kJ/mol and Eβ = 87 kJ/mol for sucrose and

Structural relaxation times were fitted to the Vogel−Fulcher− Tammann equation (eq 1) in Origin program. It is worth mentioning that fitting in Origin is based on the Levenberg−

Table 3. Fitting Parameters from the VFT and Arrhenius Equation for Sucrose and Trehalose process α

βb

a

sucrose trehalose a

γb

log τ0 [s]

DT (K)

T0 (K)

log τ∞ [s]

Ea (kJ/mol)

log τ∞ [s]

Ea (kJ/mol)

−11.70 ± 0.68 −15.22 ± 0.81

997 ± 270 1959 ± 301

308 ± 6 321 ± 6

−18.76 ± 0.20 −17.20 ± 0.2

98 ± 1 87 ± 1

−15.05 ± 0.05 −16.17 ± 0.05

52 ± 0.5 51 ± 0.5

VFT. bArrhenius. 1564



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trehalose, respectively. It should be noted that similar values of the activation energies of the secondary modes in trehalose were reported by De Gusseme et al.33 In our recent paper we have demonstrated that the β-relaxation process in disaccharides is closely connected to the twisting motions of the glycosidic linkage.37,39 Therefore, it was postulated that the activation energy of the slow mode can be quite a good indicator of disaccharide flexibility. Moreover, one can suppose that the higher the activation barrier of the β-process, the greater the rigidity of the carbohydrate. Hence, following this simple dependence we can state that motions of the glycoside linkage in sucrose are significantly reduced with respect to that occurring in trehalose. Similar findings were reported in theoretical as well as NMR studies, which showed that sucrose is one of the most rigid of all disaccharides.32,56 It is also important to compare pre-exponential factors of the β- and γrelaxation processes in sucrose and trehalose. It can be seen that τ∞γ in both disaccharides lies within the range 10−15−10−16 s. Hence, these values are close to that predicted for the Debye like relaxations (τ = 10−14 s). Consequently, accordingly to the Eyring formalism we can state that the activation entropy for these processes can be negligible.57 The main outcome of these considerations is that the γrelaxation process is connected to the intermolecular motions of some part of sucrose or trehalose molecules. In fact, it was shown that rotation of the hydroxymethyl unit is a molecular mechanism of this mode. 40 On the other hand the preexponential factor determined for the β-process is much shorter 10−18−10−19 s. This means that change in entropy during motions of glycosidic linkage is rather significant. Thus, this process can be treated as the best source of information about glassy dynamics of studied disaccharides. Since amorphous disaccharides are very often used as stabilizing matrix for food, labile pharmaceuticals or proteins, one needs to investigate impact of aging on the dynamics of the secondary relaxation processes. Very recently Dranca et al.41 carried out a physical aging experiment on sucrose and trehalose. They showed that activation energies of the secondary relaxation processes changed significantly as an effect of aging of both saccharides at room temperature. To verify this finding we have carried out an aging experiment under similar conditions in sucrose. Results of our measurements are shown in Figure 7a,b. It can be seen that there are two effects accompanying this process. The first one is the change in the amplitude of both secondary relaxation processes (panel a). The second observation is that the relaxation time of the β-process increases as aging proceeds (panel b). However, it should be noted that the change in τβ is less than 0.1 decade and lies within an experimental error. In addition, further measurements on aged sample were carried out to check if activation energies of the faster and slower relaxation processes changed as it was reported by Dranca et al.41 As it can be seen in Figure 6, the relaxation times as well as activation energies of the β- and γ-processes measured for the aged and nonaged samples are almost the same. Therefore, we can conclude that the aging process does not influence dynamics of the secondary relaxation processes in such a significant way as shown in ref 41. One can also add that many dielectric studies carried out in the glassy state of different systems indicated that relaxation times of the secondary relaxation modes remain unaffected by the aging process. Furthermore, activation energy of the secondary relaxation processes did not change with the course of aging.58,59 In this context it is important to mention about

Figure 7. (a) Results of aging of the glassy sucrose performed at T = 298 K. (b) Change in β-relaxation time vs time of aging.

high pressure measurements. It was shown in many cases that application of very high pressure (up to p = 1 GPa), leading to very significant densification of the sample, influences the activation energy of the secondary relaxation processes only in a few percent.60 Thus, it is quite remarkable that investigations of the impact of aging on the dynamics of secondary relaxation processes with the use of DSC and dielectric spectroscopy differ so substantially. One can suppose that both techniques probe different kinds of motions, and this is probably the main reason for the discrepancy between calorimetric and dielectric data. However, further studies are necessary to understand this problem. Additionally, based on our dielectric data collected during aging of the sucrose we can determine structural relaxation time deeply in the glassy state. For this purpose we have applied the approach proposed by Roland and Casallini.58 These authors claimed that to calculate the relaxation time of the structural process at the given temperature in the glassy state one needs to perform isothermal time-dependent measurements of the imaginary part of dielectric permittivity ε″(f)̃ at fixed frequency f (lying in range of the JG mode, in our case (β) process), see Figure 8, upper panel. Then, one can evaluate structural relaxation time using following equation: 1565



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experimental data to eq 3 we obtained the value of log τα = 4.81 s (18 h). At this place it should be stressed that log τα = 4.81 s was calculated for the measurements carried out at 298 K for 48 h. On the other hand, it is necessary to add that although the determined τag does not necessarily represent equilibrium τα (which is surely longer than a few years), they are isostructural values. Therefore, one can conclude that the applied approach enabled us to evaluate isostructural relaxation time of the primary mode at the given temperature from the aging process. Finally, in order to fully describe glassy dynamics in trehalose and sucrose, high pressure measurements were carried out on samples being much below the glass transition temperatures. Both disaccharides were compressed up to p = 400 MPa at room temperature, see Figure 9. It can be seen that in both cases relaxation times of the β-process increase with applied pressure, while the position of the γ-mode remains almost the same. The most striking is that the application of the pressure slows down significantly (more than 1 decade) the β-process in trehalose and also in sucrose (these data are not shown in this paper). Since it has been recognized that this process originated from the motions of the glycosidic linkage, we can suppose that the structure of disaccharide becomes more rigid with increasing pressure. Additionally, the activation volume, which is defined as difference between molar volume of activated and nonactivated state of the molecule, was calculated from the following equation: ΔV = RT /log(e)(d log τ /dp)

We obtained ΔV = 15.8 cm3/mol and 17.7 cm3/mol for the βprocesses in sucrose and trehalose respectively (see Figure 9b). It is worth recalling that the activation volumes determined for the β-process in maltose and leucrose were equal to ΔV = 15.539 and 21.562 cm3/mol, respectively. Hence, we can conclude that, although activation volumes of the β-processes in the family of disaccharides differ, they are very close to each other. On the other hand activation volumes estimated for the γ-process are 3 cm3/mol and are close to those obtained for the faster secondary relaxation process in fructose63 and leucrose.62

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Figure 8. Upper panel loss spectrum of sucrose obtained after subtraction of the dc conductivity. Solid line represents the best KWW fit with the stretch exponent equal to βKWW = 0.38 . Lower panel presents dependence ε″(t)/ε″(t=0) of the β-process vs aging time at T = 298 K.

ε″(f ̃ , tag) ∼

ε″(f , tag = 0)

=

(4)

CONCLUSIONS We have performed broadband dielectric measurements at ambient and elevated pressure on two very important disaccharides, sucrose and trehalose, to gain detailed information about molecular dynamics in the supercooled as well as in the glassy state. We have shown that in the former state only one structural relaxation process can be observed. We have compared our data to those published earlier by Bhardwaj et al.42 and Kwon et al.43 We found out that freeze-drying as well as heating in a microwave oven influences dielectric spectra in a very significant way. It is clear that amorphous samples obtained from both techniques have much more complicated structure than those obtained by simple supercooling of the melt. This implies that interpretation of the dielectric data seems to be much more difficult and may lead to misinterpretation of the dielectric data. Moreover, inspired by a paper by Dranca et al. we have investigated the effect of aging of the sample on the dynamics of the β- as well as γ-relaxation processes. In contrast to the results presented in ref 41 we have found that activation energies of both modes do not change during aging carried out at T = 298 K. Based on this finding we came to conclusion that dielectric spectroscopy and DSC probe different kinds of motions (mobility) and this is probably the

⎧ ∼ ∼ ⎫ ⎡ t ⎤ βag ⎨Δε″(f , tag) exp⎢ − ag ⎥ + εeq ″ (f )⎬ ⎣ τag ⎦ ⎭ ⎩ ∼

ε″(f , tag = 0) (3)

where ε″eq(f) ≡ ε″( f,tag→∞) is an equilibrium value, Δε″( f,tag) = ε″(f,tag=0) − ε″eq is a change of ε″ during aging, tag is aging time, and βag is stretch exponent, which can be determined from the fitting of the structural loss peak to the KWW function.61 In this model we assume that parameter βag does not vary with the aging time. Unfortunately, it is difficult to evaluate the stretch exponent of the α-process of sucrose in the vicinity of the glass transition temperature from the raw data. To do this the following procedure was applied. As a first contribution of the dc conductivity was subtracted from the loss spectra of the sucrose measured at T = 353 K. Next, data obtained in such a way were fitted to the KWW function with the βKWW = 0.38; see upper panel of Figure 8. From fitting the 1566



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Figure 9. (a) Dielectric loss spectra obtained after compressing trehalose up to 400 MPa at T = 293 K. (b) Dependence of the β-relaxation times of the sucrose and trehalose as a function of pressure. Crystallization of Amorphous Sucrose below the Calorimetric Glass Transition Temperature from Correlations with Mobility. J. Pharm. Sci. 2007, 96, 1258. (6) Wang, B.; Tchessalov, S.; Cicerone, M. T.; Warne, N. W.; Pikal, M. J. Impact of sucrose level on storage stability of proteins in freezedried solids: II. Correlation of aggregation rate with protein structure and molecular mobility. J. Pharm. Sci. 2009, 98, 3145. (7) Hinrichs, W. L. J.; Prinsen, M. G.; Frijlink, H. W. Inulin glasses for the stabilization of therapeutic proteins. Int. J. Pharm. 2001, 215, 163. (8) Slade, L.; Levine, H.; Ievolella, J.; Wang, M. The glassy state phenomenon in applications for the food industry: Application of the food polymer science approach to structure-function relationships of sucrose in cookie and cracker systems. J. Sci. Food Agric. 1993, 63, 133. (9) Carpenter, J. F.; Pikal, M. J.; Chang, B. S.; Randolph, T. W. Rational design of stable lyophilized protein formulations: some practical advice. Pharm. Res. 1997, 14, 969. (10) Wang, W. Instability, stabilization and formulation of liquid protein pharmaceuticals. Int. J. Pharm. 1999, 185, 129−188. (11) Noel, T. R.; Ring, S. G.; Whittam, M. A. Dielectric relaxations of small carbohydrate molecules in the liquid and glassy states. J. Phys. Chem. 1992, 96, 5662−5667. (12) Gangasharan; Murthy, S. S. N. Study of α-, β-, and γ-relaxation processes in some supercooled liquids and supercooled plastic crystals. J. Chem. Phys. 1993, 99, 9865. (13) Tyagi, M.; Murthy, S. S. N. Dynamics of water in supercooled aqueous solutions of glucose and poly(ethylene glycol)s as studied by dielectric spectroscopy. Carbohydr. Res. 2006, 341, 650−662. (14) Champion, D.; Maglione, M.; Niquet, G.; Simatos, D.; Le Meste, M. Study of alpha- and beta-relaxation processes in supercooled sucrose liquids. J. Therm. Anal. Calorim. 2003, 71, 249−261. (15) Meissner, D.; Einfeldt, J.; Kwasniewski, A. Contributions to the molecular origin of the dielectric relaxation processes in polysaccharidesthe low temperature range. J. Non-Cryst. Solids 2000, 275, 199−209. Einfeldt, J.; Meissner, D.; Kwasniewski, A. Comparison of the molecular dynamics of celluloses and related polysaccharides in wet and dried states by means of dielectric spectroscopy. Macromol. Chem. Phys. 2000, 201 (15), 1969−1975. (16) Noel, T. R.; Parker, R.; Ring, S. G. A comparative study of the dielectric relaxation behaviour of glucose, maltose, and their mixtures with water in the liquid and glassy states. Carbohydr. Res. 1996, 282, 193−206.

main reason for the discrepancy between calorimetric and dielectric data. Finally, it was also demonstrated that compressing of the glassy sucrose and trehalose up to 400 MPa at T = 298 K leads to significant slowing down of the relaxation time of the β-process. This finding can be interpreted in view of the increasing rigidity of the glycosidic linkage in sucrose and trehalose.

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AUTHOR INFORMATION

Corresponding Author

*University of Silesia, Institute of Physics, ul. Uniwersytecka 4, 40-007 Katowice, Poland. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are deeply thankful for the financial support within the framework of the project entitled “From Study of Molecular Dynamics in Amorphous Medicines at Ambient and Elevated Pressure to Novel Applications in Pharmacy”, which is operated within the Foundation for Polish Science Team Programme cofinanced by the EU European Regional Development Fund. K.A. acknowledges financial support from the FNP 2012 (Start Programme).

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REFERENCES

(1) Saito, M.; Ugajin, G.; Nozawa, Y.; Sadzuka, Y.; Miyagishima, A.; Sonobe, T. Preparation and dissolution characteristics of griseofulvin solid dispersions with saccharides. Int. J. Pharm. 2002, 249, 71. (2) Serajuddin, A. T. M. Solid Dispersion of Poorly Water-Soluble Drugs: Early Promises, Subsequent Problems, and Recent Breakthroughs. J. Pharm. Sci. 1999, 88, 1058. (3) Champion, D.; Hervet, H.; Blond, G.; Le Meste, M.; Simatos, D. Translational diffusion in sucrose solutions in the vicinity of their glass transition temperature. J. Phys. Chem. B 1997, 101, 10674. (4) Shamblin, S. L.; Tang, X.; Chang, L.; Hancock, B. C.; Pikal, M. J. Characterization of the time scales of molecular motion in pharmaceutically important glasses. J. Phys. Chem. B 1999, 103, 4113. (5) Bhugra, C.; Rambhatla, S.; Bakri, A.; Duddu, S. P.; Miller, D. P.; Pikal, M. J.; Lechuga-Ballesteros, D. Prediction of the Onset of 1567



Article

(17) Faivre, A.; Niquet, G.; Maglione, M.; Fornazero, J.; Jal, J. F.; David, L.. Dynamics of sorbitol and maltitol over a wide timetemperature range. Eur. Phys. J. B 1999, 10, 277−286. (18) Noel, T. R.; Parker, R.; Ring, S. G. Effect of molecular structure and water content on the dielectric relaxation behaviour of amorphous low molecular weight carbohydrates above and below their glass transition. Carbohydr. Res. 2000, 329, 839−845. (19) Finegold, L.; Franks, F.; Hatley, R. H. M. J. Chem. Soc., Faraday Trans. 1989, 85, 2945. (20) Freedberg, D. An Alternative Method for Pucker Determination in Carbohydrates from Residual Dipolar Couplings: A Solution NMR Study of the Fructofuranosyl Ring of Sucrose. J. Am. Chem. Soc. 2002, 124, 2358. (21) Poppe, L.; van Halbeek, H. The rigidity of sucrose: just an illusion? J. Am. Chem. Soc. 1992, 114, 1092. (22) Baraguey, C.; Mertens, D.; Dolle, A. Anisotropic Reorientation and Intermolecular Interactions of Sucrose Molecules in Aqueous Solution. A Temperature and Concentration-Dependent 13C NMR Relaxation Study. J. Phys. Chem. B 2002, 106, 6331. (23) Van Dusschote, D.; Tracht, U.; Heuer, A.; Spiess, H. W. Site Specific Rotational Mobility of Anhydrous Glucose near the Glass Transition As Studied by 2D Echo Decay 13C NMR. J. Phys. Chem. A 1999, 103, 8359. (24) Moran, G. R.; Jeffrey, K. R.; Thomas, J. M.; Stevens, J. R. A dielectric analysis of liquid and glassy solid glucose/water solutions. Carbohydr. Res. 2000, 328, 573. (25) Miller, D. P.; De Pablo, J. J. Calorimetric Solution Properties of Simple Saccharides and Their Significance for the Stabilization of Biological Structure and Function. J. Phys. Chem. B 2000, 104, 8876. (26) Kerc, J.; Srcic, S. Thermal analysis of glassy pharmaceuticals. Thermochim. Acta 1995, 248, 81. (27) Hancock, B. C.; Zografi, G. Characteristics and Significance of the Amorphous State in Pharmaceutical Systems. J. Pharm. Sci. 1997, 86, 1. (28) Shamblin, S. L.; Tang, X.; Chang, L.; Hancock, B. C.; Pikal, M. J. Interpretation of relaxation time constants for amorphous pharmaceutical systems. J. Pharm. Sci. 1999, 89, 417−427. (29) Dudognon, E.; Willart, J. F.; Caron, V.; Capet, F.; Larsson, T.; Descamps, M. Formation of budesonide/a-lactose glass solutions by ball-milling. Solid State Commun. 2006, 138, 68. (30) Molinero, V.; Goddard, W. A., III. Microscopic Mechanism of Water Diffusion in Glucose Glasses. Phys. Rev. Lett. 2005, 95, 045701− 045704. (31) Wachner, A. M.; Jeffrey, K. R. A two-dimensional deuterium nuclear magnetic resonance study of molecular reorientation in sugar/ water glasses. J. Chem. Phys. 1999, 111, 10611. (32) Tran, V. H.; Brady, J. W. Disaccharide conformational flexibility. I. An adiabatic potential energy map for sucrose. Biopolymers 1990, 29, 961. (33) De Gusseme, A.; Carpentier, L.; Willart, J. F.; Descamps, M. Molecular Mobility in Supercooled Trehalose. J. Phys. Chem. B 2003, 107, 10879. (34) Ermolina, I.; Polygalov, E.; Bland, C.; Smith, G. Dielectric spectroscopy of low-loss sugar lyophiles: II. Relaxation mechanisms in freeze-dried lactose and lactose monohydrate. J. Non-Cryst. Solids 2007, 353, 4485. (35) Kaminski, K.; Kaminska, E.; Ngai, K. L.; Paluch, M.; Wlodarczyk, P.; Kasprzycka, A.; Szeja, W. Identifying the Origins of Two Secondary Relaxations in Polysaccharides. J. Phys. Chem. B 2009, 113, 10088. (36) Kaminski, K.; Kaminska, E.; Wlodarczyk, P.; Pawlus, S.; Kimla, S.; Kasprzycka, A.; Paluch, M.; Ziolo, J.; Szeja, W.; Ngai, K. L. Dielectric Studies on Mobility of the Glycosidic Linkage in Seven Disaccharides. J. Phys. Chem. B 2008, 112, 12816. (37) Kaminski, K.; Kaminska, E.; Hensel-Bielowka, E.; Chelmecka, E.; Paluch, M.; Ziolo, J.; Wlodarczyk, P.; Ngai, K. L. Identification of the Molecular Motions Responsible for the Slower Secondary (β) Relaxation in Sucrose. J. Phys. Chem. B 2008, 112, 7662.

(38) Ermolina, I.; Smith, G. Dielectric spectroscopy of low-losses sugar lyophiles: III. The influence of moisture on the dielectric response of freeze-dried lactose. J. Non-Cryst. Solids 2011, 357, 671− 676. (39) Wlodarczyk, P.; Kaminski, K.; Adrjanowicz, K.; Wojnarowska, Z.; Czarnota, B.; Paluch, M.; Ziolo, J.; Pilch, J. Identification of the slower secondary relaxation’s nature in maltose by means of theoretical and dielectric studies. J. Chem. Phys. 2009, 131, 125103. (40) Kaminski, K.; Wlodarczyk, P.; Kaminski, E.; Adrjanowicz, K.; Wojnarowska, Z.; Paluch, M. Origin of the commonly observed secondary relaxation process in saccharides. J. Phys. Chem. B 2010, 114, 11272. (41) Dranca, I.; Bhattacharya, S.; Vyazovkin, S.; Suryanarayanan, R. Implications of Global and Local Mobility in Amorphous Sucrose and Trehalose as Determined by Differential Scanning Calorimetry. Pharm. Res. 2009, 26, 1064. (42) Bhardwaj, S. P.; Suryanarayanan, R. Subtraction of DC Conductivity and Annealing: Approaches To Identify Johari-Goldstein Relaxation in Amorphous Trehalose. Mol. Pharmaceutics 2011, 8, 1416−22. (43) Kwon, H.-J.; Seo, J.-A.; Kim, H. K.; Hwang, Y.-H. Study of dielectric relaxations of anhydrous trehalose and maltose glasses. J. Chem. Phys. 2011, 134, 014508. (44) Lee, J. W.; Thomas, L. C.; Jerrell, J.; Feng, H.; Cadwallader, K. R.; Schmidt, S. J. Investigation of Thermal Decomposition as the Kinetic Process That Causes the Loss of Crystalline Structure in Sucrose Using a Chemical Analysis Approach. J. Agric. Food Chem. 2011, 59, 702−712. (45) Sidebottom, D. L.; Tran, T. D. Universal patterns of equilibrium cluster growth in aqueous sugars observed by dynamic light scattering. Phys. Rev. E 2010, 82, 051904. (46) Anopchenko, A.; Psurek, T.; VanderHart, D.; Douglas, J. F.; Obrzut, J. Phys. Rev. E 2006, 74, 031501. (47) Kaminski, K.; Wlodarczyk, P.; Paluch, M. Comment to “Study of dielectric relaxations of anhydrous trehalose and maltose glasses. J. Chem. Phys. 2011, 135, 167102. (48) Richert, R.; Agapov, A.; Sokolov, A. P. J. Chem. Phys. 2011, 134, 104508. (49) Kaminski, K.; Adrjanowicz, K.; Paluch, M.; Kaminski, E. Probing of structural relaxation times in the glassy state of sucrose and trehalose based on dynamical properties of two secondary relaxation processes. Phys. Rev. E 2011, 83, 061502. (50) Döss, A.; Paluch, M.; Sillescu, H.; Hinze, G. Phys. Rev. Lett. 2002, 88, 095701. (51) Novikov, V. N.; Sokolov, A. P. Universality of the dynamic crossover in glass-forming liquids: A “magic” relaxation time. Phys. Rev. E 2003, 67, 031507. (52) Havriliak, S.; Negami, S. A complex plane analysis of αdispersions in some polymer systems. J. Polym. Sci. C 1966, 14, 99. (53) Cole, K. S.; Cole, R. H. Dispersion and absorption in dielectrics I. Alternating current characteristics. J. Chem. Phys. 1941, 9, 341. (54) Hecksher, T.; Nielsen, A. I.; Olsen, N. B.; Dyre, J. C. Little evidence for dynamic divergences in ultraviscous molecular liquids. Nat. Phys. 2008, 4, 737−741. (55) Taylor, L. S.; Zografi, G. D. Sugar-Polymer Hydrogen Bond Interactions in Lyophilised Amorphous Mixtures. J. Pharm. Sci. 1998, 87, 1615. (56) Effemey, M.; Lang, J.; Kowalewski, J. Multiple-field carbon-13 and proton relaxation in sucrose in viscous solution. J. Magn. Reson. Chem. 2000, 38, 1012. (57) Eyring, H. Viscosity, Plasticity, and Diffusion as Examples of Absolute Reaction Rates. J. Chem. Phys. 1936, 4, 283. (58) Casalini, R.; Roland, C. M. Aging of the Secondary Relaxation to Probe Structural Relaxation in the Glassy State. Phys. Rev. Lett. 2009, 102, 035701. (59) Ngai, K. L.; Paluch, M. J. Chem. Phys. 2004, 120, 857. (60) Paluch, M.; Pawlus, S.; Hensel-Bielowka, S.; Kaminski, K.; Psurek, T.; Rzoska, S. J.; Ziolo, J.; Roland, C. M. Phys. Rev. B 2005, 72, 224205. 1568



Article

(61) Williams, G.; Watts, D. C. Non-symmetrical dielectric relaxation behavior arising from a simple empirical decay function. Trans. Faraday Soc. 1970;66:80−85; Kohlrausch R. Nachtrag uber die elastiche Nachwirkung beim Cocon und Glasladen. Ann. Phys. (Leipzig) 1847, 72, 393. (62) Kaminski, K.; Kaminska, E.; Hensel-Bielowka, S.; Pawlus, S.; Paluch, M.; Ziolo, J. High pressure study on molecular mobility of leucrose. J. Chem. Phys. 2008, 129, 084501. (63) Kaminski, K.; Kaminska, E.; Paluch, M.; Ziolo, M.; Ngai, K. L. The True Johari−Goldstein β-Relaxation of Monosaccharides. J. Phys. Chem. B 2006, 110, 25045.

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Sucrose and Trehalose - American Chemical Society

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