Biomacromolecules 2005, 6, 3045-3050
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Simultaneous and in Situ Analysis of Thermal and Volumetric Properties of Starch Gelatinization over Wide Pressure and Temperature Ranges Stanislaw L. Randzio* and Marta Orlowska Institute of Physical Chemistry, Polish Academy of Sciences, ul. Kasprzaka 44/52, 01-224 Warsaw, Poland Received May 25, 2005; Revised Manuscript Received June 30, 2005
A method for simultaneous and in situ analysis of thermal and volumetric properties of starch gelatinization from 0.1 to 100 MPa and from 283 to 430 K is described. The temperature of a very sensitive calorimetric detector containing a starch-water emulsion at a selected pressure is programmed to rise at a slow rate; volume variations are performed automatically to keep the selected pressure constant while the heat exchange rate and the volume are recorded. The method is demonstrated with a novel investigation of pressure effects on a sequence of three phase transitions in an aqueous emulsion of wheat starch (56 wt % water). The volume changes during the main endothermic transition (M), associated with melting of the crystalline part of the starch granules and a helix-coil transformation in amylopectin, but also with an important swelling, were separated into a volume increase associated with swelling and a volume decrease associated with the transition itself. Thermodynamic parameters for this transition together with their pressure dependencies have been obtained from four independent experiments at each pressure. The data are thermodynamically consistent, but are poorly described by the Clapeyron equation. The negative volume change of the slow exothermic transition (A) appearing just after the main endothermic transition (M) is small, spread out over a wide temperature interval, and occurs at higher temperatures with increasing pressures. This transition is probably associated with reassociation of the unwound helixes of amylopectin with parts of amylopectin molecules other than their original helix duplex partner. The positive volume change of the high-temperature, endothermic transition (N) with a small enthalpy change is probably associated with a nematic-isotropic transformation ending the formation of a homogeneous SOL phase (in the sense of Flory), and is also pushed to higher temperatures with increasing pressures. Knowledge of the state of wheat starch as a function of pressure and temperature is important in extruder processing. The data also provide a basis for the elliptic phase diagram for starch gelatinization. The method is easily adapted to determine similar data for other macromolecular materials. Introduction The transition between native and nonnative states of biomacromolecules, such as proteins and some polysaccharides, exhibits a unique elliptical or a near-elliptical shape on the pressure-temperature surface.1-5 This makes the interplay between pressure and temperature as inducing variables very important for both science and practice, especially in food and pharmaceutical industries. Temperature and water content have been extensively used as variables in physicochemical investigations of biomacromolecular materials performed with various methods. However, pressure has been used very seldom, especially as it is concerned with in situ studies.6,7 Smeller8 noted that only the experimental difficulties can explain the relative scarceness of pressure studies. There are several reasons to measure the effect of pressure on a wide variety of biomacromolecules, the most important being to separate the effects of volume and thermal energy changes, which appear simultaneously in temperature experiments.9 Molecular processes * Corresponding author. Fax: +48-22-632 52 76. E-mail: randzio@ ichf.edu.pl.
that alter the volume of the system are especially sensitive to pressure. Several phenomena are worth studying in both temperature and pressure experiments.8 Fundamental understanding of the influence of processing on food products requires studies to elucidate how the physical properties of food materials vary as a function of conditions encountered in processing and handling. These conditions range from the relatively mild environments associated with food storage to the extreme conditions encountered during extrusion. An understanding of the effects of processing on the physical properties of food materials should allow prediction of the formulation of raw materials and processing conditions, to achieve desired end-product properties. To achieve this goal, systematic studies must be done to develop a database on the physical properties of food materials as a function of variables relevant to processing.10 There has previously been no instrument or method suitable for measuring simultaneously and in situ both the thermal and mechanical properties of biomacromolecular systems on a pressure-temperature plane over wide ranges of variables. The present contribution presents a method that uses pressure and temperature as independent variables and
10.1021/bm0503569 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/26/2005
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high-temperature endothermic transition (N) occurring at water contents around 50 wt % and higher is associated with destruction of amylose-lipid complexes or with a nematicisotropic transition that ends the formation of the isotropic SOL phase. The present contribution is aimed at a novel in situ investigation of the pressure influence on the above transitions in the wheat starch-water system. A mixture of native wheat starch with 50 wt % added water (56.0 wt % total water content) has been selected for the present study to demonstrate the performance of the new method. Experimental Section Figure 1. DSC traces obtained at atmospheric pressure at a rate of 16.67 mK s-1 for aqueous native wheat starch emulsions at selected concentrations of water (total water contents in weight percent).11 For the description of other symbols see the text.
simultaneously records both the heat effect and the volume variations of a system from 283 to 430 K at pressures up to 100 MPa. The performance of the method is demonstrated with an in situ analysis of pressure effects on the phase transformations occurring during thermal gelatinization of a 0.44 wheat starch + 0.56 water emulsion, previously observed with a differential scanning calorimeter (DSC) at atmospheric pressure.11,12 Starch is one of the most important natural macromolecules. Its importance stems from the fact that the starch granule is an almost universal form for packaging and storing carbohydrate in green plants. It is also one of the main components of food materials, especially those submitted to extruder processing, where pressures up to 100 MPa can be encountered. The process of preparing a homogeneous SOL phase (in the sense of Flory13) from a mixture of native starch and water is called gelatinization. Starch gelatinization is a combined process consisting of hydration of amorphous regions and subsequent melting of crystalline arrays. It was demonstrated recently11,12 that all the transformations occurring during starch gelatinization can be observed with highsensitivity DSC done at a low rate of temperature scanning at atmospheric pressure. Typical results are shown in Figure 1 for pastes or emulsions of wheat starch with various total water contents. The main endothermic transition (M), occurring from 319 to 333 K independently of the water content, is likely14,15 associated with melting of the crystalline part of the starch granules followed by a helix-coil transformation in amylopectin, the main component of starch. This endothermic transition is followed by a water-dependent, slow, exothermic transformation (A), which is probably related to reassociation of the unwound helixes of amylopectin with parts of amylopectin molecules other than their original helix duplex partner, forming physical junctions and creating more general hydrogen bonded associations.15 Such an exothermic effect was previously observed by other authors.16,17 A study performed with synchrotron X-ray diffraction suggested that, after the endothermic effect of melting of the starch granule, upon further heating a crystallization of amylose-lipid complexes was observed that also should be associated with an exothermic effect.18 The
Materials. Wheat starch powder was from PROLABO, France, with amylose content of 21.0 ( 0.2%, lipid content of 0.27 ( 0.01%, and protein content of 0.36 ( 0.01%. To prepare a water emulsion or paste, a given amount of demineralized and bidistilled water was added to a weighed sample of starch powder and the mixture was blended manually until a homogeneous sample was obtained. pBromochlorobenzene and p-dibromobenzene were from Fluka (purity >99%) and further purified by CHEMIPAN through crystallizations from various solvents (ethanol, methanol, n-hexane). The final purity determined by chromatography was 99.99% for both substances, and that determined by the cryometric Skau method was 99.98% for p-bromochlorobenzene and 99.94% for p-dibromobenzene. Benzene was from HAJDUKI (purity 99.5%) and was further purified by CHEMIPAN first through chemical removal of sulfur compounds (K-Na alloy) and then by fractional distillation and preparative chromatography. The final purity determined by chromatography was 99.99%, and that determined by the cryometric Skau method was 99.97%. Benzoic acid was from POCH (purity >99.5%, used without further treatment). Gallium and indium were from SKAWINA (purity 99.999%) and used without further treatment. Method. The new method presented here is based on the basic principles of scanning transitiometry, previously described.19 Immediately after preparation, a sample of a homogeneous emulsion (about 4 g) is placed in a highpressure vessel surrounded by a calorimetric detector. A transition or change in state is induced by controlled variation of an independent variable (p, V, or T) while the other independent variable is kept constant. The variations of the dependent variable as well as the associated heat effect are simultaneously recorded as a function of time or of the scanned variable. A single transitiometric experiment always leads to simultaneous determination of a pair of thermodynamic derivatives (thermal and mechanical) or to a pair of thermomechanical coefficients [Rp and κT, γ and κT, Cp and Rp, or CV and γ, depending on the selection of the pair of independent variables].20,21 Instrumentation. A schematic of the instrument used in this study, constructed according to the general principles of scanning transitiometry, is presented in Figure 2. It consists of a calorimeter equipped with high-pressure vessels, a pVT system, and LabView virtual instrument (VI) software. Two cylindrical calorimetric detectors (Φ ) 17 mm, l ) 80 mm), made from 622 thermocouples (chromel-alumel) each, are
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Figure 2. Schematic of a transitiometric installation for simultaneous and in situ analysis of thermal and volumetric properties of starch gelatinization at pressures up to 100 MPa from 283 to 430 K.
mounted differentially and connected to a nanovolt amplifier. The calorimetric detectors are placed in a metallic block, the temperature of which is directly controlled with a 22-bit digital feedback loop (∼10-4 K), which is part of the transitiometer software. The calorimeter block is surrounded by a heating-cooling shield. The temperature difference between the block and the shield is set constant and is controlled by an analog controller. The temperature measurements, both absolute and differential, are made with calibrated Pt 100 sensors. The heaters are embedded in the outer surfaces of both the calorimeter block and the shield. The whole assembly is thermally insulated and enclosed in a stainless steel body fixed on a mobile stand, which allows the calorimeter to be moved up and down over the calorimetric vessels. When performing measurements near 273 K or below, dry air is pumped through the apparatus to prevent water condensation. The calorimetric vessels are made from 0.8 cm internal diameter 316 SS tubing and are fixed on a mounting table attached to the mobile stand. A flexible ampule containing the sample is placed in the measuring vessel on top of a spring, ensuring placement of the sample in the center of the calorimetric detector. Another technique is to use mercury as the hydraulic fluid and place a sample of prepared material directly on the mercury. Only the measuring vessel is connected to the pV line. The reference vessel acts only as a thermal reference; a stainless steel bar of appropriate dimensions is placed in it to balance the baseline of the differential calorimetric signal. The tubing of both measuring and reference vessels are connected to reducers, placed inside the calorimeter when it is in the lowered (measuring) position. The connections from the reducers to the manifold are made with thin stainless steel capillaries to reduce heat losses to the environment. The vessels are closed with a cone plug fixed in place by an internally threaded cover, which
also acts as a heat exchanger between the calorimetric vessel tubing and the calorimetric detector. Two sleeves are also fixed on the calorimetric vessel tubing below the cover to help control the heat exchange between the calorimetric vessel tubing and both the calorimeter block and the shield. The piston pump (9 cm3 of total displaced volume) is driven by a stepping motor controlled by the transitiometer software (manual control is possible during preparatory operations). The pressure detector is a Viatran 245 transducer, with 100 MPa full range and precision of 0.15% fsd (full scale deflection). The pressure detector, the output of the calorimetric amplifier, and the stepping motor are connected to a NI PCIMIO-16XE-50 multifunction board through an NI SCB-68 shielded connector block. The temperature measurements and digital control of the calorimetric block are performed through a serial port. The software, elaborated with the use of LabView language, performs as a virtual instrument (VI). It consists of 90 subVIs, each responsible for a particular functionspressure measurement, temperature measurement, counting the motor steps for recording the volume variations, measuring the calorimetric signal, etc.sand each performs independently. However, all the subVIs form a hierarchical structure with a top window, where the experimenter can see simultaneously all four variables (pressure, p, volume variations, V, temperature, T, and the heat exchange rate, q) associated with the process under investigation and the current status of the temperature and pressure control loops. The experiments are performed by starting thermal and mechanical stabilization over at least 5000 s; then the temperature scanning starts, which is accompanied by automatic volume compensation to keep the pressure constant. At the end of the scan the temperature is kept constant also for at least 5000 s. Any static baseline shift of the calorimetric signal between the low- and high-temperature
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stabilizations is corrected. No corrections are made for the calorimetric signal recorded during the temperature scan. The method presented here is rather simple and safe in practice. The total volume of the liquid phase under pressure is only about 20 mL, and the energy accumulated in it is rather small and not dangerous. The mercury used as a hydraulic fluid is always contained in a closed space. In case of a leak, the mercury is collected on a special protecting plate. The calorimetric vessels are conveniently and reproducibly closed and opened with a torque wrench with the vessels placed in a specially designed holder. Because of the high sensitivity of the instrument, some precautions must be taken to ensure valid measurements. In the case of the calorimetric signal, the main precaution is to carefully compensate the thermal balance of the differential calorimetric vessels. It is also important to keep the initial mercury level always in the same position, just above the entry to the calorimetric detector zone. Adjustment of the mercury level is easily done with the motorized pump. With respect to the volumetric component, it is very important that displacement of the mercury during calibration experiments be sufficiently slow to avoid overpressures in the flow lines, and differentiation of the piston displacement must be carefully done to avoid excessive noise on one hand and excessive damping on the other. Calibration. The temperature and energy scales of the differential calorimetric detector were calibrated under atmospheric pressure with the fusion of the following: gallium, Tm ) 302.91 K, ∆Hm ) 5.59 kJ mol-1; pbromochlorobenzene, Tm ) 337.73 K, ∆Hm ) 18.760 kJ mol-1; p-dibromobenzene, Tm ) 360.45 K, ∆Hm ) 20.530 kJ mol-1; benzoic acid, Tm ) 395.55 K, ∆Hm ) 18.062 kJ mol-1; and indium, Tm ) 429.75 K, ∆Hm ) 3.28 kJ mol-1. The calibration experiments were done by enclosing a calibration substance in a 75 mm long thin glass tube placed in the center of the calorimetric vessel. The remaining inner space of the calorimetric vessel was filled with dried starch. The precision of the temperature scale is (0.2 K. The energetic calibration constant kc of the calorimetric detector depends on temperature and is described by kc/(W V-1) ) 3.423 × 10-3 + 9.993 × 10-6T/K
(1)
The mean deviation between eq 1 and the calibration data is 1.4%. The reproducible resolution of the calorimetric detector varies from 1.3 × 10-7 W at 303 K to 1.6 × 10-7 W at 430 K. As reported previously, for properly designed experimental vessels, the energetic calibration constant of the calorimetric detector does not depend on pressure.22 The volumetric calibration of the high-pressure pump was performed by weighing 11 mercury samples displaced by known numbers of motor steps. Each motor step corresponds to a displacement of (5.22 ( 0.03) × 10-6 cm3. The volumetric calibration of the high-pressure pump and both energetic and temperature calibrations of the calorimetric detector were verified by test measurements using isobaric fusion of benzene.23 Figure 3 presents an example of such measurements performed at a scanning rate of 2.5 mK s-1 at 100 MPa. The mean results from eight independent
Figure 3. Example of simultaneous transitiometric traces (heat flux and volume variations) of isobaric melting at 100 MPa of 0.3858 g of benzene used as a verification test for thermal and volumetric calibrations of the transitiometer used in the present study.
Figure 4. Volume variations during isobaric temperature scans at various pressures with only hydraulic liquid (mercury) present in the system. The data at 10 and 100 MPa are shifted by +0.3 and -0.3 mm3 K-1, respectively, to avoid overlapping.
measurements gave the following: ∆fusV(100 MPa) ) 0.1038 ( 0.0028 cm3 g-1 (the respective literature value is ∆fusV(100 MPa) ) 0.1026 cm3 g-1),23 ∆fusH(100 MPa) ) 131.1 ( 2.1 J g-1 (the respective literature value is ∆fusH(100 MPa) ) 126.3 J g-1),23 Tfus,onset(100 MPa) ) 304.2 ( 0.3 K, and Tfus,peak(100 MPa) ) (305.7 ( 0.5) K (the literature value is Tfus(100 MPa) ) 305.6 K,23 given without any specification). The agreement with volumetric data is very good. Small differences from the thermal data probably are caused by internal heat exchange conditions in the calorimetric vessel. In test measurements only 0.4 g of benzene was floating on the mercury, while in the calibration experiments the calibration substances were placed in the center of the calorimetric vessel and were surrounded by a dry starch powder. Another test was performed on the temperature and pressure dependence of the thermal expansion of the mercury used as hydraulic liquid. The hydraulic liquid was displaced to the top of the empty calorimetric vessel, and temperature scanning measurements were performed at a rate of 2.5 mK s-1 at various pressures. Results are presented in Figure 4. The thermal expansion of the hydraulic fluid is almost constant at a given pressure and only very slightly decreases with temperature. The mean values are 0.949 ( 0.033 mm3 K-1 at 10 MPa, 0.895 ( 0.038 mm3 K-1 at 60 MPa, and 0.888 ( 0.050 mm3 K-1 at 100 MPa.
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Figure 6. Division of dV/dT for the main endothermic transition (M) into a positive dV/dT due to swelling and negative dV/dT due to the transition itself.
Figure 5. Transitiometric traces (heat flux and dV/dT per gram of dry starch) obtained simultaneously and in situ by scanning temperature at a rate of 2.5 mK s-1 at various pressures for a starch-water emulsion (56.0 wt % total water content).
Results and Discussion Figure 5 presents results obtained in isobaric experiments by scanning temperature at a low rate of 2.5 mK s-1 (0.15 K min-1) at pressures of 10, 60, and 100 MPa. Results at each pressure present two output signals recorded simultaneously as a function of temperature, heat flux (rate of heat exchange), and dV/dT (thermal expansion), with the last two quantities expressed per gram of dry starch. The most important observation is that all the transitions recorded previously at atmospheric pressure at a temperature scanning rate of 16.7 mK s-1 (1 K min-1) with a high-sensitivity DSC (see the respective trace at 56.0 wt % total water content in Figure 1) are also observed in the transitiometric traces in Figure 5 performed at elevated pressure at a much lower temperature scanning rate of 2.5 mK s-1 (0.15 K min-1). The present method also measures simultaneously the volume changes at those transitions. In Figure 5 the right ordinate presents the dV/dT of the sample; the dV/dT values from the hydraulic liquid (see Figure 4) have been subtracted from the experimental data. The dV/dT of the sample at the main endothermic transition (M) decreases over the pressure range
under investigation (10-100 MPa). Also note that the changes of dV/dT at the particular transitions are rather small, while the general tendency is for dV/dT to rise considerably with temperature over the whole temperature range. The last phenomenon is associated with swelling of starch granules during gelatinization, even at elevated pressures. This allows a more detailed analysis of the main transition (M). For several degrees prior to and after the transition, dV/dT increases linearly with temperature with the same or a very similar slope. Assuming this, dV/dT during the transition can be divided into two contributions, one due to the assumed linear swelling and one due to the phase transition itself. Figure 6 presents an example of such a division of the results obtained at 10 MPa. Once the division is made, the two contributions can be integrated separately to give volume changes occurring in the transition: a positive change for the swelling and a negative change for the transition itself. Integrated volumetric data, together with thermal data, all obtained for each pressure from at least four independent experiments, performed each time on a freshly prepared sample, are given in Table 1. The errors are the standard deviations from the mean values of all experiments performed at each pressure. Table 1 also contains data at 0.1 MPa obtained previously11 with a DSC. From linear approximations of the pressure dependence of the parameters presented in Table 1, the following slopes could be obtained: dHtrans/dp ) -(9.85 ( 2.25) mJ MPa-1 g-1, dVtrans/ dp ) (2.27 ( 0.37) × 10-3 mm3 g-1 MPa-1, dVswelling/dp ) -(9.28 ( 2.05) × 10-3 mm3 g-1 MPa-1, and dTtrans/dp ) -(24.6 ( 6.9) mK MPa-1. Assuming the main transition (M) is an equilibrium first-order transition (Ehrenfest classification24), the last slope can be also obtained from the Clapeyron equation and data from Table 1; from 10 to 100 MPa, (dTtrans/dp)Clapeyron ) -(78.3 ( 2.5) mK MPa-1. Although the slopes are both negative, the agreement is poor, implying that the mechanism of the transition is more complicated than the first-order transition assumed by the Clapeyron equation. Also, the pressure dependence may not be linear, especially at low pressure. Future studies will focus on that problem. Despite a large number of pressure studies on starch gelatinization, only the results of Rubens and Heremans7 were obtained from in situ studies and are in agreement with the present results. In Figure 4 of their study, performed with the use of infrared spectroscopy and a
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Table 1. Thermodynamic Data for the Main Transition (M) in an Aqueous Emulsion of Wheat Starch (56 wt % Total Water) Expressed per Gram of Completely Dry Starch pressure (MPa) g-1)
∆transH (J ∆transV (mm3 g-1) ∆swellingV (mm3 g-1) Ttrans,onset (K) a
0.1a
10
60
100
3.52 ( 0.07
3.12 ( 0.12 -0.788 ( 0.038 1.53 ( 0.10 319.5 ( 0.5
2.65 ( 0.07 -0.647 ( 0.040 1.22 ( 0.05 318.1 ( 1.0
2.45 ( 0. 7 -0.586 ( 0.035 0.683 ( 0.036 317.9 ( 0.6
320.5 ( 0.5
Data at 0.1 MPa have been taken from Randzio et al.11
diamond anvil cell, Rubens and Heremans7 show that the temperature of the transition is lowered by pressure increase. Opposite the pressure effects on the main endothermic transition (M), the pressure influence on both the exothermic transformation (A) and the high-temperature endothermic transition (N) is positive. At 10 MPa, the exothermic transformation starts at 348.6 ( 0.6 K and is shifted by pressure to higher values at a mean rate of 38.9 ( 9.9 mK MPa-1. Also at 10 MPa the high-temperature endothermic transition starts at 382.7 ( 0.2 K and is shifted by pressure to higher values at a mean rate of 96.1 ( 3.4 mK MPa-1. These observations are in agreement with a general thermodynamic approach to these transitions. In Figure 5, in transition A, the exothermic effect is always associated with a decrease of thermal expansion, which is most probably caused by a negative volume change at that transition, similar to the observation made on the main transition (M). In transition N, the endothermic effect is associated with a small increase of thermal expansion, which is most probably caused by a positive volume change at that transformation. Thus, the Clapeyron equation would also give in both cases positive dT/dp slopes. The uncertainty limits given for the above results contain both a purely instrumental contribution, 1-5%, and a contribution from the preparation of the emulsion that can be several percent.
-(24.6 ( 6.9) mK MPa-1. Also at 10 MPa the exothermic transformation (A) starts at 348.6 ( 0.6 K and is shifted by pressure to higher temperatures at a rate of 38.9 ( 9.9 mK MPa-1 and the high-temperature endothermic transition (N) starts at 382.7 ( 0.2 K and is shifted by pressure to higher temperatures at a rate of 96.1 ( 3.4 mK MPa-1. This is thermodynamically consistent, because the negative enthalpy change (exothermic) for transition A is associated with a negative volume change, and in the case of transition N, the positive enthalpy change (endothermic) is associated with a positive volume change. These data supply knowledge on the state of wheat starch at given conditions of pressure and temperature, which can be of importance in extruder processing. Acknowledgment. We acknowledge the valuable comments and remarks of the reviewers. The instrument used in the present study was made in the Institute of Physical Chemistry, but it can immediately be available commercially on a solid prototype basis. References and Notes (1) (2) (3) (4) (5) (6)
Conclusions A novel method described here allows simultaneous and in situ investigation of thermal and volumetric properties of starch gelatinization over extended ranges of pressure and temperature. The heat flux and dV/dT for a sequence of phase transformations during thermal gelatinization of an aqueous emulsion of wheat starch (56 wt % total water) were performed at various pressures. At 10, 60, and 100 MPa, the dV/dT of the main transition (M), associated with melting of the crystalline part of the starch granules followed by a helix-coil transformation in amylopectin and swelling, was divided into a positive volume change associated with swelling and a negative volume change associated with the transition itself. Mean values of thermodynamic parameters of that transition together with the slopes of linear pressure effects are thermodynamically consistent, although the agreement with the Clapeyron equation is poor. At 10 MPa the main endothermic transition (M) starts at 319.5 ( 0.5 K and is shifted by pressure to lower temperatures at a rate of
(7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
Hawley, S. A. Biochemistry 1971, 10, 2436. Heremans, K. Annu. ReV. Biophys. Bioeng. 1982, 11, 1. Balny, C.; Masson, P. Food ReV. Int. 1993, 9, 611. Kunugi, S. Prog. Polym. Sci. 1993, 18, 805. Douzals, J. P.; Perrier-Cornet, J. M.; Coquille, J. C.; Gervais P. J. Agric. Food Chem. 2001, 49, 873. Rubens, P.; Snauwaert, J.; Heremans, K.; Stute, R. Carbohydr. Polym. 1999, 39, 231. Rubens, P.; Heremans, K. Biopolymers 2000, 54, 524. Smeller, L. Biochim. Biophys. Acta 2002, 1592, 11. Weber, H. D. G.; Drickamer, Q. ReV. Biophys. 1983, 16, 89. Kaletunc¸ , G.; Breslauer, K. J. J. Therm. Anal. 1996, 47, 1267. Randzio, S. L.; Flis-Kabulska, I.; Grolier, J.-P. E. Macromolecules 2002, 35, 8852. Randzio, S. L.; Flis-Kabulska, I.; Grolier, J.-P. E. Biomacromolecules 2003, 4, 937. Flory, P. J. J. Am. Chem. Soc. 1941, 63, 3083. Cooke, D.; Gidley, M. J. Carbohydr. Res. 1992, 227, 103. Waigh, T. A.; Gidley, M. J.; Komanshek, B. U.; Donald, A. M. Carbohydr. Res. 2000, 328, 165. Shogren, R. L. Carbohydr. Polym. 1992, 19, 83. Schiraldi, A. In Chemical Thermodynamics; Letcher, T. M., Ed.; Blackwell Science: London, 1999; pp 251-264. Le Bail, P.; Bizot, H.; Ollivon, M.; Keller, G.; Bourgaux, C.; Bule´on, A. Biopolymers 1999, 50, 99. Randzio, S. L. Chem. Soc. ReV. 1996, 25, 383. Randzio, S. L. Thermochim. Acta 1997, 300, 29. Randzio, S. L. Annu. Rep. Prog. Chem., Sect. C 1998, 94, 433. Randzio, S. L.; Grolier, J.-P. E.; Quint, J. R. ReV. Sci. Instrum. 1994, 65, 960. Bridgman, P. W. Phys. ReV. 1914, 3, 153. Ehrenfest, P. Proc. K. Ned. Akad. Wet. 1933, 36, 153.
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