Structural Evolution of the Dihydrate to Anhydrate Crystalline

Jan 23, 2009 - Wide-angle X-ray scattering measurements were performed to record structural changes during the transition from trehalose dihydrate to ...
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J. Phys. Chem. B 2009, 113, 2201–2206

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Structural Evolution of the Dihydrate to Anhydrate Crystalline Transition of Trehalose as Measured by Wide-angle X-ray Scattering Duncan Kilburn* and Paul E. Sokol Indiana UniVersity Cyclotron Facility, 2401 Milo B. Sampson Lane, Bloomington, Indiana 47404 ReceiVed: August 29, 2008; ReVised Manuscript ReceiVed: December 8, 2008

Wide-angle X-ray scattering measurements were performed to record structural changes during the transition from trehalose dihydrate to crystalline anhydrous R-trehalose. The results show that large dihydrate crystals rearrange into smaller sized R crystals; from the peak widths we calculate a crystallite size of typically ∼40 trehalose molecules. We find that the dehydration probably takes place in a two-step process with different time scales for both the water removal step and the molecule rearrangement step. This suggests that there is crystal rearrangement in the dry state some 60 °C below the dry glass transition temperature of trehalose, which is unusual for a relatively large and strongly interacting molecule. 1. Introduction The influence of temperature and water content on the structure of bulk trehalose-water matrix has been investigated extensively with various probes over the last couple of decades. This is in large part because of the obvious connection these two variables have with what is arguably trehalose’s most intriguing property: the ability to preserve the viability of biological matter after desiccation and rehydration. It has been reported many times that some organisms synthesize trehalose to allow them to survive periods of extreme drought. Although trehalose is by no means unique in this regard, a significant number of experiments have focused on it as opposed to other disaccharides. This is probably because of suggestions that it was the most effective.1 The bioprotectant effect has been ascribed broadly to the ability of trehalose to form a dry frame that is relatively immobile and stabilizes the biological components when dehydrated. In the literature, two hypotheses are often presented to account for this stabilization: (1) the water replacement hypothesis whereby trehalose hydrogen bonds to the cellular components, thus directly replacing water molecules; (2) the vitrification hypothesis whereby the trehalose forms a dehydrated amorphous solid significantly below its glass transition temperature (110 °C). These effects are not mutually exclusive and in fact are probably both important in making trehalose an effective stabilizer of cells. For an overview of the development of these ideas see refs 2-6. In common with other saccharides, amorphous trehalose is strongly plasticized by water. When stored at humidities of approximately 40% amorphous trehalose readily absorbs water and forms a dihydrate crystal. At lower humidity it probably forms a local crystal structure. In fact, the local formation of hydrated crystal necessarily removes water from the surrounding amorphous medium, hence making the surrounding amorphous trehalose less mobile and more structurally stable. This has been discussed as a possible mechanism to allow trehalose to remain structurally sound even under gently changing humidity levels. Local crystalline regions form and act as sinks for water, thus * Corresponding author. Phone: (410) 516 6776; e-mail: [email protected]. Current address: T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland, 21218-2685.

Figure 1. Schematic diagram of the transitions between phases of trehalose due to water addition/removal and temperature change. This diagram represents a broad overview of the accepted phases but is not claimed to be definitive. Subtle variations in heating rate and water activity result in more complex phase relationships. There is much ongoing discussion with respect to these specifics.

the water in the rest of the trehalose is regulated at a low level.1 The easy and reversible transport of water into this crystal via channels in the trehalose frame has been suggested to be of fundamental importance to the anhydrobiosis.7 Upon gentle drying from solution, trehalose forms a stable dihydrate crystal. This crystal can be dehydrated in several ways, leading to different end products. For example, an amorphous, anhydrous trehalose is formed if the dihydrate is heated rapidly to above about 110 °C under vacuum or dry nitrogen. If, however, the dihydrate is gently heated at 50 °C under vacuum, the water is removed, leaving behind a morphologically different crystal structure, called R-trehalose. A schematic view of the hydrated/anhydrous phases of trehalose is shown in Figure 1. The path along the top of the diagram is the focus of this work. The gentle drying of solution forms a dihydrate, which upon further gentle drying forms R-trehalose. Anhydrous amorphous trehalose can be formed from any of these phases: the solution can be freeze-dried; the dihydrate crystal can be heated rapidly to high temperatures; and the R-trehalose crystal can be melted. The direct conversion of dihydrate crystal to anhydrous amorphous form is particularly sensitive to the temperature of dehydration. It is thought that if it is heated quickly the dihydrate can melt, forming a hydrated amorphous form, which then dehydrates leaving an amorphous

10.1021/jp807704n CCC: $40.75  2009 American Chemical Society Published on Web 01/23/2009

2202 J. Phys. Chem. B, Vol. 113, No. 7, 2009 anhydrate. Incomplete melting, or partial dehydration to the R form before the melting occurs can result in an anhydrous solid with a mixture of amorphous and R-crystal structure. In addition, it is worth noting that heating the anhydrous amorphous form above approximately 160-170 °C,8,9 depending on heating rate and conditions, results in the stable anhydrous β-trehalose crystal. Unlike R-trehalose, this does not absorb water from the atmosphere. In fact, careful experimentation using a diverse array of techniques has revealed many subtleties in the full phase diagram of trehalose. Many of these subtleties, it is claimed, play an integral role in explaining the effectiveness of trehalose as a biopreservant. The most frequently reported techniques are wideangle X-ray scattering (WAXS), differential scanning calorimetry (DSC), or a combination of the two.1-17 These are augmented by combustion calorimetry,18 Fourier transform Raman spectroscopy,19 optical microscopy,20 dielectric relaxation spectroscopy,1 and positron annihilation lifetime spectroscopy,9 as well as others. Each study seems to uncover a new subtlety in the phase diagram. The complexity of the interaction of this molecule with water and itself means that for a full molecular understanding of the transformations and the resultant phases one must account for thermodynamic, topological, and dynamic informationsremembering, always, that these three are interdependent. The importance and versatility of water molecules in biological-type settings, as reviewed by Chaplin,22 seems to be well demonstrated in this relatively simple system. Indeed, despite the uncertainty specifically concerning the extent of trehalose’s importance as a bioprotectant, the complexity of its phases and their relation to water, temperature, and time make trehalose an ideal model system for studying these relations in biological matter. The work described in this study is focused on the gentle dehydration of the dihydrate crystal, which forms an anhydrate crystalline state. This is an extreme version of the dehydration that is likely to go on in the natural state. The dehydration in this experiment occurs at moderate temperatures and low (effectively zero) humidity. Pure dihydrate crystal is dehydrated; the aim was to be able to accurately picture or model the dynamics of water passing through a strongly interacting biological matrix. We are particularly interested in the interplay of dehydration and crystallization; why does the act of removing the water under these conditions necessarily leave the R form, and is the formation of the R form locally simultaneous with the removal of water? Previous work indicated that this simultaneity is indeed a feature of the transition.12 This is reasonable as it is well-known that mobility in such systems is strongly related to water content. At similar temperatures the amorphous trehalose anhydrate is well below its glass transition. Experimental Details 2.1. Materials. R-R-Trehalose dihydrate (R-D-glucopyranosyl, R-D-glucopyranoside) with a purity of over 99% was purchased from Sigma Aldrich. It was used without further purification. The crystals were ground into a powder for the scattering measurements; observation under a microscope confirmed that the powder had a maximum grain size of ∼200 µm, which is comparable to those used in a previous study.21 2.2. X-ray Scattering. The X-ray diffraction patterns were obtained with an RU-200 X-ray generator using a Copper rotating anode source operated at 40 kV and 80 mA. A standard θ-2θ scattering geometry was employed. A graphite monochromator on the scattered beam was used to select the unresolved Cu KR with wavelength 1.541 Å. The powder sample

Kilburn and Sokol was loaded into a quartz capillary tube with diameter 1 mm. The capillary was sealed via a rubber O-ring to a “fill” line that could either be open to atmosphere or under vacuum (less than ∼0.5 mbar). The capillary containing the sample was slotted into a tight-fitting hole drilled through the central axis of a steel cylinder. This steel cylinder then acted as a heat sink, and its temperature was controlled by water flow from a heat bath. A thermocouple embedded in the cylinder was used to confirm excellent thermal conduction between the flowing water and the sample holder. A slice the height of the X-ray beam (1.5 cm) was cut out of the cylinder at the required angles to allow the X-ray beam to illuminate the capillary at the center of the cylinder and for those scattered X-rays to have a clear path to the detector. We believe that the above steps lead to precise control of the sample’s temperature and atmosphere. 2.3. Experimental Procedure. Because of the sensitivity of the phase of trehalose to humidity, temperature, and time, it is important to be as precise as possible in the description of the experimental procedure. It is a relatively simple procedure, but we describe it explicitly in stepwise fashion for clarity. The trehalose as delivered was ground gently using a mortar and pestle to try to achieve a better powder sample. A gentle grinding was used, stopping with a maximum grain size of approximately 200 µm. We did not grind further due to concerns about inducing amorphization;23 we bear this in mind although it is worth pointing out that that amorphization is extremely unlikely given that we ground gently for a couple of minutes, whereas that described in ref 23 occurred in a ball mill over (for full amorphization) 23 h. All experiments reported in this paper used trehalose from the same grinding batch. The powder was loaded into the capillary tube and, after recording the mass of the sample and capillary tube, diffraction patterns were taken with the sample at room temperature (22 °C) and humidity (>25%, high enough to remain dihydrate). The vacuum and heating were activated simultaneously with the start of the X-ray data collection. Heating to 50 °C took less than 20 min as measured on the sample container (also acting as heat reservoir). This is completed within the first two angle sweeps for the X-ray data collection. After the full phase transition had been measured we recorded the mass of the sample and capillary tube again. This last step was to confirm a 9.5% mass loss, corresponding to the loss of two water molecules per trehalose, and therefore the complete dehydration of the dihydrate. This also acted as a post hoc confirmation that we had a fully hydrated dihydrate to begin with. Results and Discussion Diffraction patterns of trehalose dihydrate and the anhydrous form of trehalose prepared by slow dehydration, the R form, are shown in Figure 2. The spectra are similar to previously reported patterns.11,14,16 To obtain the patterns in Figure 2 six scans were taken, with the sample being rotated around its central axis by 0.2° after each scan. It is notable that the peak heights vary significantly between sample orientations for the dihydrate but do not for R-trehalose, suggesting that for the R form we have what can be considered a perfect powder sample, but the dihydrate cannot be considered a perfect powder. This has been observed before11,16 and indicates that the dihydrate grains contain large single crystals that are large enough to have their preferred orientation reflected in the diffraction pattern. We see no evidence for reduced grain size upon dehydration, so the loss of a preferred direction for R-trehalose must come about by rearrangements within the grains themselves. One other

Structural Evolution of Trehalose

Figure 2. X-ray scattering patterns for trehalose dihydrate (lower spectrum) and sample fully dehydrated at 50 °C (upper spectrum). Characteristic peaks are marked.

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Figure 4. Evolution of the peak at ∼16° as trehalose dihydrate dehydrates and becomes the R anhydrate form.

batch they have identical grain size distributions. A measure of the crystallite size can be determined using the Scherrer formula:24

D)

Figure 3. Evolution of peak at ∼23.8° as trehalose dihydrate dehydrates and becomes the R anhydrate form.

striking feature of the peak transformations that occur between the spectra in Figure 2 is the general broadening of the diffraction peaks. Again, this observation has precedent and is caused either by a strained crystal phase or a reduction in the spatial extent of the crystallites.12 A reduction in mean crystallite size would provide an explanation for the above observation of powder peak height invariance for the R form if the new, smaller, crystallites formed had random orientations. We assume that the extent of dihydrate-like crystal can be characterized by the height of the peak at 23.8°. In so doing, we are regarding this peak as being a δ-function broadened by resolution. Figure 3 shows the peak height decreasing as a function of time for a sample heated at 50 °C under vacuum. This plot actually uses the sum of two experimental runs, each normalized prior to summing, both of which are peak heights achieved by averaging over 20 sample angles. This is necessary because, as noted above, the dihydrate crystal’s diffraction pattern shows a preferred orientation. The preferred orientation appears to change during dehydration as rearrangements occur and possibly some grains dehydrate before others. We observe that, for a single sample angle, the peak at 23.8 ° does not simply monotonically decrease. Even averaged in the way described, it can be seen in Figure 3 that there are fluctuations outside that expected from statistical error. The important conclusion from Figure 3 (and the individual plots from which it is constructed) is that the dihydrate structure, and therefore the water, is gone after about 6 h. This is similar to the time scale previously observed for grain sizes of commensurate size under similar heating and vacuum conditions.21 Much shorter dehydration times have been reported in the past12 at the same temperature, but the grain size was not reported in this instance. It is well-known that particle size has a significant effect on dehydration kinetics,20 so this may be the origin of the discrepancy. The question presents itself: what is the crystallite size reached in the R phase, and is it the same in all cases, given a constant grain size? First, in relation to the starting grain and crystallite size, we assume that as all samples come from the same grinding

Kλ β cos θ

(1)

where D is the mean size of the crystallite, λ is the scattering X-ray’s wavelength, β is a measure of the line width broadening, θ is the scattering angle, and K is the so-called shape factor and depends critically on the shape of crystallite assumed. One of the experimental runs is shown in Figure 4. The data shown in Figure 4 are collected between 15 and 17° with a step size of 0.02° and a collection time of 8s at each point, giving a complete angle sweep time of 13 m 28s. This figure shows in more detail the time dependence of the change of the characteristic features described above, namely: (1) the intensity of the peak centered in the region of 15.9° increases. This simply indicates the growth of the R-phase crystallite fraction; (2) the full width at half-maximum (fwhm) of the growing peak appears to decrease. This is a less clear observation and requires quantitative confirmation; (3) the background intensity appears to increase. Again, this requires quantitative confirmation. It is noted that the line-width broadening in eq 1 is the linewidth broadening due to the crystallite size only. The instrumental resolution function, g(θ), must be deconvoluted from the measured line shape, h(θ) (shown in Figure 4) in order to be used in eq 1. Because of the complexity of the linewidths measured (we have no instances of both the sample and resolution function both being, for example, Gaussian functions) it was necessary to deconvolute using Fourier transforms (FTs) of the lineshapes. The resolution line shape was determined from the diffraction pattern from a -325 mesh Silicon powder. It has nonsymmetric features that were determined to be due to the sample arrangement; when the powder was held in an isolated capillary tube a Gaussian line shape resolution function was observed. For all spectra there are two steps performed before the deconvolution. The first of these is to calculate the background intensity in the spectrum, assumed to be the lowest intensity measured in an individual angle sweep, and subtract this value from the intensity at all angles. The background value is related to an amorphous scattering component and does increase as the transformation to the R form progresses. The second step is to examine the spectra for rogue spikes of data and then remove these. For some of the spectra during the initial stages of transformation it was found that the peak shown at 16.9° in Figure 2 encroached on the angle range to be analyzed (typically 15.3-16.4°). For the data presented in this paper it was always in the tails of the desired peak (>3σ from the mean value). The

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Figure 5. Peak height at ∼16° as a function of time for dihydrate f R anhydrate dehydration at 50 °C for several powder samples.

Figure 6. The fwhm values at ∼16° as a function of time for dihydrate f R anhydrate dehydration at 50 °C for several powder samples.

few spectra where it substantially obscured the peak were discarded and are not presented here. A process of apodization25 with a carefully chosen function smoothed out the undesired intensity without affecting the bulk of the main peak to be analyzed. In each case this condition was confirmed visually. The deconvolution was performed in the usual manner.24 The FT of the measured line shape, H(ζ), was divided by the FT of the (measured) resolution function, G(ζ), to give the FT of the “pure” diffraction line shape, F(ζ) ) H(ζ)/G(ζ). In principle, the only subsequent step is to inverse FT F(ζ), to obtain the “pure” line shape f(θ). In practice, however, it is common to remove some of the higher frequency terms in H(ζ) and/or G(ζ) as they describe noise in the data for smoothly varying functions. We retained the lowest 7 or 9 frequency components out of a total of typically 60 components (as a discrete FT was performed, the exact number of components depended on the angular range analyzed). This is justifiable because our interest in this paper is to calculate the fwhm of the resultant diffraction peak, and it was found empirically that removing these components does not alter the FWHMs calculated, it merely reduces their associated error. It was confirmed by comparison with peaks reconstructed using higher numbers of components that the final number used here represented the width of the peak accurately while reducing the statistical noise reproduced from the high-frequency components. We found that seven components was usually sufficient for the initial stages of the transformation with broader peaks but that nine were required later for the narrower peaks as the R phase reached completion. The fwhm was then calculated from the deconvoluted peaks. The resultant data are shown in Figures 5 and 6. Figure 5 shows the peak height of the R-trehalose peak centered at ∼16°, and Figure 6 shows the “pure” fwhm (postdeconvolution) of the same peak, both as a function of time. The first thing to notice is that the transformation does not occur over the same time scale, despite the dehydration conditions being identical for all these runs: 50 °C, vacuum on, as detailed above. This is most obvious in Figure 5 where, for example, run 7 reaches a constant peak height after ∼3.5 h (this has also the lowest final peak height), and run 3 requires 12.5 h to reach a constant

Kilburn and Sokol

Figure 7. Peak area and fwhm at ∼16° as a function of time for dihydrate f R anhydrate dehydration at 50 °C for run 10. The arrows and fractions indicate the percentage of the initial water present in the dihydrate that is left at the indicated time.

height. What is clear from this is that R growth seems to occur even after the water is removed from the dihydrate phase and that this growth stage varies between experiments. This is possibly surprising, but then it should be noted that the dry glass transition temperature of amorphous trehalose is ∼100 °C, and so the system in question here (at least once fully dehydrated) is certainly a nonequilibrium system. The nonreproducibility of this dehydration is also evident in the differing values for the ultimate peak-height for R-trehalose and the final fwhm of these peaks. In fact, the reproducibility is marked only by the angle position of the peak in the final R-trehalose state. The fwhm values as shown in Figure 6 exhibit a large amount of scatter, but it is certainly possible to draw general conclusions. This figure supports the prediction from visual examination of peaks such as those in Figure 4 that the width of these peaks decreases as the height grows. The decrease in width is a result of increasing crystallite size, quantified in eq 1. For the experiments here, the following values are used in eq 1: λ ) 1.541 Å; the peak is at 2θ ) 16°; and we use K ) 0.9, which is the appropriate value when the thickness of a crystallite is sought perpendicular to the reflecting planes, leading to the diffraction peak in question.24 The peak fwhm, β, expressed in radians, can then be used to calculate the crystallite dimension, D. Taking the maximum and minimum limiting values from Figure 6 of 0.6 and 0.25 degrees, crystallite dimensions of 135 and 400 Å, respectively, are calculated. The size of a trehalose molecule can be calculated using the crystallographic data from ref 26. We calculated the modulus of the vector connecting two extremal hydrogen atoms (H(C6) and H(C6′) from Figure 2 in ref 26), derived for trehalose in its dihydrate form. This distance was 9.3 Å, making the peak widths above correspond to on the order of 15 and 43 molecules. The total area under the peak is a metric that can be used to more accurately represent the extent of crystallinity than the peak height. This is shown for run 10 alongside the fwhm for that run in Figure 7. For some of the experimental runs, including this one, the experiment was stopped after 2, 4, and 6 h, and the mass of the sample chamber was measured. In this way the extent of water loss could be determined for these experiments. It is certainly true that in the ∼2 min that the sample was exposed to the air for measurement water could be readsorbed. We assume that only superficial sorption occurs, and this is confirmed by the fact that the peak height continues to increase monotonically with no discernible influence of the nonvacuum exposure. In Figure 7 both the peak area and the time are shown on logarithmic scales; and it can be seen that, at least in the region corresponding to 0-10 h, the plot follows a straight line. It can also be seen that the fwhm of the peak decreases, although there is a significant dip in the data at around

Structural Evolution of Trehalose

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TABLE 1: Fit Parameters Derived from X-ray Scattering Patterns during Trehalose Dihydrate Dehydration and Conversion to r-Trehalose sample 1 2 3 4 5 6 7 8 9 10 11

log (A0)

m

tmin (hr)

tmax (hr)

2.31 2.76 2.89 2.62 3.43 2.74 3.52 2.27 3.36 1.11 2.92

2.17 1.86 1.99 2.33 1.49 3.09 1.37 2.57 1.46 3.32 1.57

1.8 2.5 0.7 3.1 1.1 2.0 0.7 3.4 0.9 2.2 0.2

10.8 8.2 13.0 7.1 3.8 4.5 3.1 8.1 5.4 12.6 8.5

8.5 h, which appears to be an artifact of the deconvolution/ fwhm determination method used. The plots of log(peak height) vs log(time) were fitted to a straight line. This models the crystal growth according to eq 2:

A ) A0tm

(2)

and values for A0 and m are shown in Table 1. Also shown in Table 1 are the time ranges used for fitting, with tmin and tmax being the minima and maxima of the time ranges used in each individual fit. The fits to the above equation for all the samples are all as good as shown in Figure 7, although as indicated by the time ranges in which the fits were performed, the growth described by eq 2 does not always commence at the start of the experiment. It is worth noting that there is no correlation between the mass of trehalose in the experiment and any of the fit parameters. This was an important confirmation that varying masses are irrelevant for the physical processes involved. Another striking feature of Figure 7, which is confirmed by other experiments, is that the extent of R-trehalose, as measured by the area under the peak, is not directly proportional to the amount of water lost. In fact, there seems to be crystalline rearrangement even after all of the water has gone, which corroborates the discrepancy between the times in Figure 3 and Figure 5. Certainly there is no slowing of the transformation as the water content decreases. For example, at the point marked as corresponding to 80% water loss, the R-trehalose peak is only at 11% of its final, maximum value. During another run (11) the peak height was at 46% of its final value when 93% of the water had been lost. As the water fraction is determined from mass differences that are small compared with the total sample-cell arrangement, the error on these levels is high. Nevertheless, this strongly indicates significant crystal rearrangement ∼60 °C below the dry glass transition temperature of trehalose, which is unusual for such a large, strongly interacting molecule. This points to a very specific and local rearrangement, possibly due to the instability created by removing water molecules. This suggests that the conversion to R-trehalose from dihydrate is a two-step process with two associated timescales. It is known from previous experiments that the removal of water leaves a more open structure.21 A mechanism suggests itself whereby the structural transition that occurs in the dry state is facilitated by the open structure. That is, the free volume left behind in a trehalose frame (exactly the same molecular orientations, but without the stabilizing water) when the water molecules are removed allows greater mobility than the dry amorphous glass. The extra free volume was observed previously using positron annihilation lifetime spec-

troscopy. The free volume between molecules was found to be greater for R-trehalose than for dry amorphous trehalose (prepared by freeze-drying and then equilibriating at zero relative humidity for ∼4 weeks).9 Other previous experiments have concluded that this removal is exactly commensurate with R formation,12 the data presented here, however, indicate that this is not the case. Previous in situ X-ray dehydration experiments reported complete dehydration in approximately 2 h.12 Clearly, the mean trehalose grain size will influence this time scale strongly, but Willart et al. do not report this dimension in their paper and so we cannot be sure as to the origin of this discrepancy. What we do know, however, is that a discrepancy exists between the data presented in this paper when we are sure that the experimental procedures are consistent. The exponent m in eq 2 from fits to the peak area date are shown in Table 1. The values are distributed about 2 (max. 3.32; min. 1.37). One mechanism that immediately suggests itself is the seeding of crystallites, followed by growth out from these with a constant rate of crystallization per unit surface area of the crystallite. This mechanism would lead to an exponent of 2, but we find values distributed (outside error limits) around 2. We must repeatedly bear in mind, however, that the dry system is significantly below its glass transition, so normal molecular mobility leading to the formation of layers on a crystallite would be very surprising. It is puzzling that the end points of the dihydrate f R transition seem to vary. The final peak height at ∼16 °, as well as the time to complete this transition, varies between experiments. One explanation is that this is not a conventional phase transition and that the final state, including possible variation in crystallinity, therefore shows variation between different systems. We view this as being consistent with a model in which the water is removed from the dihydrate, and then the resulting structure collapses or rearranges much below its Tg and so is far from equilibrium. In this case we expect the specific rearrangements in a sample to be greatly influenced by specific grain shapes and boundaries. We would expect a repeatable time for crystal growth at a given temperature and pressure. That we do not observe this is potentially a cause for concern regarding the experimental setup. It is therefore worth recapitulating the salient checks and observations necessary for interpreting this data. First, when preparing the samples we know that there is a distribution of grain sizes. It is reasonable to ask whether this will affect the dehydration/crystallization kinetics. As noted above in the description of sample preparation, all samples were taken from the same grinding batch; we therefore assume that they all have identical grain size distributions. We have no direct measurement of the grain sizes in each experiment but note that the dehydration kinetics are likely to be strongly dependent on grain size, with larger grains taking longer to dehydrate than small ones. It is therefore reassuring that the dehydration times were the same in three separate experiments (summarized in Figure 3 as the decrease of a dihydrate crystal peak height). We assume that the initial grains are single crystals but have no independent measure of the initial crystal dimensions other than to observe that the diffraction lines from the dihydrate show no broadening beyond resolution. We also note that, as mentioned before, the diffraction peaks from the dihydrate sample vary in intensity with sample orientation. In a grannular sample this strongly suggests single crystal grains. If each grain consisted of smaller crystallite domains then the preferred orientation would be lost and the diffraction peaks would not vary with sample orientation,

2206 J. Phys. Chem. B, Vol. 113, No. 7, 2009 as in the case of R-trehalose. We recognize that as a conclusion from this work we are unable to identify a single value for the rate of crystal transformation from the dihydrate to the anhydrous R-trehalose. We are, however, able to make perhaps a more important conclusion about the underlying process. We have shown that the dehydration and crystal transformation are not simultaneous, and we have demonstrated that the crystal growth follows a time dependence as would be found for a growing crystal surface. These observations fit into a picture of the dehydration mechanism whereby water leaves trehalose dihydrate following channels in the trehalose structure.7,17,21 That water can then reversibly enter the structure that is left, the R-trehalose, is key in the structurally stabilizing nature of this process. Given that we have described the dehydrated sample as being far from equilibrium, it is worth asking: what is the equilibrium state of dehydrated trehalose at 50 °C? Recent results have shown that in the temperature range 105-117 °C R-trehalose is in a kind of superheated crystal.15 That is, the amorphous form has lower free enthalpy than the R-crystal, but because the glass transition of dehydrated trehalose is at a higher temperature still, vitrification occurs over hundreds of hours. No lower boundry was put on the “real” melting temperature of R-trehalose. It could be, in light of our data, that R-trehalose is not, in a strict sense, an equilibrium phase of dehydrated trehalose but is a meta-stable collapsed form of dehydrated trehalose dihydrate. The real equilibrium state is actually β-trehalose, but this is reached via the amorphous state with extremely slow steps both to vitrify from R, and then crystallize to β. The latter assertion that β-trehalose is the true equilibrium phase is supported by X-ray patterns from ref 15 that show clear nucleation of the β phase after vitrification at 110 °C, which is far below the usually reported “cold-crystallization” temperature of 150-160 °C. It should be emphasized that the latter point is speculative and that further studies are required to establish the real nature of the phase diagram. Conclusion It has been shown in this work that the phase transition induced by the removal of water molecules from trehalose dihydrate is a complex process. Repeated dehydrations under the same conditions do not show the same results from X-ray scattering patterns. From this it is shown that the kinetics of dehydration vary between runs and that although the final crystal structure is consistent, the crystallite sizes also vary. This points to a two-step process with different time scales for both the water removal step and the molecule rearrangement step. What is surprising and particularly interesting about this observation is that a step in this phase transition, mediated by water removal, involves a crystalline phase with greater free volume and mobility than the corresponding amorphous phase. Typically this is not observed for normal crystalline/amorphous materials prepared using pressure and temperature, as opposed to water, as the controlling parameter.

Kilburn and Sokol Trehalose is one of several sugars of interest to the scientific community due to their biopreservant properties. It is clear that understanding the close coupling of water and the sugar molecules and the structural and dynamic properties that follow from this is key in understanding the biopreservation and utilizing it in, for example, drug delivery systems. It is now understood that in most biological settings water is not merely a background solvent but is an active agent. The results presented here indicate that for the case of water and trehalose the intimate interactions are far from being understood and may require a rethink of how best to describe a hydrated molecular phase diagram. Acknowledgment. This work was supported by the Department of Commerce under Grant No. 70NANB5H1163A04. We would like to acknowledge valuable discussions with J. Ubbink. References and Notes (1) Crowe, J. H.; Carpenter, J. F.; Crowe, L. M. Annu. ReV. Physiol. 1998, 60, 73. (2) Crowe, J. H.; Crowe, L. M.; Chapman, D. Science 1984, 223, 701. (3) Crowe, J. H.; Leslie, S. B.; Crowe, L. M. Cryobiology 1994, 31, 355. (4) Crowe, J. H.; Hoekstra, F. A.; Nguyen, K. H. N.; Crowe, L. M. Biochim. Biophys. Acta 1996, 1280, 187. (5) Branca, C.; Magazu`, S.; Maisano, G.; Migliardo, P. J. Chem. Phys. 1999, 111 (1), 281. (6) Clegg, J. S. Comp. Biochem. Physiol. B. 2001, 128, 613. (7) Sussich, F.; Skopec, C; Brady, J.; Cesa`ro, A. Carbohydr. Res. 2001, 334, 165. (8) Sussich, F.; Bortoluzzi, S.; Cesa`ro, A. Thermochim. Acta 2002, 391, 137. (9) Kilburn, D.; Townrow, S.; Meunier, V.; Richardson, R; Alam, A; Ubbink, J. Nat. Mater. 2006, 5, 632. (10) Sussich, F.; Princivalle, F.; Cesa`ro, A. Carbohydr. Res. 1999, 322, 113. (11) Nagase, H.; Endo, T.; Ueda, H.; Nakagaki, M. Carbohydr. Res. 2002, 337, 167. (12) Willart, J. F.; De Gusseme, A.; Hemon, S.; Descamps, M.; Leveiller, F.; Rameau, A. J. Phys. Chem. B 2002, 106, 3365. (13) Willart, J. F.; Dane´de, F.; De Gusseme, A.; Descamps, M.; Neves, C. J. Phys. Chem. B 2003, 107, 11158. (14) Furuki, T.; Kishi, A.; Sakurai, M. Carbohydr. Res. 2005, 340, 429. (15) Willart, J. F.; He´doux, A.; Guinet, Y.; Dane´de, F.; Paccou, L.; Capet, F.; Descamps, M. J. Phys. Chem. B 2006, 110, 11040. (16) Rani, M.; Govindarajan, R.; Surana, R.; Suryanarayanan, R. Pharm. Res. 2006, 23 (10), 2356. (17) Nagase, H.; Ogawa, N; Endo, T.; Shiro, M.; Ueda, H.; Sakurai, M. J. Phys. Chem. B 2008, 112, 9105. (18) Pinto, S. S.; Diogo, H. P.; Moura-Ramos, J. J. J. Chem. Thermodyn. 2006, 38, 1130. (19) Taylor, L. S.; Williams, A. C.; York, P. Pharm. Res. 1998, 15 (8), 1207. (20) Taylor, L. S.; York, P. J. Pharm. Sci. 1998, 87 (3), 347. (21) De Gusseme, A.; Carpentier, L.; Willart, J. F.; Descamps, M. J. Phys. Chem. B 2003, 107, 10879. (22) Chaplin, M. Nature ReV. Mol. Cell Biol. 2006, 7, 861. (23) Willart, J. F.; De Gusseme, A.; Hemon, S.; Odou, G.; Danede, F.; Descamps, M. Sol. State Comm. 2001, 119, 501. (24) Klug, H. P.; Alexander, L. E. X-ray Diffraction Procedures For Polycrystalline and Amorphous Materials, 1st ed; John Wiley and Sons: New York, 1954; Ch 9. (25) Jansson, P. A. (Ed.) DeconVolution with Applications in Spectroscopy; Academic Press, Inc.: London, 1984. (26) Taga, T.; Senma, M.; Osaki, K. Acta Crys. 1972, B28, 3258.

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