1. IntroductionHydration and dehydration reaction processes

of many organic and inorganic compounds ac-company phase changes, and X-ray diffraction(XRD) is a very powerful technique for studyingthe reaction processes. Thermal analysis is an-other important technique often used to obtaininformation needed for understanding the reac-tion processes. Commercially available instru-ments for XRD and thermal analysis had beendeveloped separately, and an XRD instrument iscommonly used only for XRD analysis and aTG-DTA (thermogravimetry-differential thermalanalysis) instrument for thermal analysis. Acomparison between XRD data and thermalanalysis data will provide a deep understandingof the hydration and dehydration reactionprocesses. However, these reaction processesare generally kinematical, depending on theirthermal history and environmental factor suchas humidity, as will be shown later in the resultsand discussion section [1, 2]. Therefore, it is offundamental importance to measure XRD dataand thermal analysis data at the same time andunder the same environmental conditions, inparticular, when the humidity is involved as oneof the environmental factors in the reactionprocesses.

Fawcett et al. reported the construction of aninstrument for simultaneous measurements of

XRD data and differential scanning calorimetry(DSC) data under the same temperature and at-mospheric conditions. The instrument wasequipped with a position sensitive proportionalcounter (PSPC) for rapid scanning of whole pat-terns during elevation or falling of the tempera-ture, and it was used for studying performancesof polymers and catalysis [1, 3]. Other instru-ments, developed for XRD-DSC studies, wereequipped with a rotating anode generator and ascintillation counter [4] or a combined systemof synchrotron radiation with PSPC [5].

Recently, we developed a new XRD-DSC in-strument, as a commercially available labora-tory system, by assembling a DSC unit on astandard XRD goniometer in order to achieve adesign concept of reliable and simple in opera-tion [6, 7]. A humidity control unit could also beequipped with the present system, and it can beused for studying hydration and dehydrationprocesses of organic and inorganic materialssuch as pharmaceutical compounds, some ofwhich are unstable under high humidity condi-tions. In this paper, special features of the instrument are described, and examples onstudying hydration, dehydration and rehydra-tion processes of trehalose dihydrate under var-ious humidity conditions are presented.

THE RIGAKU JOURNALVOL. 21 / NO. 1 / 2004, 25–30

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SIMULTANEOUS MEASUREMENTS OF X-RAY DIFFRACTION(XRD) AND DIFFERENTIAL SCANNING CALORIMETRY (DSC)DATA UNDER CONTROLLED HUMIDITY CONDITION:INSTRUMENTATION AND APPLICATION TO STUDIES ON HYDRATION, DEHYDRATION, AND REHYDRATIONPROCESSES OF PHARMACEUTICAL COMPOUNDS

A. KISHI AND H. TORAYA

Application Center, Rigaku Corporation, Matsubara, Akishima, Tokyo 196-8666, Japan and X-ray Research Laboratory, Rigaku Corporation, Matsubara, Akishima, Tokyo 196-8666, Japan

An instrument for the simultaneous measurement of X-ray powder diffraction (XRD) dataand differential scanning calorimetry (DSC) data under a controlled humidity condition hasbeen developed in order to achieve a design concept of reliable and simple in operation. Exam-ples of the application to studies on hydration, dehydration, and rehydration processes ofpharmaceutical compounds under various humidity conditions are presented. The usefulnessof the present XRD-DSC system will be demonstrated.

2. Instrumentation

2.1. XRD SystemFigure 1 shows the Rigaku/XRD-DSC system,

consisting of (A) a sealed X-ray tube, (B) a go-niometer system, (C) a scintillation counter forXRD measurement, (D) a heat-flux DSC unit atthe sample position, (E) a DSC control circuit,and (F) a humidity control unit. In order to keepthe specimen surface horizontal during a DSCmeasurement, one of the Rigaku q–q goniome-ter systems (ex., ULTIMA, ULTIMA III andTTRAX) is used. The goniometer system canuse either the Bragg-Brentano focusing or a parallel-beam geometry. The parallel-beamoptics, consisting of a parabolic multiple-layermirror for collimating the incident beam and aparallel- slit analyzer on the diffracted beamside, is free from a specimen-displacementerror, and, therefore, it has an advantage overto the parafocusing system [8]. This advantageof the parallel-beam optics is of great impor-tance when peak positions are significantly dis-placed by the thermal expansion of the speci-men material at high temperatures.

A rotating-anode (18 kW) can be attached, in-stead of a sealed-tube (2 kW), onto the q arm onthe incident-beam side, when the goniometersystem TTRAX is chosen. A measurable 2qranges is from 2 to 55° when the DSC unit ismounted, and typical scan rates are 0.5° to5°/min and 10° to 40°/min for slow and fastscanning, respectively. The diffractometer sys-tem can be used also as a conventional typeXRD system if the DSC unit is replaced with anormal type specimen attachment.

2.2. DSC UnitA schematic diagram of a DSC unit is shown

in Figure 2. Two aluminum specimen containerswith dimensions of 7�7�0.25 mm3 are set sideby side: one is used for the XRD measurementand the other one is used as a reference mater-ial for the DSC measurement. These containersare surrounded with a cylindrical-shaped fur-nace made of silver. Two windows are openedfor incident and diffracted X-rays, and they arecovered with thin aluminum foils to preventheat convection and heat loss. The furnace isplaced inside of a ceramic thermal insulatorwith two openings, and it is further surroundedwith an aluminum container (Figure 1). Twowindow of the outermost container are alsocovered with aluminum thin foils.

The specimens, kept stationary during themeasurement, can be heated in the temperatureranges from room temperature (RT) to 350°C at

a heating/cooling rate of 0.5 to 10°C. Any com-binations of heating/holding/cooling stages caneasily be programmed on a PC, and they can becycled. The specimen can also be cooled fromRT to �40°C if a low temperature attachment isused. In this case, the DSC unit can be cooledby the heat conduction of cold nitrogen gas, va-porized from liquid nitrogen.

2.3. Humidity Control UnitIn the humidity control unit, nitrogen gas,

coming into the unit, is divided into twobranches, and two mass-flow controllers con-trol their flow rates. The nitrogen gas in one ofthe branches is introduced into a water bath toproduce water vapor. The two nitrogen gases,one is dry and the other one of high humid, arejoined together, and then they are introducedinto the DSC unit. Signals of temperature andhumidity, which are detected with sensors lo-cated near the specimen, are feed-backed tocontrol mass-flow controllers, keeping presetvalues of the temperature and humidity. The

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Fig. 1. Rigaku/XRD-DSC system. A) X-ray sealedtube, B) goniometer (ULTIMA), C) scintillationcounter, D) DSC unit, E) DSC control unit and F) hu-midity control unit.

Fig. 2. Schematic diagram of a DSC unit.

humidity generator can provide wet gas in thehumidity range from 5% relative humidity (RH)at 25°C to 90% RH at 60°C (from 0.16% to 17.7%H2O–N2.). The humidity control unit is availableas an optional component.

2.4. Functions for Data OutputAs will be shown later, multiple XRD patterns

can be plotted as a function of temperature. In-tegrated intensities of some selected reflectionsin the XRD pattern can also be plotted as thefunction of temperature. They can be replacedwith peak height intensities or full width at half-maximum (FWHM).

3. Experimental

3.1. SpecimenPowder of commercially available trehalose

dihydrate (Sigma, Lot No. 111K377) was usedThe powder specimens, each of which has aweight of approximately 10mg, were mountedon aluminum specimen containers describedearlier, and their surfaces were gently made flat.The specimen used in the present experimentwas identified as virtually pure trehalose dihy-drate by a preliminary XRD measurement.

3.2. XRD-DSC MeasurementsThe XRD-DSC system, used in this study, con-

sists of a goniometer of Rigaku/ULTIMA, a DSCunit based on Rigaku/ThermoPlus DSC8230 anda humidity control unit of Rigaku/Hum-1A.

XRD experimental conditions were as follows:CuKa radiation generated at 40 kV and 50 mA, afixed-angle divergence slit of 0.5°, an anti-scat-ter slit of 0.5°, a receiving slit of 0.3 mm, agraphite monochromator, and a scintillationcounter. Profile intensities in the 2q-range of 5–

40° were step-scanned at a scanning speed of20–40°/min.

In order to supplement the experiment, theTG analysis was conducted with Rigaku differ-ential thermobalance Model ThermoPlus 8210.

4. Results and Discussion

4.1. Dehydrations of Trehalose Dihydrate underDry Nitrogen Gas Flow

The dehydration process of trehalose dihy-drate was first observed under dry nitrogen gasflow. The heating rate was 3°C/min and the XRDscan speed was 20°/min, and thus the tempera-ture was raised by about 6°C during the obser-vation of one XRD pattern. In Figure 3, powderdiffraction patterns observed during the dehy-dration process are plotted as a function of tem-perature. Figure 4 shows variations of inte-grated intensities of selected X-ray reflections inFigure 3 and a variation of the DSC curve, plot-ted on the same temperature axis as that usedin Figure 3. A clear correlation between XRDand DSC data can easily been seen in Figure 4.

Structural changes of trehalose dihydratewith increasing temperature can be divided intofive stages. No appreciable change was first ob-served in trehalose dihydrate up to the temper-ature of 63°C (stage I). As the temperature wasfurther increased, an endothermic peak was ob-served on the DSC curve, and the intensities ofXRD peaks of trehalose dihydrate gradually de-creased. Along with these variations, a new in-termediate phase appeared together with asmall amount of amorphous phase as repre-sented as the presence of halo in the diffractionpatterns, and intensities of the new phase wereincreased. At the temperature of �89°C, which

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Fig. 3. Powder diffraction patterns observed during the dehydration process of trehalose di-hydrate plotted as a function of temperature. Numbers represent (I) dihydrate, (II) anhydrate,(III) amorphous, (IV) anhydrate 2, and (V) liquid.

is just after the observation of the endothermicpeak maximum on the DSC curve, the XRDpeaks of dihydrate disappeared, whereas the in-tensities of the new intermediate phase reachesalmost the maximum. From a supplemental ob-servation of the TG curve, this event was identi-fied as the dehydration of trehalose dihydrateand the formation of anhydrate (stage II). Aftera small endothermic DSC peak at 120–133°C,this intermediate anhydrate phase changes intoan amorphous phase, as revealed by the XRDpattern (stage III). Around an exothermic DSCpeak region of 175–200°C this amorphous phasewere further changed into a crystalline anhy-drate, as shown by the XRD patterns (stage IV).Finally, it melts at 205–218°C, indicated by theendothermic DSC peak and the loss of crys-talline peaks in the XRD pattern (stage V). Thetotal time for the XRD�DSC measurement wasabout an hour.

4.2. Dehydrations of Trehalose Dihydrateunder Highly Humid Nitrogen Gas Flow

The dehydration behavior of trehalose dihy-drate has dramatically been changed when theenvironmental atmosphere was switched fromdry nitrogen gas to highly humid nitrogen gas.In this experiment, the nitrogen gas had a par-tial water vapor pressure of 12.0 kPa (equivalentto 77% RH at 55°C), and its temperature wasraised at a heating rate of 2°C/min. XRD pat-terns were recorded at a scan speed of 40°/min,corresponding to the elevation of temperatureby 2°C during the observation of one XRD pat-tern.

Figures 5 and 6 show XRD and DSC patterns,

presented as those shown in Figures 3 and 4.As demonstrated in these diagrams, dehydra-tion began at 80°C, and it was followed by theformation of a well-crystallized anhydrate phaseat about 95°C (stage II). In contrast to the dehy-dration process in the case of dry nitrogen gasflow, the well-crystallized anhydrate trehalose,which is identical to that which appeared above170°C in Figure 3, was formed even at a temper-ature as low as 100°C. Two small endothermicpeaks appeared on the DSC curve at 115–128°C,but no significant change was observed in XRDpatterns. A broad DSC profile in the tempera-ture range below melting point suggested apossible progress of the decomposition of theanhydrate phase, as observed as the decreasein XRD intensities. Variations of the DSC curveand the XRD intensities in Figure 6 show thatwell crystallized anhydrate phase was formed inthe process of dehydration and decomposednear the melting temperature.

It may be very interesting to note that the dehydration of trehalose dihydrate in highlyhumid atmosphere derived well-crystallized an-hydrate phases at lower temperatures thanthose in a dry or low humid atmosphere.

4.3. Dehydration and Rehydration of Treha-lose Dihydrate

In this experiment, trehalose dihydrate wasfirst heated up to 101°C, and then cooled downnear RT under the dry nitrogen gas flow. In thesucceeding stage, the specimen was kept at36°C and low humid nitrogen gas, having ini-tially a partial water vapor pressure of 1.7 kPa(29% RH at 36°), was introduced. After 180 min

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Fig. 4. Variations of integrated intensities of some selected reflections in Figure 3 and theDSC curve plotted as a function of temperature.

from the start of the experiment, the humiditywas slightly increased to 2.2 kPa.

As was demonstrated in Figure 7, XRD resultsshowed that trehalose dihydrate was first dehy-drated under the dry nitrogen gas flow at the el-evated temperature. The anhydrate phase oftrehalose (stage II), thus produced, was, how-ever, rehydrated following the introduction oflow humid nitrogen gas (stage III). On the DSCcurve in Figure 8, two exothermic peaks wereobserved, and they appeared when (1) the lowhumid nitrogen gas of 1.7 kPa was first intro-duced and (2) the humidity was then elevated to2.2 kPa. Correspondingly to these exothermicpeaks, XRD intensities of the dihydrate phase

were rapidly increased, indicating that the in-crease of humidity accelerates the formation ofcrystalline dihydrate.

5. SummaryAn instrument for the simultaneous measure-

ment of XRD and DSC data under a controlledhumidity condition has been developed as acommercially available system. It was designedin order to achieve both reliable and simple inoperation. Experimental results of the dehydra-tion and rehydration process of trehalose dihy-drate are presented as examples of applicationof the present XRD-DSC system. Phase changesof the trehalose dihydrate are kinematical,

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Fig. 5. Powder diffraction patterns observed during the dehydration process of trehalose di-hydrate plotted as a function of temperature. Numbers represent (I) dihydrate, (II) anhydrate,(III) liquid.

Fig. 6. Variations of integrated intensities of some selected reflections in Figure 5 and theDSC curve plotted as a function of temperature.

greatly depending on the environmental condi-tions of temperature and humidity, and useful-ness of the XRD-DSC system are summarizedas follows: (1) XRD and thermal data can simultaneously be measured under the sameenvironmental conditions of temperature andhumidity; (2) XRD and DSC data can comple-ment each other for understanding reactionprocesses; (3) reaction processes can be ob-served under controlled humidity conditionsand it is especially very useful for the studies onhydration and dehydration processes; and (4)specimens in a very small amount of �10 mgcan be measured within 1 or 2 h during one rununder temperature and humidity variations.

References

[ 1 ] T. G. Fawcett, C. E. Crowder, L. F. Whiting, J. C. Tou,W. F. Scott, R. A. Newman, W. C. Harris, F. J. Knoll andV. I. Caldecourt, Adv. X-ray Anal., 28 (1985) 227–232.

[ 2 ] H. Yoshida, Bunseki, 6 (2000) 336–341 (in Japanese). [ 3 ] R. A. Newman, J. A. Blazy, T. G. Fawcett, L. F. Whiting

and R. A. Stowe, Adv. X-ray Anal., 30 (1987) 493–502.[ 4 ] H. Yoshida, R. Kinoshita and Y. Teramoto, Ther-

mochim. Acta, 264 (1993) 173–183.[ 5 ] I. Hatta, H. Takahashi, S. Matuoka and Y. Amemiya,

Thermochim. Acta, 253 (1995) 149–154.[ 6 ] T. Arii, A. Kishi and Y. Kobayashi, Thermochim. Acta,

325 (1999)151–156. [ 7 ] A. Kishi, M. Otsuka and Y. Matsuda, Colloids Surfaces

B: Biointerfaces, 25 (2002) 281–291.[ 8 ] J. B. Hastings, W. Thomlinson and D. E. Cox, J. Appl.

Crystallogr. 17, (1984) 85–95.

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Fig. 7. Powder diffraction patterns observed during the dehydration-rehydration process oftrehalose dihydrate plotted as a function of temperature. Numbers represent (I) dihydrate, (II)anhydrate, (III) dihydrate (initial state) and (III�) dihydrate (final state).

Fig. 8. Variations of integrated intensities of some selected reflections in Figure 7 and theDSC curve plotted as a function of temperature.