commentary: considerations in the measurement of …...g values, conventional and modulated...

Commentary

Commentary: Considerations in the Measurement of Glass TransitionTemperatures of Pharmaceutical Amorphous Solids

Ann Newman1,3 and George Zografi2

Received 7 October 2019; accepted 7 October 2019; published online 17 December 2019

Abstract. An increased interest in using amorphous solid forms in pharmaceuticalapplications to increase solubility, dissolution, and bioavailability has generated a need forbetter characterization of key properties, such as the glass transition (Tg) temperature.Although many laboratories measure and report this value, the details around thesemeasurements are often vague or misunderstood. In this article, we attempt to highlightand compare various aspects of the two most common methods used to measurepharmaceutical Tg values, conventional and modulated differential scanning calorimetry(DSC). Issues that directly impact the Tg, such as instrumental parameters, samplepreparation methods, data analysis, and “wet” vs. “dry” measurements, are discussed.

KEY WORDS: glass transition temperature; wet Tg; dry Tg; glass; differential scanning calorimetry.

INTRODUCTION

The Amorphous State and Glass Transition Temperature

In recent years, there has been an increased interest inutilizing the amorphous solid form of active pharmaceuticalingredients (API) to overcome the poor aqueous solubility,dissolution, and bioavailability of many correspondingcrystalline forms being developed as solid oral dosage

forms. Such amorphous API often are combined withamorphous small molecule coformers (1) or polymers (2) toform “miscible” mixtures, which can inhibit solid-state andsolution-mediated crystallization during handling, storage,and administration, thus providing greater supersaturationin aqueous solution relative to its crystalline form. As such, itis extremely important to understand the principles underly-ing the structural, thermodynamic, and kinetic properties ofindividual API and excipient molecules, and their mixtures, inthe amorphous state, and to provide physical measurementsthat can characterize a particular amorphous system (3).

From a thermodynamic perspective, we can begin byexamining in Fig. 1 a schematic representation of the freeenergy vs. temperature profile associated with a typicalorganic crystal and its corresponding liquid (4). Here, weobserve that the free energy of the crystal decreases as thetemperature increases until it reaches the melting tempera-ture (Tm). At this point, the free energy of the liquid andcrystal are equal and the phase transition from crystal toliquid takes place until only the liquid exists above Tm. If wenow slowly decrease the temperature of the liquid phase,under conditions that allow time for crystal nucleation andgrowth to occur, we see that the crystalline state should returnat and below Tm. However, if we decrease temperature belowTm, at cooling rates that do not allow time for crystalnucleation, the free energy of the liquid will increase withoutthe discontinuity ordinarily reflective of some type of phasechange. Thus, the super-cooled liquid formed retains theequilibrium properties of the liquid below Tm. However, asalso depicted in Fig. 1, the “super-cooled” material maintainsthe equilibrium properties of the liquid as temperaturedecreases only until an abrupt discontinuity occurs at atemperature defined as the “glass transition temperature”

1 Seventh Street Development Group, PO Box 251, Kure Beach,North Carolina, USA.

2University of Wisconsin-Madison, Madison, Wisconsin, USA.3 To whom correspondence should be addressed. (e–mail:[email protected])

Abbreviations: AAPS, Amorphous-amorphous phase separation;AFM, Atomic force microscopy; API, Active pharmaceutical ingre-dient; ASD, Amorphous solid dispersion; Cp, Heat capacity; DSC,Differential scanning calorimetry; DDSC, Derivative differentialscanning calorimetry; DMA, Dynamic mechanical analysis; H,Enthalpy relaxation; HPMC, Hydroxypropylmethylcellulose; IGC,Inverse gas chromatography; kJ/mol, Kilojoule/mol; mDSC, Modu-lated differential scanning calorimetry; mg, Miligram; nm, Nanome-ter; η, Structural viscosity; Pas, Pascal seconds; PECH, Poly(epichlorohydrin); PMMA, Poly (methylmethacrylate); PVP, Polyvi-nyl (pyrrolidone); PVP/VA, Polyvinyl (pyrrolidone)/vinyl acetate,copovidone; RH, Relative humidity; SFM, Scanning force micros-copy; SSNMR, Solid-state nuclear magnetic resonance; Tf, Onsettemperature; Tg, Glass transition; Tg′, Glass transition due to freeze-concentrated solution containing ice; Tg1, Glass transition of compo-nent 1; Tg2, Glass transition of component 2; Ti, Inflection temper-ature; Tm, Melting temperature; Tgmix, Glass transition of mixture;TSC, Thermally stimulated current; Tx, Crossover temperature; w1,Weight fraction of component 1; w2, Weight fraction of component 2;XRPD, X-ray powder diffraction.

AAPS PharmSciTech (2020) 21: 26DOI: 10.1208/s12249-019-1562-1

1530-9932/20/0100-0001/0 # 2019 The Author(s)

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(Tg). Thus, below this temperature the system is in athermodynamically unstable “glassy” state, with greater freeenergy relative to both the super-cooled liquid and crystal.This increased free energy leads to not only greater apparentsolubility (supersaturation) than expected for the crystal butalso to an increased thermodynamic potential for crystalliza-tion and loss of this solubility advantage. It is also thermody-namically likely that molecules in the glassy state underappropriate conditions can lose free energy and approach thesuper-cooled equilibrium state without crystallizing. This isthe basis for the well-described tendency of glasses toundergo “physical aging” below Tg (5). Thus, it appearsimportant to know whether the operating temperature forvarious pharmaceutical processes is above or below Tg, i.e.,whether one is dealing with the super-cooled equilibriumliquid or the glassy state.

To further understand the underlying molecular basis forthe “glass transition,” it should be recognized that whiledecreasing temperature increases the free energy of thesystem, the structural viscosity (η) of the system, reflectiveof translational and rotational diffusivity, increases signifi-cantly between Tm and Tg, i.e., from about 10−2 Pa at Tm to1012 Pa at Tg (6). In addition, studies of the moleculardiffusivity of these systems above Tg, have revealed atemperature, Tx, the “crossover temperature” at which theviscosity of the super-cooled liquid begins to change fromabout 102 Pa at Tx to 1012 Pa at Tg (6). It is at thistemperature, and below, that significant changes in thestructure of the super-cooled liquid occur because of theinitiation of changes in the arrangements and interactions ofmolecules, which produce a highly viscous “pre-glassy” state.Once at Tg, the equilibrium super-cooled liquid state cannotbe maintained as cooling occurs and viscosity increases, andthe thermodynamically unstable glassy state is formed. Thus,the glass transition temperature is not a thermodynamicparameter, but rather, it is a reflection of the diffusivedynamics associated with the changing viscosity as

temperature decreases, caused by significant structuralchanges. Therefore, it is not surprising that small differencesin values of Tg determined experimentally at different coolingrates, or by different methods, are often obtained. Forexample, as a rough “rule of thumb,” a one-order change inthe cooling rate produces a 3–5 K change in Tg (7). Inaddition to rapid cooling of liquid, very similar amorphousforms, but often with slightly different Tg values, can beobtained by rapid cooling of molecules in the vapor state,rapid precipitation from solution, mechanical milling of acrystalline sample, and dehydration of some crystal hydrates(8). Consequently, when reporting a value for Tg, it isimportant to report the exact conditions under which theamorphous solid was formed and the conditions by which Tg

is measured. One additional important reason for knowingthe Tg of any amorphous solid is that it provides a basis for apriori estimation of the temperatures at which variousimportant physical events occur. For example, it has beenshown that spontaneous crystallization and physical aging,can be inhibited for a period of years i.e., ~ 108 s (900 days) byreducing the temperature to about 50°C below Tg (9). Inaddition, it has been established that for most amorphousorganic materials and polymers, Tx is ~ 1.2 Tg (6), and formany organic molecules, Tm is ~ 1.5 Tg (10). Since, it is at thistemperature that there is a major change in the heat capacityand thermal expansion coefficient of the system, thermalanalytical techniques, such as differential scanning calorime-try (DSC) and dynamic mechanical analysis (DMA), areuseful tools for determining Tg experimentally (3).

Glass Transition Temperature of Binary Amorphous Mixtures

As mentioned above, amorphous APIs are often mixedwith amorphous excipients as part of pharnmaceutical dosageforms. If the two components are completely immiscible and,therefore, phase separated, we would expect the mixture toexhibit two Tg values each equal to the individual compo-nents (11,12). In many cases, small molecule or polymericexcipients can be processed to produce a “miscible” mixture,very much as what one might expect from two miscible liquidsof relatively similar chemical structure. Here, we generallyobserve a single Tg value dependent on the API-excipientcomposition and intermediate to the two individual values(11). There are situations where in immiscible systems each ofthe components have Tg values which are too close to bedetected separately, i.e., within about 10°C, or because thedomain size of the separated phases are less than 30–100 nmand not detected by thermal techniques such as DSC (11). Insuch cases, evidence for the presence of such phase separateddomains of individual components has been obtained usingX-ray powder diffraction (XRPD) (11) and solid-state nuclearmagnetic resonance (SSNMR) spectroscopy (13).

In general, it is known that solutions of two liquids willform if the free energy of mixing is negative. Since theentropy of such mixing is generally positive, leading to anegative free energy, the enthalpy of mixing, arising fromintermolecular interactions, will determine whether themixture is phase separated, or is thermodynamically close toideal (a regular solution), or non-ideal. Larger enthalpies ofmixing, either positive or negative, generally lead to signifi-cant non-idealities, and in terms of amorphous solids, we

Fig. 1. Free energy vs. temperature of a molecule reflecting differentequilibrium and non-equilibrium states (4)

26 of 13 AAPS PharmSciTech (2020) 21: 26

would expect the value of Tg for a mixture relative to theindividual Tg values, would reflect such thermodynamicdifferences. For example, it has been been shown that theTgmix of an ideal solution will be directly related to theindividual Tg values (Tg1, Tg2) weighted by the weightfraction (w) of each component (w1, w2) as shown in Eq.(1), the Gordon-Taylor equation (14). Here, temperaturemust be expressed in Kelvin units.

Tgmix ¼ w1Tg1 þ w2Tg2� �

= w1 þKw2f g ð1Þ

where, the constant K is,

K ¼ ρ1Tg1� �

= ρ2Tg2� � ð2Þ

Equation (1) has also been derived by Couchman andKaracz (15) on a thermodynamic basis, where,

K ¼ ΔCp2=ΔCp1� � ð3Þ

and, ΔCp is the heat capacity change for each componentat its Tg.

In some cases as with a variety of polymer blends, thedensities of the individual compents are close to being equal,so that Eq. (1) can now be written as the Fox equation (16),

1=Tgmix� � ¼ w1=Tg1

� �þ w2=Tg2� �� ð4Þ

This equation is useful as an approximate “back-of-the-envelope” prediction of Tgmix only knowing the weightfraction and Tg of each component.

Generally speaking, if the chemical structures of theindividual components are reasonably close, we would expectclose to ideal conditions and experimental agreement with theapplication of Eqs. (1) and (2), as for example, that observedin Fig. 2, for mixtures of indomethacin and PVP/VA(polyvinyl (pyrrolidone)/vinyl acetate, copovidone) (Fig. 2)(17). In those cases where intermolecular interaction betweenthe individual components themselves is greater than thatbetween the two different molecules, we can expect asignificant deviation from ideality and experimental valuesof Tgmix that are less than predicted by Eq. (1). This type ofbehavior, illustrated in Fig. 3, has been noted with very polarmolecules, such as sucrose, when mixed with PVP, because ofthe very strong hydrogen bonds that exist between the sugarmolecules (18). On the other hand, as shown in Fig. 4, whenintermolecular interactions between the components arequite strong relative to bonding of the components withthemselves, experimental values of Tgmix will be greater thanpredicted. This, indeed, has been reported for amorphousmixtures of an organic API salt and various grades of PVP(19).

Effcts of Absorbed Water on the Glass TransitionTemperature

It is well established that amorphous solid forms ofmolecules containing hydrogen bond donors and acceptors

can absorb significant amounts of water from the vapor stateinto the bulk phase as a function of relative humidity andtemperature (20,21). As such, water molecules essentiallydissolve within the glass or super-cooled liquid to form a“solution,” where water molecules break intermolecularhydrogen bonds between the molecules in the solid and formhydrogen bonds with these molecules. Based on the discus-sion above, it is not surprising that such absorption of waterand the formation of a miscible solution within the amor-phous form should influence the structure and thermodynam-ics of the system so as to produce a distinct change in suchproperties as structural viscosity, free volume, and the glasstransition temperature. As observed from the parameters inEqs. (1) and (4), the very low Tg of water, 136 K, shouldgreatly reduce the “dry” Tg of an amorphous solid. Indeed, asshown in Fig. 5, for a PVP-water system, there is a significantreduction in Tgmix with increasing water content (22).Consider, also, as an example given in Fig. 6, the Tgmix ofan indomethacin-water miscible mixture, produced by expos-ing amorphous indomethacin to various relative humidities(RH) at 30°C (23). Note that as little as 0.01, weight fraction

Fig. 2. Glass transition temperatures of miscible indomethacin- PVP/VA samples (17):solid line from the Gordon-Taylor equation (14),dotted line from Couchman-Karasz equation (15)

Fig. 3. Glass transition temperature of sucrose-PVP amorphous soliddispersions (18); solid line expected from the Gordon-Taylorequation (14)

of 13 26AAPS PharmSciTech (2020) 21: 26

of water reduces the dry Tg from 315 to about 296 K, belowroom temperature. In Fig. 6, we include a plot of datapredicted for an ideal mixture by the application of Eqs. (1)and (2) and data obtained experimentally at various watercontents. With each plot, we give the K constant calculatedfor ideal mixing from Eqs. (1) and (2), and the K constantobtained by allowing it to be an arbitrary fitting constantconsistent with the experimental data. Clearly, the absorptionof water into indomethacin has a much greater “plasticizingeffect, i.e., lower Tgmix, than would be predicted for an idealmixture. Indeed, this suggests that clustering of watermolecules occurs within the relatively nonpolar indomethacinmatrix, resulting in a large increase in free volume and muchmore efficient plasticization than might be expected (23).

Similarly, one would expect water absorbed into amiscible amorphous mixture of API and coformer to reducethe Tgmix of the binary system, in a manner described above.In such cases therefore, we would expect the absorbed waterto produce an increase in molecular mobilty sufficient to bringabout physical change unless the operating temperature issignificantly less than the resulting Tgmix, e.g., about 50°Cbelow Tgmix. Depending on the strength of the interactionbetween API and coformer in relation to Tgmix and the extentof increased molecular mobility caused by absorbed watermolecules, a number of scenarios are possible, including:separation into drug-rich and polymer-rich amorphous phasesfollowed by crystallization of drug, or crystallization of drugdirectly from the miscible dispersion (24,25).

Thus, it is apparent that in studying the properties ofamorphous solids it is very important to know exactly whatthe relationship is between water content and Tg, and howthis might change various physical properties. Such under-standing will be enhanced if, as a routine, accurate Tg valuesare obtained and an appropriate Tg vs. water content profileis experimentally determined.

TG MEASUREMENTS FOR AMORPHOUS SOLIDS

Techniques Available for Measuring Tg

One of the most common techniques for measuring Tg isDSC. A modification of this method is modulated DSC(mDSC), which can be more sensitive than conventionalDSC. Several other analytical methods have been used toinvestigate Tg values, and some of these are summarized inTable I. Minimally, these techniques are not as common asDSC but are becoming more integrated into pharmaceuticallaboratories. These methods can be used as confirmation ofthe value obtained by DSC or as a secondary method if there

Fig. 4. Glass transition temperature of amorphous solid dispersionsof MK-0591 with different molecular weight grades of poly(vinylpyr-rolidone) (19); dashed lines represent ideal mixing determined by theGordon-Taylor equation (14)

Fig. 5. Glass transition temperature of poly(vinylpyrrolidone) as afunction of absorbed water content (22)

Fig. 6. Glass transition temperature of amorphous indomethacin as afunction of water content (23); K = 0.33 represents ideal mixing fromthe Gordon-Taylor equation (14), K = 0.11 represents non-idealempirical fit to the Gordon-Taylor equation using experimental data

AAPS PharmSciTech (2020) 21: 2626 of 13

are issues that make the DSC method difficult or impractical,such as thermal degradation or a lack of sensitivity.

In one comparative study, four methods (DSC, DMA,TSC, and dilatometry) were used to measure the Tg ofchitosan (28). The sample preparation and hygroscopicity ofthe material made analysis difficult, and Tg values rangingfrom 150 to 203°C had been reported in the literature. In thiscontrolled study, the four techniques resulted in Tg valuesranging from 140 to 150°C, showing good overall agreementfor this compound. For some applications, a range of 10°Cmay be acceptable, but other applications may need moreprecise Tg values, such as determining physical stabilityconditions and handling conditions when values of Tg − 50are close to ambient conditions.

This paper will concentrate on issues to be consideredwhen measuring Tg values of pharmaceutical samples usingconventional and modulated DSC. A number of instrumentaland analysis parameters need to be considered when mea-suring and reporting a Tg value and these are discussed inmore detail in the following sections. Before discussingamorphous systems containing water, we will examine thegeneral process of determining Tg by thermal analysis.

Experimental Tg Values Obtained with DSC

The glass transition temperature is considered thetemperature where an amorphous solid undergoes an appar-ent second-order transition defined as a step change in theheat capacity (ΔCp) as a function of temperature andobserved as a baseline shift (Fig. 7a). This shift representsthe change from a glass to a supercooled liquid upon heating(as represented in Fig. 1). There are numerous ways to reportthe Tg with the most common being the onset and inflectiontemperature (Fig. 7a). It is important to understand whichtemperature is being reported, since they can vary by severaldegrees, with significant impact on use and storageconditions.

While a classic Tg signal is represented in Fig. 7a, a smallendotherm may also be observed for a glass transition, asshown in Fig. 7b. The small endotherm represents enthalpicrelaxation (ΔH) due to aging or relaxation of the amorphoussample. The enthalpic relaxation endothermic transition willincrease as the sample continues to age/relax over time (33).It is possible to minimize/eliminate this endotherm byremoving the thermal history of the sample. This involves

heating the sample past the Tg, cooling the sample, andreheating the sample past the Tg (34).

Glass transition temperatures can be used to evaluatethe miscibility of amorphous mixtures, such amorphoussolid dispersions (ASDs) or polymer blends. When twoamorphous materials are miscible, one Tg will usually beobserved in the DSC curve, whereas when the samplesare immiscible, two Tg values will usually be observed(11). The width of the Tg region can also provideinformation about the extent of miscibility, i.e. broad Tg

transitions indicate tendencies towards immiscibility. Anexample with miscible polymer blends is given in Table II;as the amount of poly (methylmethacrylate) (PMMA)increases, the Tg width increases from 26 to 100°C,indicating less miscibility in samples with more PMMA(35). The width was measured by taking the derivative ofthe Tg and measuring the onset and final temperature ofthe derivative peak. Again, it is important to know if theonset or inflection point is being reported when the Tg

signals are very wide. In the case of the 30/70 PECH poly(epichlorohydrin)/PMMA sample, for example, a differ-ence of up to 50°C would be reported for the Tg onset orinflection temperature.

Table I. Analytical Methods to Measure Glass TransitionTemperatures

Technique Reference

Scanning force microscopy (SFM) (26)Atomic force microscopy (AFM) (27)Dynamic mechanical thermal analysis (DMA) (28–30)Thermally stimulated current spectroscopy (TSC) (28)Dilatometry (28)Water diffusion (30)Density (30)Relative humidity (RH) (31)Inverse gas chromatography (IGC) (32)

Fig. 7. a Glass transition in DSC showing onset temperature (Tf) andmidpoint (inflection) temperature (Ti). b Glass transition temperatureshowing enthalpy relaxation endotherm (ΔH) (3)

AAPS PharmSciTech (2020) 21: 26 of 13 26

Tg measurements and relaxation enthalpy can be used tostudy more complex properties of amorphous materials, suchas molecular mobility (4,9,36) and fragility (37). Collecting Tg

values at different heating rates was also used to calculate the“true” Tg of corn starch containing different amounts ofwater; this value was the extrapolated Tg obtained uponregression and represents Tg when the heating rate is slowand approaching 0°C/min (38).

Conventional DSC

Conventional DSC instruments measure the heat flow ofa sample compared with a reference when both samples areheated with the same controlled temperature program(39,40). Instruments are calibrated with compounds havingaccurately known melting points and heats of fusion. Acommon standard used for pharmaceutical applications isindium (melting point 156.6°C, enthalpy of fusion 3.25 kJ/mol). It should be noted that instruments need to becalibrated whenever a different scan rate is employed toensure that accurate values for temperature and heats ofreaction are obtained.

Glass transitions are generally low in energy and can bedifficult to see under routine conditions in a conventionalDSC. Increasing the scan (heating) rate will typically improvesensitivity and enhance the appearance of Tg because the flowof energy increases over a shorter period when using a fasterscan rate (41–43). This is demonstrated in Fig. 8 foramorphous lactose (43) and glassy felodipine (41) wherefaster scan rates result in larger signals. Increasing the scanrate will also significantly increase the value of the Tg

temperature, as illustrated with felodipine, which shows adifference of up to 12°C when the heating rate is increased.This example demonstrates the importance of knowing theheating rate when comparing Tg values since significantdifferences can be observed.

Thermal events detected by conventional DSC can resultbecause of overlapping transitions, such as Tg overlappingwith enthalpic relaxation, desolvation, or crystallizationtransitions. Generally, it can be difficult or impossible toseparate or eliminate the overlap using conventional DSC,such as preheating the sample to remove solvent or thermalhistory, without changing the sample that is being analyzed.

Modulated DSC

The second technique commonly used for Tg measure-ments is mDSC (44,45). It is a technique that uses a

combination of a conventional DSC linear heating with atemperature modulation superimposed over the heating rate.The result of this temperature modulation is that the heatingrate is no longer constant, but will change in a modulatedmanner. This allows multiple heating rates to be measuredsimultaneously, which, in turn, increases resolution andsensitivity, as well as providing a direct measurement of heatcapacity (45). It also allows deconvolution of the data intoreversing and non-reversing components of thermal events.Reversing transitions include Tg, heat capacity, and melting,while non-reversing events include enthalpic relaxation,desolvation, crystallization, and decomposition. The mDSCplot obtained will contain three curves, including both thereversing and non-reversing curve, as well as a conventionalcurve, which is similar to that obtained with conventionalDSC conditions (Fig. 9).

Most of the mDSC calibration procedures (baselinecorrection, temperature scale, and sensitivity) are the sameas those for conventional DSC using a standard such as

Table II. Tg Width for PECH/PMMA Polymer Blends (35)

PECH/PMMAa Tg width (°C)

100/0 2085/15 2670/30 6550/50 8030/70 1000/100 40

PECH, poly(epichlorohydrin); PMMA, poly (methylmethacrylate)

Fig. 8. Variation of Tg with heating rate for a amorphous lactoseexhibiting Tg (adapted from (43)) and b glassy felodipine withenthalpic relaxation; heating rate: (a) 10.0; (b) 20.0; (c) 40.0; and(d) 80.0 K/min (adapted from (41))


indium. An additional calibration for heat capacity is requiredfor mDSC, and sapphire is commonly used for this step. Thiscalibration should be performed using the same modulationprogram used for analysis (42). For sensitive measurements, itis also important to have sample and reference pans that arethe same initial weight to minimize background heat capac-ities (47), and additional weight corrections may be neededfor hermetically sealed pans (48).

Several parameters need to be optimized for the mDSCrun, including heating rate, modulation amplitude, modula-tion period, and sample size (45,49). Slow heating rates,ranging from 0.1 to 5°C, are commonly used for mDSC,resulting in longer run times compared with conventionalDSC. As observed with conventional DSC, a faster scan rateresults in more sensitivity (Fig. 10a). It is suggested that thereshould be at least five modulation cycles during the transition,therefore the width of the Tg will impact the modulationamplitude chosen. Modulation periods of 20–80 s areacceptable for many samples, and, as shown in Fig. 10b, themodulation period will influence the sensitivity of themeasurement. Sample sizes of 2–20 mg are commonly testedto find the optimal weight for the analysis (45). It should benoted that all parameters need to be optimized for the samplebeing analyzed (50). A standard set of conditions will notwork for all materials, and artifacts due to improperdeconvolution can emerge in the reversing and non-reversing curves when the wrong parameters are used. Theseartifacts could lead to misinterpretation of the data, and,ultimately, affect the use and stability of the amorphoussample during development. Additional information onsetting instrumental parameters will be specific to the mDSCequipment being used and can be obtained from themanufacturer.

It has been suggested that “mDSC should be viewed as acomplimentary approach to conventional DSC, rather than areplacement. Rather, the best approach for characterizingnew materials is to start with conventional DSC and thenswitch to mDSC if its advantages are required” (45).Collecting the conventional DSC curve under appropriateconditions for Tg values can also provide useful informationwhen determining mDSC parameters, such as approximate

temperature range and transition width, while the mDSC canprovide more sensitivity and additional data (such as the heatcapacity) if needed for the system being studied.

Wet vs. Dry Tg Measurements

In this section, some of the unique issues that arise whenattempting to measure the Tg values of an amorphous systemwhen it contains a specific amount of absorbed water will beexamined. It is important to understand the type of datarequired when designing the thermal experiments, and, toassure that the water content in the sample is maintainedduring the measurement. As summarized in the “INTRO-DUCTION,” amorphous materials can contain water thatmay significantly decrease the Tg of the sample. Collectingdata with water contained in the sample is described as a“wet” Tg. In some cases, a Tg without the water is needed,and this is commonly called a “dry” Tg. It is important tounderstand the type of data needed in order to pick theappropriate sample preparation and instrumental conditions.

Wet Tg measurements can be collected using conven-tional or modulated DSC instruments. There are numerousexamples in the literature demonstrating the effect of wateron the wet Tg of amorphous samples, excipients, anddispersions (22,38,51–54). For wet Tg measurements, thesample pan is hermetically sealed to retain the water in thesample. Crimped, pinhole, and open pans should not be usedto determine wet Tg values. It is important that a truehermetic seal is obtained, since any break in the seal will leadto water loss and an inaccurate Tg temperature. If significantwater is contained in the sample and a large sample size isused, pressure buildup in the pan may be an issue, leading todeformed pans, sample leakage, and reduced contact with thecell, which can result in poor reproducibility and potentialartifacts in the data (43). It has been reported that theintegrity of the seal can be an issue above approximately150°C (55). Smaller sample sizes can help reduce pressurebuildup and its issues when using routine pans or commercialhigh-pressure pans (~ 150 Bar) are available from instrumentvendors. An easy test to confirm that the seal is intact is toweigh the sample after the run and confirm that no change inweight has occurred when compared with the initial sampleweight (55). Small sample sizes in a hermetically sealed panscan also cause issues. It has been reported, for example, thatwater loss into the headspace can result in a dehydrationendotherm when using hermetically sealed pans with smallamounts of hydroxypropylmethylcellulose (HPMC) films.Weighing the pan after the run showed no loss in weight,indicating the water was retained in the pan (56). Note thatthe dehydration temperature might be slightly higher due tothe more confined area in the DSC pan. Increasing thesample size in the pan, using an inverted lid, or using asmaller pan will minimize or prevent this from occurring.Other considerations, such as contact of the sample with thesample pan and baseline corrections, may also be needed forcertain compounds (57).

For dry Tg measurements using a conventional DSC, thesample can be dried before the analysis or during the run. If itis dried before the analysis, it is important to understand howexposure to ambient conditions during DSC sample prepara-tion may change the water content of the sample, and this

Fig. 9. Modulated DSC curves for amorphous saquinavir, showingthe reversing, non-reversing, and total heat flow. Note the Tg in thereversing heat flow curve is separated from the enthalpic relaxationevident in the non-reversing curve (46)


preparation method is not recommended for hygroscopicsamples. A second method to collect dry Tg values is to use anopen pan, collect data at ~ 20°C/min to a temperature abovethe Tg (to erase thermal history and remove water orvolatiles), quench cool to 50°C below the Tg, hold for about10 min, and then reheat through the Tg (34). A third cyclethrough the Tg is recommended. If the Tg is reproducible inthe second and third cycles, then the transition is confirmed asa Tg. If different values are obtained in the second and thirdcycles, then the transition may not be a true Tg and othercharacterization of the sample may be needed. The use of adry purge gas, such as nitrogen, in the DSC instrument shouldmaintain a dry environment throughout the heat/cool process.

An open pan can also be used to collect dry Tg

measurements using mDSC. Crimped or pinhole pans willallow egress of water out of the pan, but the rate ofdehydration will not always be consistent, due to the variableextent of crimping or size of a manual pinhole in the lid, andthis inconsistency could significantly affect the thermal curvesobserved (49,58). If mDSC parameters are chosen correctly,the Tg obtained should be separated from the water loss, withthe Tg in the reversing curve and the dehydration in the non-reversing curve, as shown in Fig. 11. Multiple cycles withmDSC have also been reported to remove thermal history(46), and multiple mDSC heating cycles could also be used toconfirm dehydration and the dry Tg.

Fig. 10. a Effect of scan rate on the reversible heat flow of polystyrene; amplitude 1°C overa period of 60 s with a scan rate of (a) 2°C/min, (b) 3.5°C/min, and (c) 5°C/min. b Effect ofmodulation period on the reversible heat flow of a polystyrene sample; scan rate of5°C/min, amplitude 2°C, and a period of (a) 45 s, (b) 60 s, and (c) 100 s (50)


CASE STUDIES

Amorphous Citric Acid

A study with citric acid, a common excipient used inlyophilization and oral dosage forms, was performed toinvestigate the wet and dry Tg of the amorphous material(60). Conventional DSC data were collected, using scan ratesof 10 K/min, and additional studies were performed usingscan rates of 5–40 K/min. Amorphous samples were made byheating and quenching the crystalline materials in pinhole(anhydrate) or hermetically sealed (monohydrate) pans andsubsequently measuring the Tg. KF values of the crystallinematerials resulted in less than 0.05% water in the anhydrateand 8.6% water in the monohydrate.

The Tg value measured for the dry citric acid was 11°C,while the wet citric acid (containing 8.6% water) wassignificantly lower at − 25°C (Fig. 12a). An additionalexperiment was performed with the monohydrate in apinhole pan, which resulted in Tg and ΔCp values similar tothe dry amorphous citric acid material, indicating that thepinhole pan resulted in loss of water during the DSCexperiment. The low Tg values explained the difficultiesencountered trying to produce and maintain these amorphousmaterials when made by traditional methods, such as quenchmelting on a larger scale. The addition of water sorbed by theamorphous material under ambient conditions, therebylowering the Tg even further, added to the stability issues.The Gordon-Taylor equation was used to calculate the Tg formixtures of citric acid and water. Excellent agreement wasfound between the calculated and experimental values. Thisanalysis allowed accurate estimations for the Tg of amorphouscitric acid water mixtures for water contents up to 8.6%water.

The DSC curve for a 10% (w/v) citric acid aqueousfrozen solution (Fig. 12b) exhibited two thermal eventsbetween − 90 and − 40°C. The lower temperature is attributedto the Tg of a freeze-concentrated solution with high watercontent but without ice, and the higher transition was due to a

freeze-concentrated solution containing ice and is representedas Tg′. For successful freeze drying, the sublimation of iceshould be at or below the Tg′. In this case, the Tg′ of citricacid solutions was substantially lower than − 45°C, the lowesttemperature that could be attained with their laboratoryequipment, therefore, producing amorphous citric acid bylyophilization without crystallization was not successful.

Immiscibility in Amorphous Solid Dispersions with Water

The phase behavior of amorphous solid dispersionscontaining the hydrophilic polymer PVP and various hydro-phobic drugs (nifedipine, indomethacin, ketoprofen,droperidol, and pimozide) was investigated after exposureto elevated RH conditions at room temperature (24). Sampleswere analyzed using DSC and infrared (IR) spectroscopy todetermine miscibility. For all initial ASD samples, analysisconfirmed complete miscibility by DSC (one Tg) and IR(specific drug-polymer interactions).

Storage at elevated RH conditions (75–94% RH)resulted in two different phenomena. Two dispersions(indomethacin-PVP and ketoprofen-PVP) were found tomaintain miscibility after water exposure. The other threesystems were found to separate into immiscible drug-richand polymer rich amorphous phases, in a process calledamorphous-amorphous phase separation (AAPS). Asshown in Fig. 13a, for the pimozide-PVP system, the dryTg of the amorphous pimozide was reported as 60°C (dryPVP K12 was reported around 112°C). The DSC curvesclearly showed one Tg initially (94°C) for the ASD, whiletwo Tg values were evident after exposure at 94% RH for42 h (− 21 and 44°C). The marked decrease in both Tg

values, compared with the initial Tg values for the individualcomponents, was due to the water (22.4%) absorbed by theASD. Upon drying the samples (after RH exposure), two Tg

values are still evident, but are now reported at highertemperatures (61 and 112°C). The sample remains amixture, but the Tg values of the individual componentsincrease due to the water lost upon drying.

Fig. 11. A polymeric drug substance showing the loss of water in the broad non-reversibleendotherm and the underlying Tg in the reversible curve (59)


The IR spectra confirmed the separation of thepimozide-PVP system. After RH exposure the relativeintensity of the peak at 1709 cm−1 (assigned to free carbonylmoiety of the drug) was found to decrease, which suggestedan increase in the drug-drug hydrogen bonding (Fig. 13b). Asimilar decrease was observed in the intensity of the peak at1661 cm−1, which was assigned to the carbonyl group of PVPwhen it is hydrogen bonded to drug molecules. Theseobservations were consistent with phase separation of thesystem into drug- and polymer-rich amorphous regions andconfirmed the DSC results.

Physical stability and crystallization were also studied forthe various ASDs. Drug crystallization in systems exhibitingAAPS was found to occur earlier (< 6 days at 94% RH) whencompared with systems that remain miscible (> 46 days at94% RH). Evidence of water-induced phase separation wasobserved after storage at RHs as low as 54% for thepimozide-PVP system. It was evident that the drug

crystallization was much faster following the AAPS, sincethe crystallization inhibitory influence of the polymer wouldbe reduced. These studies highlight one issue with acceleratedstability testing at high RH conditions, which may not berepresentative of physical stability that could be achieved atlower RH or with protective packaging to limit exposure towater.

CONCLUSIONS

As with any solid-state characterization method, it isimportant to understand the instrumental parameters andsample preparation methods used to collect Tg data. Thechoice of conventional vs. modulated DSC instruments willbe dependent on the amorphous system being analyzed, and,while mDSC can help improve sensitivity and separateoverlapping transitions, it may not be the best choice for allsamples. Other parameters, such as sample pan configuration

Fig. 12. DSC curves for a dry (anhydrous) and wet (monohydrate) amorphous citric acidsamples showing the Tg values for each sample; b 10% (w/v) citric acid aqueous frozensolution. Insert shows two thermal events between − 90 and − 40°C. The derivative DSC(DDSC) curve clearly shows two transitions (60)


Fig. 13. Analysis of pimozide-PVP ASDs before and after RH exposure and subsequentdrying a Tg values before and after exposure to 94% RH with and without drying. The Tg

of pure amorphous pimozide (top) is included for comparison. b IR spectra of the carbonylregions for pimozide-PVP ASD. A reduction in the intensity of peaks assigned to the freepimozide carbonyl (2709 cm−1) and PVP carbonyl when hydrogen bonded to pimozide(1661 cm−1) was observed as the storage RH was increased and the resulting spectra werefound to be more similar to the calculated spectrum of the physical mixture composed ofamorphous drug and PVP (24)


and scan rate, can drastically change the value and should bespecified for all reported Tg values. When collecting data, it isimportant to understand if the effect of water is required, andparameters should be chosen to optimize the “wet” Tg value.When reporting “wet” Tg values, it is important to provideaccurate water values. When reporting dry Tg values, it isimperative to remove all water in the samples to provide anaccurate value.

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