Transformation of microstructure and phase of disodium guanosine 5′-monophosphat

Qiao Chen ,Fengxia Zou ,Pengpeng Yang ,Jingwei Zhou ,Jinglan Wu ,Wei Zhuang ,*,Hanjie Ying ,*

1 State Key Laboratory of Materials-Oriented Chemical Engineering,Nanjing Tech University,Nanjing 210009,China

2 College of Biotechnology and Pharmaceutical Engineering,National Engineering Research Center for Biotechnology,Nanjing Tech University,Nanjing 210009,China

3 College of Chemistry and Chemical Engineering,Nantong University,Nantong 226019,China

Keywords:Solid form transformation Thermodynamics Disodium guanosine 5′-monophosphate Solubility Crystallization Intermolecular force

ABSTRACT Microstructure and phase transformation ofdisodiumguanosine 5′-monophosphate(5′-GMPNa2)are extremely important for controlling the process and understanding the mechanism of crystallization.In this work,the thermodynamic properties of polymorphous 5′-GMPNa2 especially the solubility were studied,the solubility results show that 5′-GMPNa2 is more soluble in ethanol-water(E-W)than in isopropanol-water(I-W).The amorphous form of 5′-GMPNa2 is more soluble than the crystalline form at the same mole fraction and temperature.Meanwhile,the crystalline forms and morphologies of the residual solids were characterized by PXRDand SEM.The results indicate that solid forms of 5′-GMPNa2 transformed spontaneously from amorphous to crystalline when the ethanol proportion is≥20%.In addition,increasing the pH facilitates the dissolution of 5′-GMPNa2 and helps to maintain the crystalline form.The associated Gibbs free energy values were calculated to verify the trend of transformation from amorphous to crystalline 5′-GMPNa2.These results should help to guide the industrial crystallization process and to obtain the crystalline form of 5′-GMPNa2.

1.Introduction

Polymorphism exists in most solid chemical compounds.In the amorphous solid form,the molecular arrangement is chaotic and completely different from that in the crystalline form.Different solid forms have varying physical and chemical properties,such as solubility,dissolution rate,melting point,density,compressibility,and hygroscopicity.These characteristics further in fluence the bioavailability,stability,crystallization process,and preservation condition of the compounds[1-3].Therefore,it is essential to investigate properties of all polymorphic forms in detail in order to understand the process of transformation between polymorphs.During polymorphic transformation,metastable forms with high Gibbs free energies transform into stable forms with low Gibbs free energies[4].Hence,the Gibbs energy,although sometimes difficult to obtain,can be used as a criterion for determining the stability ofsolids.The obtained information,in addition to energy of two crystalline forms,can be used to judge the relationship of polymorphic transformation via comparing their solubilities;the metastable form with high solubility transforms into the stable form with low solubility.In general,polymorphic transformations can be classified into monotropic transformations without intersections and enantiotropic transformations with intersections about the energy or the solubility of the polymorphs.There are two different mechanisms for polymorphic transformations:solid-state transition(SST)and solution-mediated transition(SMT).For SSTs,comparing the melting pointand heatoffusion oftwo crystalline forms can allow the polymorphic transformation to be predicted according to the“heat of fusion rule”of Burger and Ramberger[5,6].For SMTs,the system can be effectively studied using slurry experiments and spectroscopic methods[7,8].No matter which method is adopted,it is essential to characterize the crystalline forms[9,10]using techniques such as thermogravimetry(TG),differential scanning calorimetry(DSC),powder X-ray diffraction(PXRD),scanning electron microscopy(SEM),and Raman spectroscopy.

Studies of polymorph transformation can proceed along two directions,namely from dynamic and thermodynamic perspectives.Maher et al.[11]investigated the in fluence of temperature and solid mass loading on the stable phase nucleation rate.Kulkarni et al.[12]found that the bonding of the solute could decide the crystalline form.Kitano et al.[13]studied the transformation of L-citrulline crystals in different solvent proportions.However,until now,there have been few studies on solid form transformations from the thermodynamic viewpoint,even though the thermodynamics of polymorphs is important for further understanding their differences.Furthermore,the thermodynamic information about these polymorphic transformation processes could guide the design of industrial crystallization steps.

As an industrialpurification technique,crystallization relies strongly on accurate information about the thermodynamic equilibrium.Therefore,solubility is generally considered essential for all works regarding crystallization processes[14].While the solubilities of important pharmaceutical and chemical compounds are routinely reported,there are fewer studies on the solubilities of polymorphic compounds.In the literature,Jim and Kim[15]studied the solubility of Forms I and II of clopidogrel hydrogen sulfate in several solvents.Bon filio et al.[16]analyzed the polymorph dissolution properties ofchlorthalidone.Hirai etal.[17]analyzed the dissolution behavior of metastable crystalline or amorphous drugs by mathematical models.

Disodium guanosine 5′-monophosphate(5′-GMPNa2)is one of the four common ribonucleotides that constitute ribonucleic acids(RNAs).It is a product widely used in the food,pharmacological,and health product industries.5′-GMPNa2is an amphoteric compound containing a phosphate group and amine group,and therefore the solvent pH will affect its ionic form and crystallization process.The main solid forms of5′-GMPNa2include one amorphous formand fourcrystalpolymorphs(I,II,III,and IV)[18,19],and both the stable seven hydrous I-form and anhydrous amorphous forms are sold.5′-GMPNa2is usually processed from a fermentation broth using methanol or ethanol as an antisolvent.The amorphous form is readily prepared during crystallization processes with immature processing conditions,while the crystal products tend to be poor quality in general,for example,they often contain small amounts of amorphous 5′-GMPNa2,the crystal water is unstable,and the crystal products easily deteriorate or agglomerate.Severalpapers have reported the solubility of 5′-GMPNa2with different anti-solvents(such as E-W,methanol-water),temperatures,and pH values[20,21].However,these studies did not elaborate upon the polymorph being used.Furthermore,there have been no reports on the solubility of polymorphic 5′-GMPNa2.Zou et al.[22]investigated the nucleation and growth mechanisms of 5′-GMPNa2in anti-solvent/water mixtures,and,more recently,the amorphous-to-crystal transformation of 5′-GMPNa2from a kinetic viewpoint[23].From the point of view of complementary,in this work we examine the transformations between 5′-GMPNa2polymorphs fromthe thermodynamic perspective.

This study used a series ofstructuraland morphological characterization methods(TG-DSC,PXRD,and SEM)to prove that the different solid forms of 5′-GMPNa2possess different thermodynamic properties,structural arrangement,and crystal habits.The solubilities of the amorphous and I forms were studied,which will provide further understanding for the control of the amorphous product in future industrial crystallization processes.The solubilities of the crystalline and amorphous forms of 5′-GMPNa2in binary I-W and E-W solvent mixtures between 298.15-318.15 K and pH=8.5-10.0 were studied.Meanwhile,the residual solids were examined by PXRD and SEM for possible polymorphic transformation.The modified Apelblat equation was used to model the solubility of 5′-GMPNa2.Factors affecting the polymorphic transformation are proposed from the view of intermolecular forces.Furthermore,the Gibbs free energies,dissolution enthalpies,and dissolution entropies of the two polymorphs were calculated and compared.

2.Experimental

2.1.Materials

5′-GMPNa2(mass fraction purity N99%)was manufactured at Nanjing Tongkai Biological Technology Co.(China).Its crystalline form was prepared by recrystallization,where ethanol was slowly dripped into an aqueoussolution of5′-GMPNa2,with supersaturation nucleation and growth gradually occurring.The amorphous form was obtained by pouring excess ethanol into an aqueous solution of 5′-GMPNa2,which caused large quantities of white amorphous solids to precipitate quickly.Both preparation process used ethanol as an anti-solvent(ethanol:water volume ratio=1.6:1)at 303.15 K,and products were filtered and air-dried at 303.15 K.The two solid forms were sifted to 200 mesh(aperture 0.075 mm).Ethanol and isopropanol(analytical research grade,mass fraction purities N99.7%)were provided by Shanghai Shenbo Chemical Co.(China).Sodium hydroxide(mass fraction purity≥99.6%)was purchased from Xinlong Scientific Co.(China).De-ionized water was prepared in the laboratory.

2.2.Characterization methods for solid forms

The polymorphic forms of 5′-GMPNa2were identified using TG,DSC(NETZSCH STA449-F3),and PXRD(Bruker D8,Cu Kαradiation)[24].The morphologies were observed using SEM(HITACHI TM3000).The crystallinities were calculated using JADE 5 analysis software.The concentrations were measured using high-performance liquid chromatography(HPLC)on an Agilent 1200 HPLC system equipped with a Zorbax SBAq C18 column.

2.3.Experimental procedure

The solubility data were obtained by the static method[25].20 ml mixed solution with certain molar fraction of alcohol and water was injected in a 25 ml triangular bottle,and then the triangular bottle was added excess amount of 5′-GMPNa2.The experiments were implemented in water bath under magnetic stirring.Solid-liquid mixing was stirred for at least 4 h with 100 r·min-1.The equilibrium time was determined by measuring the concentration of 5′-GMPNa2every 45 min untila constantconcentration was obtained.Subsequently stirring was stopped and keep still the upper layer was clear,the undissolved 5′-GMPNa2solid about0.5 g is laid atthe bottom ofthe bottle.The residual solids are tiny compared with the whole system,and the solubility experiment(stirring and statics)lasted at least 8 h,so we think there is plenty time to ensure that the system achieves solid-liquid-solid equilibrium.After that,the upper clean portion of a saturated solution(1 ml)was filtered through a 0.22 μm organic film,weighed,diluted,and the concentration was measured.All solubility experiments were conducted in triplicate,and the average values were employed to calculate the mole fraction solubility(x1).The values of x1and the alcohol fraction in the solvent mixture(x2)were defined using Eqs.(1)and(2),respectively.

where m1,m2,and m3represent the masses of 5′-GMPNa2,alcohol,and water,respectively;and M1,M2,M3the corresponding molecular weights.

3.Results and Discussion

3.1.Characterization of solid forms

Fig.1.The raw materials of 5′-GMPNa2 used for the experiments:(a)PXRD patterns;(b)SEM images of crystalline form;(c)SEM images of amorphous form.

Fig.2.TG-DSC curves of the experimental materials of 5′-GMPNa2:(a)crystalline form;(b)amorphous form;↑the upward arrows indicate the endothermic.

The as-synthesized crystalline and amorphous forms of 5′-GMPNa2were analyzed by PXRD,SEM,and TG-DSC,and the results are shown in Figs.1 and 2.The PXRD patterns indicate that crystalline 5′-GMPNa2has obviously different characteristic peaks at 7.9°,12.7°,15.9°,20.4°,22.6°and 37.9°;amorphous 5′-GMPNa2has only one characteristic peak at27.6°and a swellfrom 13°to 20°.This is consistentwith the literature[26,27].The crystallinities of the crystalline and amorphous forms were 95.3%and 3.26%,respectively.The two forms also have differentappearance morphology in the SEM images:the crystalline form appears as regular rectangles,while the amorphous form is constructed from disordered rod-like shapes[27].The TG-DSC results show the different thermodynamic characteristics of the two solid forms.The TG curve of the crystalline form includes four separate sections due to the stepwise loss of crystal water molecules(indicated by broken lines),giving a total water content of 22.71%,which is consistent with literature[28].Meanwhile,the curve of the amorphous form is smoother,with a total water content of 17.53%.From DSC pattern,the different molecular arrangements bring about different heat absorption and release behaviors,the response signal of the endothermic peak of crystalline 5′-GMPNa2is obviously higher than amorphous 5′-GMPNa2,which means that crystalline 5′-GMPNa2is missing crystal water,however amorphous 5′-GMPNa2is missing amorphous water.Combined with the results of PXRD,SEM and TG-DSC the conclusion can be obtained that amorphous 5′-GMPNa2contain little crystalline 5′-GMPNa2.They are two different forms of 5′-GMPNa2.

3.2.In fluences of temperature and solvent on solubility

The modified Apelblat equation,shown in Eq.(3),is a semiempirical model that is widely used for predicting solubilities at different temperatures[29,30].

where A,B,and C are empirical parameters,and T is the absolute temperature(K).

In this work,the Apelblat model used to analyze and correlate the experimental data.The experimental data and fitting parameters are displayed in Tables S1 and S2,the fitting figures are shown in Fig.3.The modified Apelblat model was found to perform well when fitting the solubilities of 5′-GMPNa2in I-W and E-W,with total R-squared values of greater than 99.9%.

The fitting results can intuitively illustrate the trends in solubility.The solubility of 5′-GMPNa2increases with increasing temperature and decreasing alcohol mole fraction.It is more soluble in ethanol than in isopropanol at the same mole fraction and temperature.5′-GMPNa2is a macromolecule with many asymmetrical polar functional groups,while ethanol has a higher dielectric constant(24.3 F·m-1)than isopropanol(18.3 F·m-1).As solvent polarity increases with the dielectric constant,in other words,ethanol has a higher polarity than isopropanol.Therefore,the behavior of 5′-GMPNa2in various solvents follows the rule of“like dissolves like”[31].The amorphous form was markedly more soluble than the crystalline form in aqueous isopropanol and ethanol mixtures[1].The disorganized spatial molecular arrangementin the amorphous form makes ita metastable form with higher energy;it was easier to dissolve.Meanwhile,the solubility data in Fig.3 show that the solubilities between crystalline and amorphous forms possibly had a monotropic relationship(the crystalline form being less soluble than the amorphous one)under almost all experimental conditions;while there was acrossing at approximately 308.15 K when using a 20%mole fraction of ethanol(Fig.3(b)),crystalline and amorphous forms may had a enantiotropic relationship.Further studies are necessary to validate these phenomenon.

3.3.In fluence of pH on solubility

The effects of pH on the solubility was also studied.Fig.S1 shows that the solubility of 5′-GMPNa2clearly increases with increasing pH in both I-W and E-W,with the amorphous form being more soluble than the crystalline form.5′-GMPNa2has different ionic forms[32]in aqueous solutions due to it is amphoteric nature,which can be described by Eqs.(4)-(7)[21].

Fig.3.Temperature and alcohol dependence of the solubility of 5′-GMPNa2 in binary alcohol-water solvent mixtures:(a)10%mole fraction;(b)20%mole fraction;(c)30%mole fraction;(d)40%mole fraction;black denote crystalline;red denote amorphous.

From the ionization balance we can see thatthe isoelectric point(pI)of 5′-GMP is 1.5.The equilibriums moving to the right as the pH increases,that is to say the solubility of 5′-GMP ionic cluster increases with the increase of pH,GMP3-is easier to dissolve in aqueous.This can explain the positive correlation between the solubility and the pH in a given solvent composition.When the pH is from 8.5 to 10,both GMP2-and GMP3-exist in the aqueous solution.The solubility of sodium guanosine 5′-monophosphate is obviously the highest at pH=10.0,because guanosine monophosphate exists in the formoftrisodium guanosine monophosphate.

3.4.Verification of residual solids

According to relevantliteratures,the amorphous formof5′-GMPNa2transforms into the crystalline form,in a SMT[26,33].We carefully examined whether the solid 5′-GMPNa2changed its form during the solubility experiments.The undissolved residual solids were filtered,air-dried at 303.15 K,and then their structures were identified using PXRD(2θ =5°-60°with steps of 0.02°)and the material morphology was observed using SEM.

The PXRD patterns are shown in Figs.4 and S2-S6.In the I-W,the amorphous solid kept its form(Fig.S2),while the crystalline form was partly transformed into the amorphous one,as shown in Figs.4 and S3.In contrast,the crystalline form was completely maintained in the E-W(Fig.S4),while the amorphous solid was transformed to crystals under most of the conditions(except at 10%E-W mole fraction),as shown in Figs.S5 and S6.

In order to quantify the transformation processes occurring during the solubility experiments under different conditions,the crystallinities of the materials were calculated,and the results are shown in Fig.5.In the I-W(Fig.5(a)),the crystallinity of the residual solid exceeded 85%when the mole fraction of isopropanol was 30%and 40%,while it was below 10%in solutions with a 10%mole fraction of isopropanol.In 20%mole fraction isopropanol,the residualsolid had a high crystallinity at 298.15 and 308.15 K,and low crystallinity at 313.15 and 318.15 K.Therefore,the crystalline form tended to transform to the amorphous one at higher temperatures and lower mole fractions of isopropanol.In the E-W,shown in Fig.5(b),the crystallinity of the residual solids exceeded 85%in 20%-40%mole fraction ethanol,while it was below 10%in 10%mole fraction ethanol.In other words,the amorphous form was transformed into the crystalline form when the mole fraction of ethanol exceeded 20%.

Even after increasing the pH to 10.0,the amorphous form could not transform into the crystal one in solutions containing I-W and a 10%mole fraction ofethanolsystem.The crystalline form partly transformed into the amorphous form in 10%and 20%mole fractions isopropanol in Fig.5(c),while the amorphous form was maintained itself in 10%mole fraction ethanol in Fig.5(d).It is worth noting that the transformed temperature of at which the crystalline form transformed into the amorphous form clearly increased as the pH of the isopropanol system was increased,as shown in Fig.5(a)and(c).This is because addition of NaOH increases the sodium and hydroxyl ion concentrations,which helps to assemble the crystalline structure[27,32,34],as Na+promotes the stacking of the G-Quartet structure,which is necessary for forming the crystal structure;in addition,OH-can improve the solubility of 5′-GMPNa2,which is favorable for SMT.In other words,the crystalline form is preferred at slightly higher pH values;therefore destroying the crystalline structure under the same conditions requires more energy(i.e.,a higher temperature).

Furthermore,SEM images were also employed to characterize the morphologies of the residual solids,as shown in Fig.6.Fig.6(a)and(b)show that the crystalline form was stable at 303.15 K,but transformed to the amorphous form at 308.15 K,which is consistent with the PXRD results in Fig.S3(b).The amorphous form in 10%mole fraction ethanol kept its form(Fig.6(c)),while in 20%mole fraction ethanol the amorphous form transformed to the crystalline form(Fig.6(d)),which is consistent with the PXRD results in Fig.S5.

Fig.4.PXRDpatterns ofcrystal5′-GMPNa2 from derived from differentexperimentalparameters:10%mole fraction I-W(a)pH=9.5,(b)pH=10;20%mole fraction isopropanolsystem(c)pH=9.5,(d)pH=10.

Fig.5.Crystallinity of residual solid of 5′-GMPNa2 derived from the PXRDpatterns of the samples from different experimentalparameters:(a)crystalline of residualsolid ofcrystalline 5′-GMPNa2 in I-W withoutadjusting pH;(b)crystallinity of residual solid of amorphous 5′-GMPNa2 in E-W withoutadjusting pH;(c)crystallinity of residual solid of crystalline 5′-GMPNa2 in I-W with different pH;(d)crystallinity of residual solid of amorphous 5′-GMPNa2 in E-W with different pH conditions.

Fig.6.SEMimages ofdifferentexperimentalconditions for5′-GMPNa2.Crystalline form in I-Wwith 20%mole fraction atdifferenttemperatures:(a)303.15 K,(b)308.15 K;(c)amorphous form in E-W with 10%mole fraction at 308.15 K;(d)amorphous form in E-W with 20%mole fraction at 308.15 K.

To summarize,our experiments confirmed that transformations between the crystalline and amorphous forms occurred during the solubility experiments.

According to the PXRD patterns of residual solid,we know that polymorphic transformation has different directions in isopropanol and ethanol,respectively.In I-W system,when the isopropanol fraction was 10%and 20%,the relationship between amorphous and crystalline 5′-GMPNa2was enantiotropic transition,the crystalline was stable form under low temperature and high pH,and the amorphous was considered as the stable form under high temperature and low pH;when the isopropanol fraction was 30%and 40%,the relationship between amorphous and crystalline 5′-GMPNa2was monotropic transition,the crystalline was deemed as the stable form.However in E-W system,when the ethanol fraction was 10%,the relationship between amorphous and crystalline 5′-GMPNa2was monotropic transition,the crystalline was regarded as the stable form;when the ethanol fraction was 20%,30%,and 40%,the relationship between amorphous and crystalline 5′-GMPNa2was enantiotropic transition,the crystalline was considered as the stable form.

The partial solubility data is biased because that polymorphic transformations result that different form turn into same crystal form,while they have the different crystallinity and particle sizes between before and after solid transformation.Note that this does not mean that the experimental measurements are inaccurate.The solution is always saturated during the transformation process,so there is no driving force to promote new crystal growth,while the smaller particles are more soluble than the large ones,according to the Kelvin equation[35],so amorphous materials have higher solubility than crystalline materials.At same time there is an unavoidable error in the experiment.As these factors result in solubility differences of same crystalline form.

3.5.Hypothesis of transformation mechanism

There have been reports of solventenvironmentaffecting crystalline form[12,36,37].It is known that 5′-GMPNa2easily binds to recipient molecules as it contains many electrophilic groups(C,N,P,O).The different association capacities(hydrogen bond,Van Edward force etc.)between 5′-GMPNa2and the solvents results in different intermolecular interactions,which helps to explain the polymorphic transformations that took place under different conditions.We suppose that the association capacity is mainly affected by the solvent(polarity,molecular size,proportion etc.);the molecular structure and cluster rearrangement of 5′-GMPNa2could change in different solvent environments.

At low alcohol mole fractions,when the solution achieve saturated due to that the solubility is higher,a dynamic balance between dissolution and precipitation is highly frequent.There is not enough time for 5′-GMPNa2molecular rearrangement,5′-GMPNa2molecular is precipitated in an amorphous form when amorphous as materials.However when 5′-GMPNa2crystal as materials,5′-GMPNa2molecular is easily form hydrogen bond with ethanol than isopropanol so 5′-GMPNa2molecular has great possibility self-assembled to amorphous in I-W as shown in Fig.7(a)-(c);on the one hand hydrogen bond between ethanol and 5′-GMPNa2hindered intermolecular coalescence,on the other hand crystalline materials as nucleus is advantageous to keep crystal structure.So crystalline maintain itself form in E-W.At high alcohol mole fraction,when the solution achieve saturated due to that the solubility is low,a dynamic balance between dissolution and precipitation is slow-motion.There is enough time for 5′-GMPNa2molecular rearrangement to crystalline in E-W as shown in Fig.7(d)-(f),5′-GMPNa2molecular attach to the nucleation of the existing crystal to form crystalline in I-W.Therefore,we speculate that 5′-GMPNa2has a high nucleation energy barrierin I-W,and add crystalseed can effectively promote amorphous transform into crystalline.Continue to increase the proportion of alcohols,5′-GMPNa2molecular almost insoluble in alcohol,at same time alcohol molecular and 5′-GMPNa2molecularcompare to watermolecular,aftera period of time crystalline transformed into amorphous.

Fig.7.Transformation process between crystalline form and amorphous form in different solvent environment:(a)-(c)crystalline form transform into amorphous form in I-W;(d)-(f)amorphous form transform into crystalline form in I-W.(a)and(d)5′-GMPNa2 solid dissolved;(b)and(e)polymorphous transformation;(c)and(f)the system achieve stable.molecular of 5′-GMPNa2;amorphous of 5′-GMPNa2 solid;crystalline of 5′-GMPNa2 solid;isopropanol molecule;ethanol molecule;water molecule.

3.6.Prediction of thermodynamic properties

In order to predict these conversion processes,the associated thermodynamic properties were also analyzed.The van't Hoff equation[29]correlates logarithmic solubility with the reciprocal of the absolute temperature(Eq.(8)).The molar dissolution enthalpy and the molar dissolution entropy were obtained by fitting the Van't Hoff equation,and the results are displayed in Table 1.

Since the two solid forms have different molecular arrangements,they are also expected to differ in△G.The stable form usually has lower solubility and energy than the metastable form,and this difference provides the driving force for polymorphic transformation.The difference between the Gibbs free energy[38]of the two polymorphs reflects the ratio oftheir fugacity(f).The fugacity is proportional to the thermodynamic activity(a),where the proportionality depends on the choice ofstandard state.Furthermore,the thermodynamic activity can be approximated by the solubility.These relationships are expressed in Eqs.(9)-(11).

where△Hdand△Sddenote the standard enthalpy and entropy of dissolution,respectively;R is the gas constant;and△G is the Gibbs free energy.

According to PXRD patterns,during the process of solubility experiment crystalline partly transform into amorphous 5′-GMPNa2in 10%and 20%mole fraction isopropanol;amorphous transform into crystalline 5′-GMPNa2in 20%30%and 40%mole fraction ethanol.Therefore,we don't calculate the values of Hdand Sdin these conditions.Table 1 shows that the molar dissolution enthalpy is positive(△HdN 0)under allexperimental conditions,meaning that this is an endothermic process.This can account for the solubility of 5′-GMPNa2increasing with increasing temperature.At the same time,△Hdand△Sdof the crystals are larger than those of the amorphous counterparts under the same conditions.Therefore,the solubility of the crystalline form tends to be more sensitive to temperature change than the amorphous form,and so the dissolution of the crystalline form needs more energy[38].According to the Gibbs free energy change in Table 2,the△G of the amorphous-to-crystalline transformation is negative in solutions with a 30%-40%mole fraction of isopropanol or 10%mole fraction of ethanol(i.e.,these are a spontaneous processes).Thus,we speculate thatthe amorphous solid would transform into the crystalline form after sufficient time.The energy calculation is consistent with the molecular level predictions.

Table 1 The mole dissolution enthalpy and,entropy of crystalline and amorphous 5′-GMPNa2 in alcohol-water mixture

Table 2 The Gibbs free energy of amorphous transformed into crystalline

4.Conclusions

In this work,the solubilities of crystalline and amorphous forms of 5′-GMPNa2in binary I-W and E-W solvent mixtures were studied by static methods.At the same mole fraction,temperature and pH,5′-GMPNa2is more soluble in ethanol than in isopropanol,and the amorphous form is more soluble.The PXRD patterns and the solubility data indicate that the transformation between the two solid forms of 5′-GMPNa2occurred during the solubility experiments;however,it proceeded in opposite directions in different solution environments.In the I-W,the crystallineto-amorphous transformation occurred at high temperatures and low isopropanol proportions.In contrast,in the E-W the amorphous form could transformspontaneously into the crystalline formwhen the ethanol proportion was≥20%.The trends in crystal transformation were interpreted from the viewpoint of intermolecular force between 5′-GMPNa2and solvent,and the association capacity was subsequently influenced by the solvent(polarity,molecular size,proportion etc.),so the molecular structure and cluster rearrangement of 5′-GMPNa2could change in differentsolventenvironments.Through theoreticalcalculation of the Gibbs free energy change of polymorphous transformation,we found that the amorphous form tends to transform into the crystalline form at 30%-40%mole fractions of isopropanol,and a 10%proportion of ethanol.The energy calculation is consistent with the molecular level predictions.

Supplementary Material

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.cjche.2018.06.003.