Quasi-Emulsion Precipitation of Pharmaceuticals. 2. Application to

Quasi-Emulsion Precipitation of Pharmaceuticals. 2. Application to Control of Polymorphism Xing

Wang#

and Donald J. Kirwan*

Department of Chemical Engineering, UniVersity of Virginia, P.O. Box 400741, CharlottesVille, Virginia 22904-4741

CRYSTAL GROWTH & DESIGN 2006 VOL. 6, NO. 10 2228-2240

ReceiVed June 29, 2005; ReVised Manuscript ReceiVed May 31, 2006

ABSTRACT: Under a number of mixing conditions, a quasi-emulsion is formed during solute precipitation in which the solvent [e.g., 60 mass % polyethylene glycol 300 (PEG300) in water] is more viscous than the antisolvent (e.g., water). 8 This microscopically segregated dispersion typically results in an increase in nucleation induction time, and this behavior can be exploited to control the polymorphic form produced during antisolvent precipitation. For three pharmaceutical solutes that form polymorphs (methylparaben, p-aminobenzoic acid, and sulfanilamide), it is demonstrated that quasi-emulsion precipitation results in the formation of the stable polymorphic form. On the other hand, when the fluid is rapidly mixed to homogeneity, a metastable polymorph form is first produced. Thus, polymorph formation can be highly mixing sensitive. When a solute is dissolved in a solvent less viscous than the antisolvent as is the case for the precipitation of glycine from water with aqueous PEG300, quasi-emulsions are not formed, and the metastable polymorph is first formed unless extreme fluid segregation (no agitation) is employed. Introduction Many pharmaceutical solids exhibit polymorphism, which is the phenomenon that the same pure substance shows at least two crystalline phases that have different arrangements of the molecules in the crystal lattice.1-3 The polymorphic solids have different unit cells and display different solid-phase physical properties.1-3 Polymorphism, therefore, significantly affects properties of the solid form of a drug, such as solubility, dissolution rate, bioavailability/bioequivalence, and chemical stability.4,5 In the pharmaceutical industry, solvate and hydrate crystals are usually called pseudopolymorphs of the anhydrous form which does not contain solvent.5 Amorphous forms also exist that have no clearly defined crystal structure and no longrange order.4 Identifying all possible solid forms of a drug is important for intellectual property protection.5,6 Further, consistent manufacturing and sale of only the approved solid form of the compound is required by FDA regulations.7 It happens quite often in pharmaceutical process development that one polymorphic form of crystals is produced in smallscale development experiments, but after scaling up the process, another form is generated. This situation has been related to effects of solvents, processing time, and temperatures, and possibly to mixing conditions. The quasi-emulsion phenomenon, described in our previous paper8 in this issue, can play a role in polymorph formation. Further, we believe that control of polymorph formation can be accomplished by the use of selected mixing modes and solutions/antisolvent combinations that favor or prevent quasi-emulsion conditions. In brief, we hypothesize that in crystallization from a quasi-emulsion, where diffusion becomes important, the nucleation and growth rate are slowed and formation of the stable polymorphic form is favored. The crystalline phase that is formed will be primarily governed by thermodynamics. On the other hand, rapid, homogeneous mixing with high supersaturation will favor the formation of a metastable form in accordance with Ostwald’s Rule of Stages,9 which hypothesizes that metastable (higher free energy) phases are first * To whom correspondence should be addressed. E-mail: [email protected]. # Present address: Crystallization Technology Laboratory, Research API, Pfizer Global R&D, 2800 Plymouth Road, Bldg 520-G346B, Ann Arbor, MI 48105.

formed in a crystallization process, i.e., the crystalline form obtained is governed by kinetics. Solvent polarity/nonpolarity is also known to affect polymorphism, especially through influences on hydrogen bond formation.5 As quasi-emulsion formation will occur when mixing solvent and antisolvent liquids that have different polar/nonpolar properties in addition to viscosity differences, care will be taken to distinguish the effect of the quasi-emulsions from other solvent effects. As described in our first paper8 in this issue, during antisolvent precipitation, quasi-emulsions (well-dispersed droplets of the more viscous, solute-containing solvent in a less viscous antisolvent) are formed when the solvent/antisolvent viscosity ratio > ∼3 or when solute can rapidly form an interfacial barrier preventing further mixing. The emulsions are termed quasi-emulsions because, in fact, the two liquid phases are thermodynamically miscible. However, they can form a suspension of microscopically dispersed droplets (100-1000 nm) stabilized by an interfacial solute barrier that is able to form because of the slow mixing of a more viscous fluid into a less viscous fluid. It was found that these emulsions can be formed when equal volumes are mixed in a confined impinging jet (CIJ) mixer or by reverse addition of a more viscous, solute-containing solution into an less viscous antisolvent. These emulsions are stable for considerable periods of time (hours to days under some conditions) if the “mixed” solution is subjected to only gentle agitation. However, they are quickly destroyed if the mixed solutions are subjected to higher agitation levels. Normal, slow addition of a less viscous antisolvent into more viscous, solute-containing solution does not result in an emulsion but in a well-mixed solution followed by rapid crystal nucleation and growth. The quasi-emulsion phenomenon differs from spherical crystallization resulting from a true emulsion (thermodynamically immiscible phases) which also is influenced by agitation because it affects the drop size distribution in the emulsion.10-12 A quasi-emulsion creates a heterogeneous system in which solute-containing solvent and antisolvent are segregated on a nano- to microscale for a significant period of time. Consequently, the observed crystal nucleation induction time or kinetics is governed by the diffusion rates of solvent/antisolvent across the interfacial region. That is, under conditions of quasiemulsion formation, the effective mixing time () the time to

10.1021/cg050305v CCC: $33.50 © 2006 American Chemical Society Published on Web 08/29/2006

Quasi-Emulsion Precipitation of Pharmaceuticals

Crystal Growth & Design, Vol. 6, No. 10, 2006 2229

Figure 1. Molecular structures of model solutes.

achieve uniform solvent/antisolvent composition) is governed by diffusion rather than hydrodynamics conditions. (This structure can be destroyed by subsequent agitation, if desired.) When a quasi-emulsion structure exists, diffusional transport is hindered and the solvent/antisolvent composition where crystallization takes place corresponds to relatively low supersaturation. Therefore, nucleation and growth rates are reduced, and the thermodynamically stable form is favored. Of course, this is a qualitative argument, and the result in any given system still may depend on the respective crystallization kinetics of the possible polymorphic forms at relatively low supersaturation. On the other hand, if the mixing conditions are such that a wellmixed solvent/antisolvent condition (especially one corresponding to high supersaturation) can be rapidly achieved on a time scale that is short with respect to the induction times of the various polymorphs, then the polymorphic form obtained should be governed by crystallization kinetics. The purpose of this paper is to provide experimental observations to test this hypothesized role of quasi-emulsion formation in controlling polymorph formation. We report on the results of a study of the effect of mixing on the crystal forms of three model (polymorphic-forming) compounds dissolved in viscous aqueous polyethylene glycol 300 (PEG300) solutions and precipitated by the addition of water. Normal and reverse mixing modes in an agitated system and with CIJ mixing of the two fluids are studied and combined with fast, slow, and no stirring after mixing to generate various mixed conditions to test our hypothesis. In addition to these model systems, the crystallization of glycine, which also forms

Figure 2. Measured solubility of MP crystals (Form I) in PEG300water solutions (25 °C).

polymorphs, but is soluble in water and does not form quasiemulsions during its precipitation from water by PEG300, is examined.8 There is limited literature about mixing effects on polymorphism. There has been a report that the formation of the polymorphs of CaCO3, vaterite, and aragonite, is influenced by the specific power input in a stirred vessel.13 Formation of different polymorphs of L-glutamic acid under different agitation conditions is also reported.14,15 No mechanism for stirring effects on polymorphism has been proposed previously. Experimental Section Materials. Solutes. To use viscous solutions of PEG300 in water as the solvent in the precipitation of pharmaceutical-like molecules that exhibit polymorphism, the model solutes selected should be relatively soluble in aqueous PEG300 and insoluble in water. Considerations of cost and availability led to the selection of the following three substances, which were used as purchased (Figure 1). Consideration of the supersaturation that could readily be achieved in precipitation led to the following common solvent and antisolvent compositions for all three solutes: solvent: 60 mass % PEG30040%H2O; antisolvent: water (deionized); final solvent composition:

Table 1. Mixing Conditions and Modes Summary

process

vessel mixing

rate of supersaturation increase by mixing

qualitative phenomena

CIJ mixing at 1:1 ratio

slow or none

CIJ mixing at 1:1 ratio

fast

reverse addition: viscous solute into antisolvent reverse addition: viscous solute into antisolvent normal addition: antisolvent into viscous solute

slow or none

mixing description

quasi-emulsion expected

emulsion drops from CIJ mixer are not destroyed by vessel mixing emulsion drops from CIJ mixer are homogenized rapidly in seconds emulsion drops are not destroyed by vessel mixing

diffusion limited

segregated

yes

seconds

rapid homogeneous

no

diffusion limited

segregated

yes

fast

emulsion drops are homogenized in seconds

seconds

rapid (or slow) homogeneous

no or limited life

fast

antisolvent mixed into the continuous phase in seconds

seconds

rapid homogeneous

no

Table 2. Methylparaben Polymorphs crystal form I.D.

habit

stability at process conditions

transition T

solubility (31% PEG300)

refs

I II

prisms platelets

stable metastable

not tested not tested

12 g/kg 18 g/kg

17, 18, this work 17, 18, this work

2230 Crystal Growth & Design, Vol. 6, No. 10, 2006

Wang and Kirwan

Figure 3. (a) Metastable platelets (Form II) and (b) stable prism (Form I) of MP crystals at S ) 4 (Form I) [∼100×, bar ) 200 µm]. 31.1 mass % PEG300 formed from the mixing of equal volumes of the two solutions. In addition, glycine (Aldrich >99%) was tested with deionized water as the solvent and 80 mass % PEG300-20% H2O as the antisolvent. Equipment. These include Harvard Apparatus PHD 2000 Syringe pump; confined-impinging-jets (CIJ) mixer; Bausch & Lomb Balplan microscope; Beckman DU640 UV/Vis spectrophotometer; TA Instruments 2920 differential scanning calorimeter (DSC); Scintag automated powder X-ray diffraction (PXRD)system. Procedures and Conditions. (1) Solubility Measurements. Solubility was measured at 25 °C except where noted. Calibration curves for the UV absorbance of each solute in solvent were developed at the following wavelengths: λ ) 220 nm (PABA), 256 nm (MP), 262 nm (SA). At constant temperature, solvent was added into a jacketed beaker or glass bottle and an excess amount of the solute crystals being tested was added. The sample was stirred for 24 h or more (24 h is long enough for our studies according to values of the solubility measured at longer times.) After centrifugation, a syringe filter (0.2 µm) was used to sample the saturated solution. The saturated solution was diluted to the proper concentration and the absorption was measured at the characteristic wavelength. If the absorption of the solute is very strong, usually the calibration curve is constructed using pure methanol as the solvent. When measuring the solubility in other solvents such as PEG300 solutions, a large amount methanol is added to dilute the saturated sample. The mass of the original samples was typically negligible compared to the mass of methanol added as diluent. The small amount of PEG300 or H2O present had no effect on the characteristic absorption of the solute. (2) Mixing Modes. CIJ Mixing. This confined impinging jet device was generously provided by Dr. Brian Johnson at Merck and Co., Inc. It was well described and characterized by Johnson and Prud’homme16 and also in our companion paper8 in this issue. Syringe pumps forced fluids through the CIJ mixer at 20-60 mL/min for each nozzle corresponding to a jet velocity of 1.6-5 m/s. The exit fluid was collected in an agitated beaker or a plastic tube or flask. Magnetically Stirred Beaker. Either 20 or 50 mL each of solution and antisolvent were used in a 100 or 250 mL beaker. Reverse addition: Solvent (with dissolved solute) is added (∼60 mL/min) into stirred antisolvent. Normal addition: Antisolvent is added (∼60 mL/min) into stirred solute-containing solvent. Post-addition mixing conditions were defined as follows: Fast stirring: 870 rpm, stir bar (25 mm × 9.5 mm) in 250 mL beaker; slow stirring: 155 rpm, stir bar (25 mm × 9.5 mm) in 250 mL beaker; no stirring: solution collected in plastic tubes (5 mL) or conical flask (150 mL). After fluid addition in a beaker or after mixing in the CIJ, three types of mixing conditions designated as “rapid homogeneously mixed”, “slow homogeneously mixed”, and “well-dispersed, but segregated” could apply and were examined. There were different combinations of mixing modes that could achieve the above three mixed-fluid conditions. For example, rapid homogeneous mixing may be achieved by normal addition plus fast stirring, CIJ mixing plus fast stirring, or slow reverse addition plus fast stirring. Since we only care about the mixing conditions, not all mixing modes were tested in each of our systems. However, all three types of mixed conditions were tested for each system. Experiments at each different mixing mode were repeated

Figure 4. Coexistence of platelet (Form II) and prism (Form I) MP crystals produced by slow homogeneous mixing conditions at S ) 4 (Form I) [∼100×, bar ) 200 µm].

Figure 5. Stable (Form I) prisms of MP crystals produced by reverse addition with slow stirring at S ) 4 (Form I) [∼100×, bar ) 200 µm]. several times. For the beaker experiments, induction time was measured from the completion of the fluid addition as no crystals were observed until some time after addition was completed. As a guide to these various mixing conditions Table 1 provides a summary of the mixing conditions and the modes under which they appear. In the case of reverse addition followed by fast stirring, a somewhat different effect was sometimes observed for methylparaben crystallization than that for fluid leaving the CIJ followed by fast stirring. As discussed below, there was an indication that the emulsion could survive for some period of time before destruction. We call this case slow



Figure 6. The transformation of MP platelets to prisms at 22.3 °C. The elapsed time from a to d is ∼30 min [∼100×, bar ) 200 µm]. homogeneous mixing. Whether there is any effect of this difference would be expected to be governed by the nucleation and growth kinetics of the system in comparison to the lifetime of the quasi-emulsion. Crystal nucleation induction times are generally observed to vary with volume of the system under study. In all our experiments, comparisons of mixing effects are made in the same volume of solution so that this variable was not altered in these tests. Finally, it is understood that the mixing in a magnetically stirred beaker is not well-characterized and not readily scaleable to larger scale vessels. However, we chose to conduct experiments at this scale to focus on the phenomenon of polymorph crystallization from quasiemulsions. Admittedly, the results shown below are qualitative in terms of mixing conditions, but they clearly demonstrate that mixing in quasiemulsion forming systems can affect polymorph formation. Larger scale studies would need to be conducted for any given solute/solvent/ antisolvent system to establish quantitatively the specific mixing conditions controlling quasi-emulsion formations and the resulting polymorph formation. (3) Temperature. Experiments except for solubility measurements were conducted at room temperature, which was typically 22.3 °C. (4) DSC. The proper amount of dried crystals (several mg) was loaded and the temperature was ramped at 10 °C/min. (5) PXRD. Details include the following: start angle: 5 deg; stop angle: 60 deg; step size: 0.05 deg; scan rate: 4 deg/min; scan mode: continuous wavelength: 1.540562 Å. (6) Supersaturation Ratio, S. For each experiment, S is the nominal measure of the supersaturation ratio defined as the ratio of the actual concentration (after mixing but before crystallization) divided by the solubility of the most stable polymorph under the experimental solvent compositions and 25 °C. For clarity, it will be written as S (polymorph form).

Results and Discussion Methylparaben. Only one crystal structure of methylparaben has been reported in the Cambridge Structural Database,17 although other forms have been reported to exist.18 Preliminary experiments indicated that different mixing conditions led to

Figure 7. DSC analyses of MP crystals: Form I ) prisms; Form II ) platelets.

different crystal morphologies so further studies were conducted as described below. Table 2 summarizes the literature information and that obtained in this study for methylparaben polymophic forms. (1) Solubility. The solubility (grams per kilogram of mixed solvent) of MP as received (Form I, prisms) was measured using the standard method and shown in Figure 2. The solubility measurement of the metastable Form II is described below in Section 3i. As noted above, considerations of solvent viscosity and achievable supersaturation dictated a solvent consisting of 60 mass % PEG300-40% H2O. The initial solute concentration in the solvent was chosen so that, after mixing with an equal volume of antisolvent (H2O), the well-mixed nominal supersaturation ratio would be S ) 4 (Form I). The corresponding


Wang and Kirwan

Figure 8. PXRD patterns of MP crystals: (a) Form I, prism and (b) Form II, platelets. Table 3. p-Aminobenzoic Acid Polymorphs crystal form I.D.

habit


transition T, °C


refs

R β

needles prisms or plates

stable metastable

15-20 15-20

30 g/kg 32 g/kg

19, 20, 21, this work 19, 20, 21, this work

supersaturation ratio for the metastable form based on our measured solubility reported in Table 2 would be S ∼ 2.7. (2) Mixing Experiments (Room Temperature). (i) Normal Addition with Fast Stirring. At room temperature, rapid homogeneous mixing was achieved by adding antisolvent water (nonviscous) into fast stirred MP/PEG300 aqueous solution (viscous). As hypothesized, the metastable crystal (Form II, platelet morphology) of methylparaben formed which could be stable in solution for 1-3 days (Figure 3a). The visibly observed induction time was ∼20 s. (ii) CIJ Mixing (50 mL/min) and Then Slow or No Stirring. Quasi-emulsions would be expected under these conditions. Stable prismatic crystals (Form I) formed as shown in Figure 3b. For slow stirring, the induction time of several minutes was longer than that in (i). If there were no stirring after CIJ mixing, large single prismatic crystals (Form I) formed in the plastic collection tubes after a much longer induction time (several hours).

(iii) Reverse Addition with Fast and Slow Stirring. The mixing in (i) and (ii) above can be considered as two extreme cases: (i) a homogeneous mixing case in which only metastable platelet crystals were generated and (ii) a “well-dispersed, segregated” case in which only the stable prism crystals were produced. If the structure of the “mixed” solution is as the quasiemulsion model suggests, we may be able to generate a “slow homogeneous mixing” between “rapid homogeneous mixing” and “segregated” conditions via reverse addition and fast stirring. The droplets formed in the beaker by reverse addition or those from the CIJ are both subject to fast stirring which should break them relatively quickly. However, we speculate that those formed in the beaker may be larger (less intense initial mixing) and, therefore, may survive longer than those smaller droplets from the CIJ device. Hence, we may see a difference under this condition depending upon the rates of diffusion, droplet destruction, and the induction time of the stable and metastable

Figure 9. Solubility (R form) of PABA in PEG300-water solutions at 25 °C.

Figure 10. Measured solubility of different polymorphic forms of PABA in 31.1 mass % PEG300.



Figure 11. Stable needle needle crystals produced by CIJ mixing (S ) 2 (R form) with (a) slow stirring or (b) no stirring. Metastable prisms (c) were formed with fast stirring. S ) 2, 22.3 °C [∼100×, bar ) 200 µm].

Figure 12. DSC analyses of PABA crystals: (solid line) needle crystals (R form) and (dashed line) prism/platelet (β form) crystals.

polymorphs. Precipitation of MP by reverse addition with fast stirring resulted in a suspension containing both crystal forms (Figure 4) while CIJ mixing followed by fast stirring always favored the metastable form. [As noted below this slow homogeneous case was not observed for other solutes at the experimental (stirring) conditions examined.] As expected, reverse addition with slow stirring (Figure 5) showed results similar to that of CIJ mixing with slow stirring (Figure 3b), i.e., the stable prismatic Form I. The quasi-emulsion model predicts that tiny droplets are produced in these two cases that slow stirring does not destroy. Therefore, crystals generated under these two conditions look similar and produce the stable polymorph. These experimental results clearly show the difference among “rapid homogeneous mixing”, “well-dispersed, segregated”, and “slow homogeneous mixing.” The effect of different mixing conditions on crystal formation rates also are in general

agreement with the induction time trends observed for salicylic acid under different mixing conditions described in part I.8 (3) Further Analysis. To understand the relationship between those two forms of MP crystals, further tests were carried out: solubility measurements, microscopic observation, and DSC and PXRD analyses. (i) Solubility Tests. Solutions saturated by precipitated platelet (II) crystals (Figure 3a) and prism (I) crystals (Figure 3b) were held for 24-48 h and then used for solubility measurements at the experimental temperature of 22.3 °C. The solubility measurements (in 31.1 mass % PEG300/water) showed that MP platelet crystals have a higher solubility (18 g/kg) than that of prismatic crystals at room temperature (12 g/kg) suggesting that the platelet (II) crystals have a higher free energy at these conditions and are the less stable form. Consistently, the measured prism solubility at 22.3 °C was a little lower than that shown in Figure 2 for 25 °C. Under quasi-emulsion conditions, the solvent composition within the droplets is controlled by diffusion and gradually changes over time. Since the free energy of the solid Form II is higher, as is its solubility, the stable form should be preferred (experience higher supersaturation) during the diffusion and subsequent nucleation process in the presence of droplets. (ii) Microscopic Observations. Microscopic observation clearly showed the transformation from metastable Form II (platelets) to the stable Form I (prisms) when these two crystal forms coexisted in the 31.1 mass % PEG solution at 22.3 °C (Figure 6a-d). This observation is consistent with the solubility measurement results that the platelets were the less stable form. (iii) DSC Analysis. The crystals shown in Figure 3a,b were filtered from the suspension with 10-µm membrane filters and washed by DI water and dried either in a vacuum at 40 °C for 2 h (prisms) or in air at room temperature for 24 h (platelets). The lower temperature for the platelets was used to minimize the possibility of a solid phase transformation. DSC analysis was then carried out with these crystals. Figure 7 clearly shows

Figure 13. PXRD patterns of PABA crystals: (a) needle crystals; (b) prism/platelet crystals.


Figure 14. The solubility of sulfanilamide (β form) in PEG300-water solution at 25 °C.

that the prism form is the stable form (Form I) exhibiting no transformations as the temperature increases to 140 °C. The platelet form (Form II) exhibits a transformation to the more stable prism form at ∼88.4 °C. It is not likely that the platelet form is a solvate or hydrate given the very small energy effect at this transition point. (iv) PXRD Analysis. Interestingly, the platelet and prism crystals had very similar PXRD patterns (Figure 8). Originally, we thought that the platelets might have transformed before the PXRD was measured, but samples measured at different times up to 24 h after the crystals were produced gave the same PXRD pattern. The microscopic observations of the geometric habit of Form II clearly suggest that it is crystalline. There is a report in the literature of two crystalline forms of aspartame having very similar PXRD patterns5 and that may be the case here. (4) Summary. Although the two forms observed in this study showed very similar PXRD patterns indicating the same crystal structure, the DSC, solubility, and transformation observations support the fact that Form II is a metastable polymorph and Form I is the stable polymorph. The results of this study with methylparaben clearly support the hypothesis that the formation

Wang and Kirwan

of a quasi-emulsion results in favoring the more stable crystalline form, while rapid mixing favors a metastable form. p-Aminobenzoic Acid (PABA). Two different PABA crystal structures (R19 and β20) have been reported. The R form crystal has a needle/fiberlike morphology, and the β form crystal is a prism/platelet. Thermodynamic stability and crystallization of PABA from different solvents have been studied recently.21 The relationship between these two polymorphs is enantiotropic with a transition temperature of 23-26 °C in water and in ethyl acetate according to solubility measurements.21 Literature information and results from this study are summarized in Table 3. It has been reported that PABA molecules form dimers in the R form of the crystal.19,21 The kinetics of this process is not known, nor is it known how dimerization is affected in different solvents. As the crystallization of PABA may be influenced by a dimer formation process, polymorph formation may be affected by parameters such as temperature, solute concentration, and solvent. However, it is recognized that in a quasi-emulsion the solute concentration will be expected to be higher within the drops which would favor dimer formation and the R form. To demonstrate the effect of mixing on polymorph formation, experiments were carried out at fixed temperature and solute concentration (supersaturation) in the same mixed solvent. (1) Solubility. The solubility of PABA as received (needle, R form) was measured with the standard method (Figure 9). The solubility in pure water shown on the figure is from the literature.21,22 By consideration of the solvent viscosity and the supersaturation that can be generated, the initial solvent was selected as 60 mass % PEG300-40%H2O. The starting concentration is prepared so that, when mixed with an equal volume of antisolvent (H2O), the nominal supersaturation ratio would be S ) 2 (R form). According to our measured solubilities of the different forms of PABA crystals (Figure 10), the R form (needle) is stable and the β form (prism/platelet) is metastable at a temperature of 22.3 °C since the former has the lower solubility at that temperature. The enantiotropic relationship is confirmed by the observation that the relative solubilities have reversed at 4 °C, and the transition temperature would lie between 15 and 20 °C.

Figure 15. (a) Needle and platelet crystals of SA at S ) 2 (β form); (b) 1 day later, needles have disappeared [∼100×, bar ) 200 µm]. Table 4. Sulfanilamide Polymorphs crystal form I.D.

habit


transition T, °C

R β γ

needle prisms platelet

metastable stable metastable

not tested not tested not tested


refs

∼37 g/kg ∼35 g/kg

29-36, this work 29-36, this work 29-36, this work


Figure 16. Platelet (γ form) crystals of SA at S ) 2 (β form) [∼400×, bar ) 200 µm].

Given the closeness of the solubility of the two forms at 22.3 °C, the supersaturation ratio for the metastable form is only some 8% lower than that of the stable form under the crystallization conditions. (2) Mixing Experiments. (i) CIJ Mixing (30 or 60 mL/ min) and then Slow Stirring (155 rpm) or No Stirring; S ) 2 (R Form) and a Temperature of 22.3 °C. Quasi-emulsion formation would be expected. Stable needle crystals formed I a few minutes under this mixing condition (Figure 11a). If there were no stirring, larger aspect ratio needles will form after 1-2 h (Figure 11b). (ii) CIJ Mixing (30 or 60 mL/min) and then Fast Stirring (870 rpm, S ) 2, 22.3 °C). Rapid (possibly slow) homogeneous


mixing is expected. The metastable β form (prism/platelet crystals) formed in a few minutes at this mixing condition (Figure 11c). (3) Further Analyses. (i) DSC. DSC results are shown in Figure 12. Crystals in Figure 12 were those from Figure 11a,c. The results confirm that the observed needle crystals were the R phase formed during quasi-emulsion conditions, while the prism/platelet crystals were the β form, formed under more homogeneously mixed conditions. The β form showed a transition in the solid state to R at about 100 °C. (ii) PXRD. The PXRD patterns for the crystals in Figure 11a,c are shown in Figure 13, panels a and b, respectively. The differences between Figure 13, panels a and b, are obvious and confirm that the two crystal forms were different polymorphs. (4) Summary. It was observed that fast stirring following quasi-emulsion formation (in the CIJ) favors the metastable form of PABA crystals, and slow stirring or no stirring of the quasiemulsion structure produces the stable form of the crystals. Therefore, the hypothesis on the influence of quasi-emulsion structure on polymorph formation again is supported. As noted above, in the PABA system the crystallization process could be complicated by the dimer formation process as solute dimers (leading to R form crystals) would tend to form in solutions at higher solute concentrations or in a less polar liquid environment such as PEG, while solute monomers are favored at low concentration and a polar environment. This effect would tend to favor the formation of the stable R form in quasi-emulsions where the dimers are favored and, therefore, could reinforce the effect of the diffusion-controlled quasi-emulsion process favoring the stable form. Sulfanilamide. The third case studied was the most complex. The three most common polymorphs of sulfanilamide reported in the literature are R, β, and γ.23-26 Other polymorphs also

Figure 17. Prism crystals of SA at S ) 2 (β form). The elapsed time from (a f c) is ∼10 min; from (c f d) it is a few days [∼100×, bar ) 200 µm].


Figure 18. Prism and platelet crystals of SA at S ) 2 (β form) [∼100×, bar ) 200 µm].

have been reported.27-29 The reports about the thermodynamic relationship between R, β, and γ are somewhat contradictory.29-33 However, it is generally agreed that the γ form is the only stable form at high temperature.30,34 The β form is reported to be stable at room temperature,29,33,35,36 and the β and γ forms are enantiotropically related.29,30,32,33 The R form is metastable and is usually reported to have a needle morphology,31,33,35,36 while the β form is prismatic.26,35 The β form is the commercially available form. Table 4 summarizes some of this information for conditions relative to our experiments. (1) Solubility. The solubility of SA from the bottle (prisms, β form) is shown in Figure 14. By consideration of the solvent viscosity and the supersaturation that can be generated, the solvent was again selected as 60 mass % PEG300-40% H2O. The concentration was prepared so that, if mixed with an equal volume of antisolvent (H2O), the initial nominal supersaturation ratio would be S ) 2 (β form). (2) Mixing Experiments (Room Temperature). (i) Fast Stirring (870 rpm) following Normal Addition, Reverse Addition or CIJ Mixing. Rapid homogeneous mixing would be expected for normal addition and CIJ mixing and possibly a slow homogeneously mixed solution with reverse addition. A mixture of both needle (R form, proved later) and platelet (γ form) crystals (Figure 15a) always appeared within one minute under all three mixing modes. Overnight, the needle crystals transformed to platelet crystals if they remained in contact with solution (Figure 15b). (ii) Reverse Addition with Slow Stirring (155 rpm). Quasiemulsions would be expected in this case. Only platelet crystals of a variety of sizes (γ form) were observed (Figure 16) after several minutes; no needle crystals (R form) were ever observed.

Wang and Kirwan

(iii) CIJ Mixing (30, 60 mL/min) with Slow Stirring (155 rpm). For this mixing condition, quasi-emulsions also would be expected. On the basis of repeated experiments, there appeared to be about a 60% chance that prism crystals (β form) will form in several minutes (Figure 17) and a 40% chance that a prism and platelet mixture (β and γ) will form in a few minutes (Figure 18.) In addition, there appeared to be three kinds of prism crystals generated during precipitation (Figure 17a): large flat prisms, rods, and normal prisms. However, as the precipitation process progressed when in contact with solution, only the flat and normal prisms remain. Rodlike crystals transformed to the other two habits (Figure 17b,c), and after several days in solution, the flat prism crystals started to transform to the normal prism crystals (Figure 17d). (iv) CIJ Mixing (20, 30, 40, 50, 60 mL/min) Collected in Tubes without Stirring. Quasi-emulsion formation was expected under these conditions. On the basis of repeated experiments, there appeared to be a ∼50% chance to get large prism crystals within 1 day, a ∼40% chance to get a mixture of crystals in several hours which slowly transformed to prism crystals, and a ∼10% chance to get large platelet crystals in ∼30 min. A photomicrograph of the mixture of crystals is shown in Figure 19a and that of their transformation is shown in Figure 19b. Usually, the transformation from crystal mixture to prism crystals took several days. Shaking the tube accelerated the process. There also appeared to be an intermediate form (identified in Figure 19a) involved in the transformation of platelets (γ form) to prisms (β form). The form labeled intermediate has a three-dimensional structure that is different from the 2-dimensional platelet. Prisms appear to grow from the intermediates. If the intermediate crystals do not exist in the mixture, as occurred in about 10% of the time, then, as shown in Figure 19c, platelet crystals (γ form) in contact with solution will be very stable at room temperature. (3) Further Analyses. Further tests of sulfanilamide crystals were carried out with DSC, PXRD, and by solubility measurements. (i) DSC. Crystals for DSC analysis are prepared by filtration through a 10-µm membrane filter, washing with deionized water, and drying at room temperature. Crystals tested were those shown in Figures 15b, 17b, and 19c. The polymorphic forms of the crystals were assigned based on their characteristic transition points as reported in the literature:29,31,37 R, 99 °C; β, 132 °C, and γ with a melting point of ∼165 °C. The DSC trace (Figure 20, solid line) confirms that the crystals shown in Figure 15b were mixtures of R and γ as the characteristic DSC peak at 99 °C of the R form is much smaller than that reported in the literature. The crystals pictured in Figure 17b were the β polymorph with a transition point at ∼132 °C (Figure 20, dash dot line). Finally, the DSC results for the platelet crystals shown

Figure 19. SA crystals from CIJ mixing without subsequent stirring at S ) 2 (β form). (a) Crystal mixture of platelets (γ form) and intermediate (b) the transformation to prisms (β form) and (c) large platelet (γ form) crystals [100×, bar ) 200 µm].



Figure 20. DSC analysis of crystals shown in (a) Figure 15b, (b) Figure 17b, and (c) Figure 19c. Table 5. Polymorphs of Glycine crystal form I.D.

habit

stability

comments

R

bipyramids

metastable?

Although R is reported less stable than γ, it is kinetically favored and there are reports that γ f R

β γ

needles trigonal pyramids

metastable stable?

in Figure 19c confirm that they are the γ polymorph (Figure 20, dash line). (ii) PXRD. The crystals used for DSC analyses were also tested with PXRD. Forms of crystals were identified based on their characteristic peaks as compared to those reported in the literature.29,31 The crystals shown in Figure 15 were confirmed to be mixtures of R and γ (Figure 21a). Crystals shown in Figure 17b were the β polymorph (Figure 21b), and the crystals shown in Figure 19c were the γ polymorph (Figure 21c). (iii) Solubility. Solubility measurements at in 31.1 mass % PEG300 in water showed that the solubility of the platelet (γ form) was slightly smaller than that of the prism (β form) [37 versus 35 g/kg], suggesting that it is a slightly more stable form. This contradicts the room-temperature transformation shown in Figure 19b, the DSC results, and the literature. It was observed that the induction time of prisms was much longer (∼12 h) as compared to the platelets (∼30 min) and that the growth rate of prisms was much lower. We suspect that the contradictory solubility measurement may reflect that the prisms take a very long time to reach equilibrium so that an erroneously high solubility value was obtained. The R form could not be readily separated from γ to obtain a solubility value. (4) Summary. An interesting phenomenon in our experiments is that the γ form crystal (the stable form at high temperature) can be produced at room temperature with PEG300/H2O solutions, while all the methods reported in the literature using other solvents had to be conducted above 90 °C to get the γ form of the crystal. This phenomenon seems to be related to a property of the PEG solution and not to the mixing condition since it occurs both with homogeneous mixing and with quasiemulsion formation. The γ form has much shorter induction time (fast nucleation and growth kinetics) than that of the β form according to our observations. Therefore, sometimes the γ form was produced where only the thermodynamically stable β form is supposed to appear according to the quasi-emulsion

Figure 21. PXRD analyses of sulfanilamide polymorphs. Crystals from (a) Figure 15 (R and γ forms), (b) Figure 17b (β form), and (c) Figure 19c (γ form).

model (Figures 15, 16, 18, and 19). The solubility difference between β and γ forms is quite small, indicating that their free energies are quite close under the experimental conditions. The pure γ form can be very stable at room temperature for a long time, although thermodynamically it should (and will) eventually transform to the β form.


Figure 22. Solubility of R glycine in aqueous PEG300 solution at 25 °C.53

For the sulfanilamide system, the stability trend was “β > γ > R” at room temperature according to literature and our observations. Rapid homogeneous mixing versus quasi-emulsions/slow homogeneous mixing showed obviously different results in polymorph formation (R + γ versus β, β + γ, or γ). The metastable R form crystals can only be produced under a rapid homogeneous mixing condition, and the stable β form crystals can only be produced from quasi-emulsions or a slow homogeneous mixing condition. However, the γ form appeared under most conditions so that, unlike the other two polymorphic systems studied, the separation of different forms of SA (γ vs R or β vs γ) is not complete. These polymorphs have quite similar crystal structures,28 and therefore thermodynamic values, so that the reduced supersaturation arising from controlled diffusion in a quasi-emulsion is not sufficient to completely separate them when the crystallization kinetics are fast for the formation of the γ form. Nevertheless, the general trend of polymorph formation in the sulfanilamide system was consistent with that predicted by the quasi-emulsion model. Glycine. All of the above studies were carried out with compounds that were soluble in the viscous solvent and precipitated by a nonviscous liquid. As shown in part I8, quasiemulsions are not likely to form in a system (e.g., glycineH2O-PEG system) where the solute is soluble in the nonviscous solvent and precipitated by the viscous antisolvent. As glycine also forms polymorphs, it was interesting to see if there were any mixing effects on polymorph formation in this system. Three polymorphs are known for glycine: R,38,39 β,40-42 γ.43,44 Well-formed R-glycine crystals are centrosymmetric bipyramids.38 β-Glycine crystals are in the form of small needles.42,45 Well-formed γ-glycine crystals are trigonal pyramidal.44 β-Glycine is the metastable form that transforms into R-glycine or

Wang and Kirwan

γ-glycine in solution or humid air.42,45 However, it remains unchanged if kept in dry atmosphere.42,46 γ-Glycine is formed from aqueous acetic acid or ammonia solution,44 by addition of compounds inhibiting the growth of R-glycine,47 or by aqueous solution irradiated with laser light.48,49 γ-Glycine is thermodynamically the most stable form at room temperature,44,50,51 and γ-glycine transforms to R-glycine when heated above 165 °C.44,47 However, the R form seems to be kinetically favored in aqueous solution48 at room temperature. Although R-glycine is slightly less stable than γ-glycine at room temperature, transformation between these two forms has not been reported in the literature except R f γ in humid air50 and γ f R in a saturated aqueous solution,49 possibly because the process is kinetically hindered.52 On the basis of these rather complex literature reports, Table 5 provides a simplified summary of the polymorph forms of glycine at room temperature in solution. (1) Solubility. The reported solubility of R-glycine in aqueous PEG300 solutions is shown in Figure 22.53 Solvent: water (deionized); antisolvent: 80% (wt) PEG300-20%H2O. The initial solute concentration in the solvent was chosen so that, after mixing with an equal volume of antisolvent, the wellmixed nominal supersaturation ratio (in the absence of crystallization) would be S ) 2 (R form). (2) Mixing Experiments (Room Temperature). (i) Stirring. [Note that the identification of polymorphic forms given here was deduced or confirmed by the PXRD analysis described in Section 3.] All stirring experiments (reverse/normal addition in a beaker) or CIJ mixed followed by fast/slow stirring generated needle crystals of β-glycine (Figure 23a,b) with a rare appearance of a pyramidal-shaped crystal of R-glycine, which might have transformed from the needle crystals. The induction time of β-glycine was 5-10 s. Gradually, all needle (β form) crystals transformed to pyramidal-shaped (R form) crystals in solution (Figure 23c). With fast stirring, the transformation process finished within ∼10 min. No γ form glycine was observed. (ii) CIJ without Stirring. Large platelet crystals were generated with an induction time ∼1-5 min (Figure 24). It was confirmed by PXRD that these plates are R-glycine. (3) Further Analyses by PXRD. Although crystals could not be dried well because of the presence of the low volatility PEG300, their PXRD pattern should not be affected by the presence of PEG300. The PXRD pattern of β-glycine is not available in the literature. By consideration of literature descriptions42,45 and comparison of photographs in ref 45 with Figure 23, panel a or b, it was concluded that the needle crystals are β-glycine. Its PXRD pattern (Figure 25a) is different from the patterns of other forms reported in the literature.54 By comparison of the PXRD patterns in Figure 25, panels b and c, to the published PXRD patterns,54 it was confirmed that γ-glycine was not produced in

Figure 23. Needle crystals generated with stirring experiments at S ) 2 (R form). (a) Normal addition (PEG into water) with slow stirring, (b) reverse addition with fast stirring, and (c) prism crystals transformed from needle crystals after 10 min [∼100×, bar ) 200 µm].



Figure 24. Platelet crystals generated by CIJ mixing without stirring at S ) 2 (R form) [∼40×, bar ) 500 µm].

our experiments, and both the prism and platelet crystal forms are R-glycine. When placed together in a saturated solution, the prism and platelet crystals will coexist without any observable changes or transformations. (4) Discussion. The glycine-H2O-PEG300 system is different from the other systems studied. The more viscous aqueous PEG300 solution now acts as an antisolvent instead of the solvent. Quasi-emulsions are not generated under the conditions of the solute being dissolved in the less viscous phase as demonstrated in part I.8 Therefore, under either CIJ or agitated beaker experiments followed by stirring (fast or slow), rapid homogeneous mixing is achieved within seconds. The metastable form (β-glycine) will then be generated first according to Ostwald’s “Rule of Stages”9 and later transforms to stable R form. For the condition of CIJ mixing without subsequent stirring, quasi-emulsion formation did not occur as was established from the absence of a Tyndall effect in the quiescent solution.8 There were, however, indications of refractive index gradients, i.e., larger regions of unmixed fluid. The induction time was now several minutes instead of the several seconds under stirring conditions. Large platelets of the R form appeared. Without stirring, molecules apparently have enough time to pack well and approach the more stable forms, probably due to transport or diffusion between larger packets of unmixed fluid reducing the rate of supersaturation generation. The results with glycine then confirm the general hypothesis that transport limitations owing to segregated fluid tends to favor the stable polymorphic form while rapidly mixing solvent and antisolvent favors metastable forms. Conclusion The quasi-emulsion model suggests that a metastable polymorphic form would appear under homogeneous mixing conditions, while the stable form would be favored under quasiemulsion conditions. The metastable form that appears would be expected to be governed by relative nucleation kinetics, while the thermodynamically stable form is presumed to appear due to diffusion limitation in a quasi-emulsion resulting in a lower supersaturation environment. To verify this prediction, studies were carried out in four different crystallization systems involving polymorphism using aqueous PEG300 and water as solvents/antisolvents. The results, summarized in Table 6, are consistent with the hypothesis that crystallization from quasiemulsions favors the formation of the stable polymorphic form.

Figure 25. PXRD patterns of crystals shown in (a) Figure 23a (β form of glycine), (b) Figure 23c (R form of glycine), and (c) Figure 24 (also R form of glycine).

This study involved only one solvent/antisolvent system, so that the hypothesis cannot be fully verified without testing in other solvent systems. It is certainly true that particular solvent/ antisolvents can favor particular polymorphs. However, we believe that there is nothing unique about PEG300/H2O as a chemical system, except the viscosity ratio, that would cause


Wang and Kirwan

Table 6. Polymorphic Forms Generated under Different Mixing Conditionsa mixing conditions solute

rapid homogeneous

MP

metastable form (Form II)

PABA SAb glycine

metastable form (β form) metastable form (R + γ forms) metastable form (β form)

slow homogeneous stable and metastable forms (Form I or mixture of I and II)

segregation (quasi-emulsion) stable form (Form I) stable form (R form) stable and metastable form (β/β + γ/γ forms) stable form (R form)

a The appearance of metastable forms suggests that crystallization kinetics are important in the process. b The γ form appeared under all conditions for this system.

the observed and hypothesized behavior in the four different solute systems studied. This statement is further supported by the observations in our companion paper8 in this issue on the crystallization of salicylic acid from quasi-emulsions that formed from various aliphatic alcohol/water systems only when the viscosity ratio exceeded ∼3. After further confirmation, the quasi-emulsion model might be used to direct the development of industrial crystallization processes involving polymorphism. During the development of a process, mixing conditions can be selected to favor quasiemulsion formation to try to ensure that the stable polymorph is formed and identified. Similarly, rapid mixing without quasiemulsion formation could help to produce and identify metastable forms that might be produced in the process. It is also important to ensure that mixing conditions do not change upon process scale-up (as they often do) as this might result in different polymorphic forms being produced. References (1) McCrone, W. C. Polymorphism, in Physics and Chemistry of the Organic Solid State; Fox, D., Labes, M. M., Weissberger, A., Eds.; Wiley Interscience: New York, 1965. (2) Haleblian, J. K.; McCrone, W. C. J. Pharm. Sci. 1969, 58, 911929. (3) Haleblian, J. K. J. Pharm. Sci. 1975, 64, 1269-1288. (4) Brittain, H. J. Polymorphism in Pharmaceutical Solids; M. Dekker: New York, 1999. (5) Bernstein, J. Polymorphism in Molecular Crystals; Oxford University Press: New York, 2002. (6) USFDA, Section 505(j)(5)(B) of the Federal Food, Drug, and Cosmetic Act. (7) USFDA, Section 505(j)(4)(A) of the Federal Food, Drug, and Cosmetic Act. (8) Wang, X.; Gillian, J. M.; Kirwan, D. J. Cryst. Growth Des. 2006, 6, 2214-2227. (9) Ostwald, W. F. Z. Phys. Chem. 1897, 22, 289-330. (10) Kawashima, Y.; et al. Congr. Int. Technol. Pharm. 5th., 1989. (11) Nocent, M.; et al. J. Pharm. Sci. 2001, 90, 1620-1627. (12) Espitalier, F.; Biscans, B.; Laguerie, C. Chem. Eng. J. 1997, 68, 95102. (13) Mersmann, A. Crystallization Technology Handbook, 2nd ed.; Marcel Dekker: New York, 2001. (14) Cashell, C.; Corcoran, D.; Hodnett, B. K. J. Cryst. Growth 2004, 273, 258-265. (15) Roelands, C. P. M.; ter Horst, Joop H.; Kramer, Herman J. M.; Jansens, Peter J. J. Cryst. Growth 2005, 275, e1389-e1395. (16) Johnson, B. K.; Prud’homme, R. K. AIChE J. 2003, 49, 2264-2282.

(17) Lin, X. Jiegou Huaxue 1983, 2, 213-218. (18) Lindpaintner, E. Microscopic Study of Polymorphic Substances. II. Mikrochemie 1939, 27, 21-41. (19) Lai, T. F.; Marsh, R. E. Acta Crystallogr. 1967, 22, 885-893. (20) Alleaume, M.; Salas-Ciminago, G.; Decap, J. J. Compt. Rend., Ser. C 1966, 262, 416-417. (21) Gracin, S.; Rasmuson, A. C. Cryst. Growth Des. 2004, 4, 10131023. (22) The Merck Index, 12th ed.; Merck and Co.: Rathway, NJ, 1996. (23) O’Connor, B. H.; Maslen, E. N. Acta Crystallogr. 1965, 18, 363. (24) Alleaume, M.; Decap, J. Acta Crystallogr. 1965, 18, 731. (25) Alleaume, M.; Decap, J. Acta Crystallogr. 1965, 19, 934. (26) O’Connell, A. M.; Maslen, E. N. Acta Crystallogr. 1967, 22, 134. (27) McLachlan, D. X-ray Crystal Structure; McGraw-Hill: New York, 1957. (28) Lin, H. O.; Baenziger, N. C.; Guillory, J. K. J. Pharm. Sci. 1974, 63, 145. (29) Sekiguchi, K.; Tsuda, Y.; Kanke, M. Chem. Pharm. Bull. 1975, 23, 1353-1362. (30) Burger, A. Sci. Pharm. 1973, 41, 290-303. (31) Toscani, S.; et al. Pharm. Res. 1995, 12, 1453. (32) Toscani, S.; et al. Pharm. Res. 1996, 13, 151. (33) Portieri, A.; et al. Magn. Reson. Chem. 2004, 42, 313-320. (34) Yakowitz, M. L. J.sAssoc. Off. Anal. Chem. 1948, 31, 656. (35) Watanabe, A. Naturwissenschaften 1941, 29, 116. (36) Watanabe, A. Yakugaku Zasshi 1942, 62, 501. (37) Lin, H. O.; Guillory, J. K. J. Pharm. Sci. 1970, 59, 972-975. (38) Albrecht, J.; Corey, R. B. J. Am. Chem. Soc. 1939, 61, 1087. (39) Marsh, R. E. Acta Crystallogr. 1958, 11, 654. (40) Fisher, E. Ber. Dtsch. Chem. Ges. 1905, 38, 2917. (41) Iitaka, Y. Nature 1959, 183, 390. (42) Iitaka, Y. Acta Crystallogr. 1960, 13, 35. (43) Iitaka, Y. Acta Crystallogr. 1958, 11, 225. (44) Iitaka, Y. Acta Crystallogr. 1961, 14, 1. (45) Ferrari, E. S.; Davey, R. J.; et al. Cryst. Growth Des. 2003, 3, 5360. (46) Drebushchak, T. N.; et al. Acta Crystallogr. 2002, E58, o634-o636. (47) Weissbuch, I.; Leiserowitz, L.; Lahav, M. AdV. Mater. 1994, 6, 953. (48) Garetz, B. A.; Matic, J.; Myerson, A. S. Phys. ReV. Lett. 2002, 89, 175501. (49) Zaccaro, J.; Matic, J.; Myerson, A. S.; Garetz, B. A. Cryst. Growth Des. 2001, 1, 5. (50) Sakai, H.; et al. J. Cryst. Growth 1992, 116, 421. (51) Boldyreva, E. V.; et al. J. Therm. Anal. Calorim. 2003, 73, 409418. (52) Boldyreva, E. V.; et al. J. Therm. Anal. Calorim. 2003, 73, 419428. (53) Fichter, S. B. S. Thesis, Department of Chemical Engineering, University of Virginia, 1997. (54) Cao, W.; et al. J. Pharm. Biomed. Anal. 2002, 30, 1111.

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