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Intergrowth of two aspirin polymorphism observed with Raman spectroscopy

Yuka Tsuri, Mihoko Maruyama, Hiroshi Y. Yoshikawa, Shino Okada,

Hiroaki Adachi, Kazufumi Takano, Katsuo Tsukamoto, Masayuki Imanishi,
Masashi Yoshimura, Yusuke Mori

PII: S0022-0248(19)30645-1
DOI: https://doi.org/10.1016/j.jcrysgro.2019.125430
Reference: CRYS 125430

To appear in: Journal of Crystal Growth

Received Date: 10 October 2019

Revised Date: 10 December 2019
Accepted Date: 14 December 2019

Please cite this article as: Y. Tsuri, M. Maruyama, H.Y. Yoshikawa, S. Okada, H. Adachi, K. Takano, K.
Tsukamoto, M. Imanishi, M. Yoshimura, Y. Mori, Intergrowth of two aspirin polymorphism observed with
Raman spectroscopy, Journal of Crystal Growth (2019), doi: https://doi.org/10.1016/j.jcrysgro.2019.125430

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Intergrowth of two aspirin polymorphism observed with Raman

spectroscopy

Yuka Tsuri,1 Mihoko Maruyama,1,2 Hiroshi Y. Yoshikawa,1,3 Shino Okada,4 Hiroaki

Adachi,1,4 Kazufumi Takano,2,4 Katsuo Tsukamoto,1,5 Masayuki Imanishi,1 Masashi

Yoshimura,6 and Yusuke Mori1,4

1
Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita City, Osaka

565-0871, Japan

2
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University,

1-5, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto City, Kyoto 606-8522, Japan

3
Department of Chemistry, Saitama University, 255, Shimookubo, Sakura-ku, Saitama

City, Saitama 338-8570, Japan

4
SOSHO Inc., 2-1, Yamadaoka, Suita City, Osaka 565-0871, Japan

5
Graduate School of Science, Tohoku University, 6-3, Aoba, Aramaki, Aoba-ku, Sendai

City, Miyagi 980-8577, Japan

6
Institute of Laser Engineering, Osaka University, 2-1, Yamadaoka, Suita City, Osaka

565-0871, Japan
Corresponding author: Mihoko Maruyama

Phone number: +81-6-6879-7705

E-mail address: maruyama@cryst.eei.eng.osaka-u.ac.jp

Abstract

Polymorphism of aspirin, one of the important pharmaceutical compounds, crystallizes

in two similar structures (form I and form II). This similarity causes the intergrowth

structure containing both form I domain and form II domains in the same crystal. The

intergrown crystal is often observed, however, the crystallization process of the

intergrown crystal was unclear. In order to reveal the crystallization process, we

performed in situ observation of the growth using the crystal obtained immediately after

the nucleation with Raman spectroscopy. The temporal change of Raman spectral

pattern showed that form I grew on the surface of the form II seed crystal. In addition,

we investigated spatially where the growth of form I was initiated. It was suggested that

the crystal growth rate of form I on form II surface depends on the local surface

supersaturation. We concluded that the crystallization of the intergrowth of aspirin was

caused by the crystal that was form II at the nucleation being covered by form I as the

growth proceeded.
1. Introduction

An organic molecule often crystallizes in different crystal structures; this

phenomenon referred to as polymorphism [1]. Each polymorph has different physical

and chemical properties including solubility, dissolution rate, stability. Therefore,

polymorph control is a significant issue in various fields, for example, in the

pharmaceutical, food and cosmetic industry.

Aspirin (acetylsalicylic acid, C9H8O4) is a well-known compound used as an

antipyretic and analgesic drug. Its polymorph control attracts enormous interest and has

often been discussed in the literature [2–5]. Aspirin has been reported to crystallize in

two similar structures: form I and form II. The structure of form I was first determined

by Wheatley in 1964 [6]. In 2005, the second structure (form II) was elucidated by

Zaworotko et al [7]. However, this form was not pure form II but rather coexisted with

the form I phase in the same crystal (intergrown structure) [3,4]. A form II crystal that

did not contain a form I domain was obtained by adding aspirin anhydrate [5]. Table 1

lists the crystallographic data of form I [3,6,8,9] and form II [4]. They crystallize to the

same monoclinic structures. The crystal structure of form II closely resembles that of

form I, and the molecular arrangements of the two forms along the b-axis direction are

identical as shown in Figure 1 [10]. In both form I and form II, a dimer is formed
between carboxyl groups on the (010) plane. Focusing on the b-axis bonding, in the

case of form I, the adjacent molecules form a centrosymmetric dimer between acetyl

groups. With regard to form II, adjacent molecules form a hydrogen bonding chain

instead of forming a dimer. Thus, there is only a slight difference in the crystal

structures of form I and form II, and an intergrowth structure containing both forms is

often obtained [3–5]. For this reason, polymorph control of aspirin is exceedingly

difficult. Understanding the intergrowth process is expected to play an important role in

enabling polymorph control.

Table 1. Crystallographic data of aspirin form I and form II crystals.

Polymorph Form I Form II

Crystal system Monoclinic Monoclinic
Space group P21/c P21/c
a, Հ 11.2776(2) 12.1515(10)
b, Հ 6.5517(1) 6.5064(5)
c, Հ 11.2741(2) 11.3677(9)
β, deg 95.837(1) 111.574(3)
V, Հ 3
828.71(2) 835.79(12)
Z 4 4
Fig. 1. The polymorphic forms of aspirin: (a) form I and (b) form II crystals.

In our previous study, we have succeeded in the selective crystallization of form II

single crystal by controlling the nucleation frequency. In addition, we attempted the

in-situ observation of the phase transformation from pure form II into a form I by

adding form I to the solution containing the pure form II [11]. Phase transformation is a

phenomenon which can be described as a result of a difference in solubility between

polymorphs [12]. Generally, when multiple polymorphs exist in the same solution, the

metastable phase completely dissolves, and only the stable phase grows thereafter

[13–15]. Therefore, observation of phase transformation process can be utilized to

confirm the presence of metastable phase. [16] When inducing the phase transformation

of aspirin from form II into form I of aspirin, change in the external shape was not
observed. However, after identifying the crystal phase through Raman spectroscopy, we

found that the original pure form II transformed into form I. It is presumed that form II

did not dissolve completely because the growth of form I and dissolution of form II

proceeded simultaneously on form II local crystal surface. It is apparent that such

phenomenon is the origin of the crystallization of the intergrowth structure. There are as

yet, however, no studies that reported in-situ observation of this phenomenon; in

particular, the phase identification during intergrowth process. In this study, therefore,

we attempted to observe in situ the crystallization of the intergrowth structure. Raman

spectroscopy allows for the in-situ observation of a new crystalline phase on the original

crystal surface [17–19]: thus, in Experiment 1, the development of the intergrown

structure was monitored through the temporal change of Raman spectral pattern during

the phase transformation. In addition, in Experiment 2, in order to investigate the site of

the intergrowth initiation, polymorphism identification after crystal growth as observed

using bright field optical microscopy.

2. Experimental

2.1. Preparation of solution

Aspirin (form I, assay 99.50–101.00%; Novacyl) was used as the growth material. The
material was confirmed to be form I via Raman spectroscopy. Acetonitrile, super

dehydrated (purity 99.8%; FUJIFILM Wako Pure Chemical Corporation) was used as

the solvent in all experiments. A solution of aspirin with a concentration of 93.4 mg/mL

was prepared in a 100 mL Teflon vessel. The solution was heated at 60°C for 3 h in an

isothermal heater (As One DO-450A) and stirred with a Teflon-coated magnetic stirring

bar (stirring speed 500 rpm) to completely dissolve the solutes. After the filtration

(ADVANTEC DISMIC 25HP020AN, 0.2 µm), the solution was dispensed in 2.5 mL

aliquots into 19 mL glass vials (Experiment 1) or 0.5 mL aliquots into an observation

cell (Experiment 2). The samples were placed in an incubator at 55°C, cooled to 25°C at

a constant cooling rate of 3°C/h, and maintained at that temperature for one day. The

supersaturation of form I was calculated by the formula σ = (C - Ce)/Ce, where C is the

aspirin concentration, and Ce is the aspirin (form I) solubility. The supersaturation of

form I at 25°C was calculated to be σI = 0.2.

2.2. Experiment 1: In-situ observation of crystallization of the intergrowth structure

with Raman spectroscopy

The seed crystals including form II (number of crystals: ~30), which were obtained

by the supercooling method, were added to the supersaturated solution prepared by the

process described above. The glass vial was then sealed (Fig. 2 (a)). One of the seed
crystals which were added to the solution was then chosen and its Raman spectrum was

measured every 2 min over a 30 min measurement period during the crystal growth (Fig.

2 (b)). Temporal change in the peak intensity of form I appearing around 290 cm-1 was

investigated.

The Raman microscope (Raman-11; Nanophoton Corp.) with an output power of 50

mW, a focused laser beam diameter of a few micrometers, and an excitation wavelength

of 532 nm was used to record the Raman spectra.

2.3. Experiment 2: Observation of crystal growth and spatial polymorph identification

of intergrown crystal

The seed crystals including form II were added to the supersaturated solution and the

crystals were grown by the same process as the crystals in Experiment 1 (Fig. 2 (a)).

The added crystals were observed under bright field optical microscopy. Bright-field

images were taken every 10 min over a 70 min measurement period. After the

observation, the crystals were taken out of the solution and dried. When removing the

crystals from the solution, residual solution was wiped off from the surface of the

crystals in order to prevent the renewed nucleation. Polymorphism of the dried crystals

was identified by Raman spectroscopy (Fig. 2 (c)). We selected two crystals from

among the crystals for measurement by Raman spectroscopy. One was the crystal that
had grown from the initial stage. The other was the crystal that nucleated at the later

stage. Here, the initial stage refers to the time from the start of the observation until a

crystal apart from the added crystal was nucleated. In the experiment, the initial stage

was the first four minutes. The later stage refers to the time thereafter. The Raman

microscope was used under the same conditions as in Experiment 1. The intensity ratio

(Pa/Pb) was compared for each crystal. Pa is the intensity of the peak appearing only on

form I around 290 cm-1, and Pb is the intensity of the peak appearing on both form I and

form II around 350 cm-1. It is expected that the intensity ratio (Pa/Pb) would be higher

for the form I crystal [20]. The Raman spectra was measured on two surfaces of each

crystal. One measurement was obtained from the surface of a crystal grown in contact

with the observation container (the bottom of glass vials), and the other measurement

was obtained from the surface of a crystal grown in contact with the solution. To

identify the polymorph, seeded crystals were measured at four points on each surface,

and later nucleated crystals were measured at two points on each surface. The number

of the measurement points of each crystal was different due to the difference in their

crystal size after growth.

Fig. 2. Schematic illustration of the experimental procedure. (a) Microcrystals were

added as seed crystals into a low supersaturated solution (supersaturation σ = 0.2), and

Experiment 1 (b) or Experiment 2 (c) was performed. (b) In-situ phase identification

was performed throughout the crystallization process with Raman spectroscopy. (c) The

crystal growth process was observed with a bright field optical microscopy. After the

growth process finished, the crystals were taken out of the solution and polymorphs

were identified by Raman spectroscopy.

3. Results

3.1. Experiment 1: In-situ observation of crystallization of the intergrowth structure

with Raman spectroscopy

Figure 3 (a) shows bright-field images of the crystal measured by Raman spectroscopy

and Figure 3 (b) shows the Raman spectrum corresponding to each image. Figure 3 (c)

shows the temporal change of peak intensity of form I from 14 to 30 min (taken every 2

min) after adding the seed crystal. If the crystal is too small, the spectral intensity is too

low to identify the crystalline phase. Therefore, the measurement was initiated only

after the crystal achieved a size sufficient for obtaining a high-intensity spectrum. As a

result, 14 min after adding the seed crystals to the supersaturated solution, the peak of

form I with low intensity was obtained was low and the crystal at this moment was

identified as form II. However, the peak intensity of form I gradually increased as the

measurement continued. This phenomenon was observed within the initial 30 min of the

measurement. The result suggests that the crystal was initially a form II crystal when

nucleated but the form I phase grew on its surface as the crystal growth proceeded.
Fig. 3 (a) Bright-field images of the growing crystal taken during Raman spectroscopy

(scale bar: 200 µm). (b) In-situ Raman analysis corresponding to the crystal shown in

(a). (Insert) Zoom-in of the range from 250 to 350 cm-1. (c) Temporal change of the

peak intensity of form I appearing around 290 cm-1 in panel (b). The intensity of form I

increased over time.

3.2. Experiment 2: Observation of crystal growth and spatial polymorph identification

of intergrown crystal

Figure 4 presents the observed progression of crystal growth. The presence of the

added seed crystals was confirmed immediately after starting observation and grew and

finally showed a plate-like morphology of about several mm in size. These seed crystals

nucleated at the initial stage in this experiment (referred to as “seeded crystals”),

contained interior many crack-like defects. As shown in a bright-field image taken 10

min after the addition of seed crystals, newly nucleated crystals were also observed

(referred to as “later nucleated crystals”). These crystals possessed a plate-like

morphology and had fewer interior defects than the seeded crystals, with steps on the

surface.

Seeded crystal and a later nucleated crystal were observed by Raman spectroscopy to

investigate the spatial distribution of polymorph. Figures 5 (a)-(d) show the Raman

spectra of each dried crystal, and Figure 5 (e) shows the intensity ratio (Pa/Pb). In the

later nucleated crystal, the intensity ratios of both the surface in contact with solution

and the surface in contact with glass bottom were high. This result indicates that the

whole crystal was form I. On the other hand, in the case of seeded crystal, the intensity

ratio of solution side surface and bottom side surface were different. The Pa/Pb was high
on the surface grown in contact with the solution, but it was low on the surface grown in

contact with the bottom of the glass vial.

Fig. 4. Bright-field images of crystal taken every 10 mins during crystallization process.

The black arrowhead indicates a seeded crystal that was observed at 0 min. The crystal

indicated by the white arrowhead was first observed at 10 min and was later nucleated

after the addition of seed crystals. Scale bar: 1 mm.

Fig. 5. (a)(b) Raman spectra of the later nucleated crystal. Spectra were measured at two

points on the surface grown in contact with the solution (a), and at two points on the

surface grown in contact with the bottom of the glass vial (b). Scale bar: 500 µm. (c)(d)

Raman spectra of the seeded crystal at the initial stage. Spectra were measured at four

points on the surface grown in contact with the solution (c), and at four points on the

surface grown in contact with the bottom of the glass vial (d). Scale bar: 1 mm. (e) The

intensity ratio (Pa/Pb) on each surface of each crystal. For the later nucleated crystal,
open circles show Pa/Pb on the surface of the solution side, and open diamonds show

Pa/Pb on the surface of the bottom side; for the seeded crystal, filled circles show Pa/Pb

on the surface of the solution side, and filled diamonds show Pa/Pb on the surface of the

bottom side.

4. Discussion

Figure 3 (a) showed that new cracks appeared inside of the seeded crystal during the

growth. In Experiment 2, the seeded crystal also contained the interior defects, as shown

in Fig. 4. These crystals showed the intergrowth of form I and form II. Whereas, the

later nucleated crystal, which was presumed as pure form I, had fewer defects inside

than the intergrown crystals. These findings suggest that we can roughly judge

intergrown crystals and pure form I crystal from the number of contained defects inside.

The intergrowth process is schematically illustrated in Fig. 6. Initially, a form II crystal

was added to the supersaturated solution (a seeded crystal). Form I and form II could

grow on this seeded crystal surface given that the solution was supersaturated for both

form I and form II polymorphs. When both polymorphs grow simultaneously, contact

between the step fronts of form I and form II polymorphs on the surface will occur. In

aspirin, it was reported that the mismatch of molecular packing between form I and
form II can cause defects within crystal [2]. Therefore, it is assumed that the observed

defects occurred by the growth of both polymorphs during the initial stage. As growth

proceeded, growth of form I appears to become dominant over the form II as growth

proceeded. The difference in supersaturation between form I and form II with the

supersaturation of stable phase being generally higher than that of the metastable phase

leads to the dominance of form I [21]. Consequently, the generated defects were likely

covered by the continual growth of form I, with the defects observed within the crystal

from the earlier stages of growth.

The result shown in Fig. 5 suggests that the crystal growth rate of form I depends on

the local supersaturation. The surface reaction of a crystal was strongly dependent on

location—i.e. whether the crystal was in contact with the solution or the bottom of the

glass vial. When a crystal was in contact with the bottom of a glass vial, the gap

between the crystal and the glass surface was narrow and the local growth can deplete

the local solution of nutrients leading to a low supersaturation in the gap regions. In

such a case, the growth of form I proceeds very slowly. Whereas, when a crystal surface

contact with the solution, the solute is readily supplied and form I grew faster than the

surface of the bottom side. In our previous study, growth of form I advanced from the

corner of the crystal surface in contact with the solution, accompanied by a phase
transformation from pure form II to form I [11]. In the last stage of the crystallization

process, the solution concentration, and hence supersaturation, probably became lower

than the equilibrium point of form II as form I growth advanced. This leads to the

initially grown form II crystal becoming covered with growth form I. We suggest that

this is the crystallization process of the intergrown crystal containing aspirin form I and

form II.

Fig. 6. Schematic illustration of the intergrowth process of two aspirin polymorphs. (a) Schematic of

the solubility curves of form I and form II. The red circle shows the concentration of solution. (b)

Stereoscopic schematics of the crystal during formation of the intergrowth. (c) The cross-sectional

illustration of the crystal shown in (b).

5. Conclusion

In this study, the in-situ observation of the intergrowth of two aspirin polymorphs

with Raman spectroscopy was presented. An intergrowth model of aspirin form I and

form II based on spatial and temporal phase identification with the Raman spectroscopy

and optical observation of crystals was proposed. Seeded form II crystal remained

inside as the intergrown crystal became covered with form I. Growth rate of form I on

form II surface varied depending on the location within the growth vessel due to

differences in the local surface supersaturation. The intergrowth of aspirin form I and

form II occurred without apparent crystal dissolution or external shape change. Thus,

this phenomenon is difficult to observe under a bright-field optical microscope. These

findings indicate that there is the possibility of unnoticed polymorphism after the

nucleation.

Given the importance of polymorph control to the pharmaceutical industry, this study

highlights the necessity for phase identification in the early stage of crystal growth (just

after crystal nucleation).

6. Declarations of interest

None.
Acknowledgement

We thank Lion Corp. for cooperating with this work. This work was supported by

Research Fellowships of JSPS (No. 18J40134), Shiseido Female Researcher Science

Grant, Caterpillar STEM award 2019 and the Osaka University Program for the Support

of Networking among Present and Future Researchers for M. M. This work was also

supported by Grant-in-Aid for Challenging Exploratory Research (B) Nos. 15K13380

and 17H02774 for Y. M. This work was partially supported by Grant-in-Aid for

Challenging Exploratory Research (B) No. 19H02613 and the joint research project of

the Institute of Laser Engineering, Osaka University (under contract subject

“2018B2-YOSHIKAWA. H” and “2019B2-YOSHIKAWA”) for H. Y. Y.

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Highlight

We observed intergrowth process of aspirin form I and II by Raman spectroscopy.

Form I grew on the surface of form II.
Intergrowth structure was composed of form II buried by form I.
Conflict of Interest Forms:

The authors declare no conflicts of interest associated with this manuscript.

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