*3.2. Design of the Seeded Cooling Crystallization for Separation of CUR*

Based on the solubility curves of CUR in presence of the main impurities and the observed nucleation behavior of pure CUR in the respective solvents, four seeded cooling crystallization processes were derived to separate CUR from the CURD mixtures as illustrated in Figure 7.

**Figure 7.** Design of the seeded cooling crystallization processes of CUR based on the solubility curves of CUR in presence of the main impurities (solid lines) and the nucleation border of pure CUR (dashed lines) in acetone (1), 50/50 acetone/2-propanol (2), 50/50 acetone/acetonitrile (3) and acetonitrile (4). Black solid/dashed lines with arrows are imaginary curves representing the variation of the CUR concentration during the crystallization process. (Tstart/Tend: start/end temperature of the cooling step; stars: temperature of seed addition; ΔcTD: maximal depletion of CUR from solution).

The starting temperatures of the crystallization processes in the 50/50 mixtures of acetone/2-propanol and acetone/acetonitrile and in acetonitrile were set at 60 ◦C. To avoid uncontrolled evaporation of acetone, 45 ◦C was chosen as the starting temperature in this solvent.

The temperatures at which seeds of pure CUR (form I) were introduced into the acetone, 50/50 acetone/acetonitrile and acetonitrile solutions were chosen to be at least 5 K below the saturation temperature of CUR (approximately in the first third of the metastable region). However, the metastable region of CUR in 50/50 acetone/2-propanol (Figure 7, purple lines) is significantly closer than for the other three solvent systems. Therefore, the seeds were added at approximately half of the metastable region. An overview of the selected process parameters for the four seeded cooling crystallization processes is given in Table 4.

**Table 4.** Overview of the selected crystallization process parameters.


Tstart/Tend: start/end temperature of cooling step; Tsat: CUR saturation temperature; Tseeds: seeding temperature.

The initial concentrations of CUR in the crude CURD mixtures were selected in accordance with the set starting temperatures to guarantee undersaturation of CUR in the starting solutions. The amounts of the crude solids, the process solvents and the calculated initial CUR content in the four starting solutions are listed in Table 5.


**Table 5.** Amount of initial substances used in the four crystallization processes.

m(CURD), m(Solvent): amounts of CURD mixture and solvent used for the starting solution; cstart(CUR): calculated concentration of CUR in the starting solution; cend=csat: concentration of CUR at the end of the cooling process, equal to the respective saturation concentration, from solubility study; ΔcTD(CUR): max. possible change of CUR concentration at the end of the cooling process, calculated based on the thermodynamic values; mmax: maximal achievable mass of CUR, calculated based on the thermodynamic values.

#### *3.3. Implementation of the Purification Process*

In the first step, the four initial crude solutions were prepared using the corresponding amount of the crude solid mixture in the respective solvent (Table 5). The seeded cooling crystallization of CUR was conducted in a second step following the four process trends shown in Figure 7. Starting at set temperatures, the unsaturated clear solutions were cooled down to 0 ◦C at a linear rate of 10 K/h. After exceeding the corresponding saturation temperature, seed crystals of pure CUR form I (ca. 50 mg) were introduced into the supersaturated solution at Tseeds (Table 4). At the end of the cooling process at 0 ◦C, the obtained product suspensions were stirred for further 0.5 h. Subsequently, solid-liquid phase separation was carried out on suction filters (pore size of filter paper 0.6 μm). To remove adhering mother liquor from the filter cake, the collected crystals were washed with about 100 g of cold acetone (< 0 ◦C, in processes 1-3) or with acetonitrile (< 0 ◦C in process 4). Then, dried at 40 ◦C, the purity of CUR and the yield were analyzed. During the washing process with acetone a visible dissolution of the filter cake was observed, caused by the high solubility of CUR in acetone (about 5 wt% at 0 ◦C). Accordingly, lower product yields could be assumed in the processes of using acetone as washing solvent (in processes 1-3).

The results of the four conducted seeded cooling crystallizations are summarized in Table 6. The maximum thermodynamically possible yield of CUR ηTD was calculated according to Equation (1), the total product yield of CUR η according to Equation (2).

$$
\eta\_{\rm TD}(\text{CLIR}) = \frac{m\_{\rm product} \cdot \text{CLIR product content}}{m\_{\rm max}} \tag{1}
$$

$$\eta(CLIR) = \frac{m\_{product} \cdot CLIR \text{ product content}}{m\_{start}(CLIR)} \tag{2}$$

Table 6 shows that in the 50/50 acetone/2-propanol mixture (process 2), the highest purity of CUR (99.4%) in the crystalline product was achieved. However, only 13% of the initial CUR content in the crude mixture was recovered. Crystalline CUR with decreasing purity of 95.7%, 92.3% and 90.1% but increasing total product yields of 31%, 55% and 62% was obtained from acetone, acetonitrile and 50/50 acetone/acetonitrile, respectively. The lower total yields from acetone and acetone/2-propanol solutions are partly associated with the enhanced CUR solubility at the final process temperature compared to the acetonitrile-containing solutions (see Figure 7), which, however, does not explain the extremely low yield achieved in the latter case.

The obtained purity results further verify that BDMC could completely be removed from the crystalline products within a single separation step, while the content of DMC was noticeably reduced. The presence of DMC as impurity in the products can be probably attributed to the most similar

molecular structure of DMC and CUR (Figure 1). It can be postulated that DMC molecules compete with CUR in the solution upon forming the main crystal lattices. To ascertain, whether DMC is present near CUR in the crystalline form or as amorphous phase, the four crystallization products were analyzed by XRPD. In Figure 8 the corresponding patterns are compared with the commercial solid standards of DMC and CUR.

**Table 6.** Results of the four seeded cooling crystallization processes. Table columns containing the products purity and yield are highlighted in grey.


mstart(CUR): calculated amount of CUR in the mixture of CURDs; mmax: max. achievable mass of CUR, based on the thermodynamic values; m(product): mass of the crystalline product gained; ηTD(CUR): max. thermodynamically possible yield of CUR; η(CUR): total product yield of CUR.

**Figure 8.** XRPD patterns of four crystallization products with increasing CUR content.

Since all XRPD reflexes in the diffractograms from the crystalline products can be clearly distinguished and are uniformly on the baseline, the presence of an amorphous fraction in the solid products cannot be confirmed. Moreover, all XRPD patterns seem to be identical to the CUR solid standard. Despite the increasing DMC content in the crystalline products (0.6–9.9%), none of the recorded patterns can be clearly assigned to the solid standard of DMC. Only a slight shift of single reflexes of crystalline products is indicated with increased DMC content in the solids.

Due to the strong similarity of CUR and DMC molecules, partial miscibility at the solid state might be a possible explanation here. However, dependent on the instrument and the structural similarity of the compounds used the limit of detection of the XRPD method is known to be 5–7 wt% and 1 wt% in best cases. Thus, incorporation of DMC molecules is not readily assessable by XRPD at these low contents. Clarifying this issue requires further work which was out of the scope of this paper.
