Crystallization of Artemisinin from Chromatography Fractions of

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Crystallization of Artemisinin from Chromatography Fractions of Artemisia annua Extract Chandrakant Ramkrishna Malwade, Hannes Buchholz, Ben-Guang Rong, Haiyan Qu, Lars PorskjæR Christensen, H. Lorenz, and A. Seidel-Morgenstern Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00399 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 11, 2016

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Organic Process Research & Development

Crystallization of Artemisinin from Chromatography Fractions of Artemisia annua Extract Chandrakant Ramkrishna Malwade1, Hannes Buchholz2, Ben-Guang Rong1, Haiyan Qu1, Lars Porskjær Christensen1, H. Lorenz2, A. Seidel-Morgenstern2,3 1

Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Campusvej 55, 5230 Odense Denmark 2

Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany

3

Institute of Process Engineering, Otto von Guericke University, 39106 Magdeburg, Germany

ACS Paragon Plus Environment


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For Table of Contents only


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KEYWORDS Artemisinin, dihydroartemisinic acid, artemisinic acid, impurities, solid-liquid equilibrium, crystallization, chromatography

ABSTRACT

Crystallization is an inevitable step in the purification of artemisinin either from the plant Artemisia annua or from reaction mixtures of semi-synthetically produced artemisinin. Rational design of crystallization process requires knowledge about the solid-liquid equilibrium in a given solvent system and effect of impurities on it. In the present work, a crystallization process was designed to purify artemisinin from fractions of a flash chromatography column effluent collected after injecting extracts of Artemisia annua leaves. The fractions from chromatography containing artemisinin were combined together into one fraction and the impurities present in this fraction were identified. The solubility of artemisinin in the mobile phase used for chromatography, i.e., n-hexane–ethyl acetate mixture of varying compositions was measured at 25 °C, 15 °C and 5 °C, respectively. The collective effect of impurities present in the combined fraction on solid-liquid equilibrium of artemisinin was evaluated by measuring the solubility of artemisinin in the combined fraction at same temperatures. The results show that the impurities present in the combined fraction increase the solubility of artemisinin. Finally, the crystallization of artemisinin from the combined fraction designed on the basis of artemisinin solubility data was carried out in two steps by adding an antisolvent and cooling crystallization. The yield of artemisinin obtained in the process was 50 % and it was found that the impurities present in the combined fraction at given concentration do not affect crystallization of artemisinin.



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1. Introduction Artemisinin is an important natural product recommended by World Health Organization (WHO) for use in combination with other drugs against Plasmodium falciparum induced malaria.1 The discovery of the artemisinin as a novel therapy for malaria was recently acknowledged by the Nobel Prize to a Chinese scientist, Youyou Tu.2 More recently, it has been found that artemisinin and its derivatives possess antiviral and anticancer activity.3,4 Artemisinin is obtained mainly from dried leaves of the plant Artemisia annua L. (sweet wormwood). Existing processes include extraction of dried leaves of A. annua by using organic solvents, ionic liquids or supercritical fluids and subsequent purification of artemisinin from crude extract. The processes described in the literature for production of artemisinin from plant biomass or produced semi-synthetically use a crystallization step for the final purification of artemisinin.5–12 The application of crystallization for separation and purification of crystalline materials requires a comprehensive knowledge of the fundamental solid-liquid equilibria, which form the thermodynamic basis of all crystallization processes. The solubility characteristics of a solute in a given solvent at different temperatures are essential for the selection of a method of crystallization. The solubility phase diagrams that are widely used in the conceptual design of crystallization processes occupy a central place in the crystallization process synthesis strategies.13–16 These diagrams describing the composition space of a given system can be partitioned into different regions by thermodynamic boundaries. Different regions of the phase diagram can be navigated by several ways e.g. cooling or heating, addition or removal of solvent, stream combination or splitting, and addition or removal of mass separating agent to obtain the desired product.17 Another important aspect of crystallization process design is the presence of impurities in the solution containing desired product and their effect on the crystallization


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process. The presence of impurities in the system can influence the efficiency of crystallization through its effect on crystallization kinetics, solution thermodynamics and crystal interface structure. The consequences of impurities present in the system can be enhancement, complete suppression, retardation of the crystal nucleation and growth or sometimes no effect at all on the crystallization.18 Moreover, the presence of impurities during the crystallization is known to modify the final product qualities such as crystal morphology, crystal size distribution and crystal habit.19,20 Therefore, a thorough understanding of the crystallization systems containing impurities is very important in order to engineer the final products of desired qualities. The effect of impurities on solution thermodynamics and kinetics can be expressed quantitatively in terms of its effect on solubility and metastable zone widths of the desired product in a particular solvent system.21 Most of the processes used for manufacturing of artemisinin report poor yield during crystallization and attribute it to the interference of impurities on the crystallization of artemisinin.9,22 However, there have been fewer efforts spent in the direction of evaluation of impurities effect on the solid-liquid equilibrium of artemisinin. Lapkin et al.22–24 investigated the effect of co-metabolites (impurities) such as camphor, dodecanoic acid, 1,8-cineol, casticin, a casticin 3’-O-glucoside and deoxyartemisinin on solubility of artemisinin in n-hexane–ethyl acetate (95:5 v/v) by using computational COSMO-RS method and reported that some cometabolites increased the solubility of artemisinin. Suberu et al.25 have also studied effect of casticin on the solubility of artemisinin in n-hexane–ethyl acetate (95:5 v/v); however, at different concentrations (two orders of magnitude lower mole fraction than Lapkin et al.22), and noticed only a marginal difference in solubility of artemisinin. This illustrates the concentration of the impurities as an important factor in assessing the effect of impurity on solubility of



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artemisinin. In addition, the effect of methoxylated flavonoids (casticin, retusin and artemetin) on crystallization of artemisinin has also been investigated by Suberu et al.25 and they found that these impurities influence crystallization yield considerably despite having no effect on solubility of artemisinin. Previously, Malwade et al.10 have proposed and evaluated a three step process for recovery of artemisinin from dried leaves of plant A. annua as shown in Fig. 1. At first, artemisinin was extracted from the leaves with dichloromethane by maceration. Dichloromethane was used as extraction solvent due to the high solubility of artemisinin in it and lower boiling point facilitating easy recovery of solvent from the extract. The crude extract obtained was fractionated with flash column chromatography to obtain fractions containing artemisinin. The fractions containing artemisinin were dried, dissolved in dichloromethane, and then subjected to two steps of antisolvent crystallization, where acetonitrile and water were used as antisolvents in step 1 and 2, respectively. An additional cooling crystallization step was used to obtain remaining artemisinin from mother liquor. However, the overall yield of artemisinin in the crystallization

Figure 1. Process flow sheet for isolation and purification of artemisinin from leaves of the plant Artemisia annua.10


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step was found to be very low (47 %), which might be due to: (1) poorly selected crystallization parameters, (2) the influence of impurities present in the fractions or (3) a combination of both. Due to the industrial importance of this process for manufacturing of artemisinin, improvement of the crystallization step on the basis of thermodynamic parameters and understanding of effect of impurities present in the chromatography fractions could be advantageous. In the present work, the solubility of artemisinin in the mobile phase used for fractionation of the A. annua dichloromethane extract, i.e., n-hexane–ethyl acetate mixtures of varying composition, was measured at different temperatures. Analysis of the chromatography fractions containing artemisinin by Malwade et al.26 revealed the presence of coumarin, casticin, artemisinic acid, dihydroartemisinic acid, a polyacetylene and artemisinin related compounds as major impurities. As mentioned earlier, the presence of impurities can alter the crystallization process significantly thereby demanding investigation of effect of individual impurities on the solid-liquid equilibrium of artemisinin. However, most of the impurities present in the combined fraction containing artemisinin are not available commercially, which is the case in most of the natural products purification problems. Therefore, the collective effect of these impurities on solid-liquid equilibrium of artemisinin was determined in this work by measuring the solubility of artemisinin as described earlier by Horváth et al.,27 in the combined fraction. Finally, a suitable crystallization strategy was devised on the basis of solubility data of artemisinin and implemented to obtain artemisinin from the combined fraction. 2. Materials and Methods 2.1 Materials Dried leaves of the plant Artemisia annua (Artemis cultivar) were provided by Aarhus University, Department of Food Science, Denmark in 2011 and stored at room temperature.



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Artemisinin (purity > 98 %) was obtained from Xiang Xi Holley Pharmaceutical Co. Ltd. in China. Since, artemisinin exists in two polymorphic forms (orthorhombic and triclinic),28,29 the polymorphic form of artemisinin used in this work was confirmed with X-Ray Powder Diffraction (XRPD) as orthorhombic. Organic solvents n-hexane (purity > 98 %) and ethyl acetate (purity > 99.8 %) purchased from VWR Chemicals Denmark were used for solubility measurement. Silica gel 60 (particle size 0.04 – 0.06 mm, 230−400 mesh ASTM) obtained from Merck KGaA, Darmstadt, Germany was used as stationary phase for flash column chromatography purification. 2.2 Experimental section 2.2.1

Composition of chromatography fraction

Chromatography column packed with silica gel 60 (300 g) as stationary phase conditioned in n-hexane was used to purify dichloromethane extract (15 g) of A. annua leaves. Height and diameter of packed silica gel in chromatography column was 17 cm and 7 cm, respectively. The column was eluted with mobile phase (n-hexane-ethyl acetate) at an average flow rate of 40 ml/min by applying constant pressure on top of the column with nitrogen gas. Gradient type of elution was used as described in the previous work.10 Total 35 fractions of 100 ml each were collected. The fractions obtained after flash column chromatography purification were analyzed with thin layer chromatography (TLC) to identify the fractions containing artemisinin. The fractions containing artemisinin were then combined together and analyzed by high-performance liquid chromatography with diode array detection (HPLC-DAD) and/or HPLC-charged aerosol detection (CAD) to determine its composition. The impurities present in the fraction were identified on the basis of their retention times obtained previously by Malwade et al.26 and liquid chromatography-mass spectrometry (LC-MS) analysis. Major impediment in the quantification


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of identified impurities from the combined fraction was unavailability of their standards except coumarin. CAD is characterized by its near universal response independent of chemical structure for nonvolatile and some semivolatile analytes at constant composition of organic solvent in the mobile phase.30 Therefore, CAD was used in this work for quantification of impurities present in the fraction except coumarin and solvent composition. Dionex UltiMate 3000 RSLC system equipped with DAD and CAD detector was used to analyze samples. ZORBAX Eclipse XDBC18 reverse phase column of dimensions 4.6 mm × 150 mm and packing material particle size of 5 µm was used for separation. The column temperature was adjusted to 35 °C. Sample injection volume of 10 µl and eluent flow rate of 0.8 ml/min was used. Isocratic elution consisting of 70 % acetonitrile added with 0.1 % formic acid as modifier was used in order to obtain universal response from CAD to all impurities present in the combined fraction. Coumarin content and solvent composition was determined on the basis of their UV response. The chromatograms and calibration curves for artemisinin and coumarin are provided in the supplementary information. Determined composition of the combined fraction and the chemical structures of artemisinin and impurities from the combined fraction are shown in Table 1 and Fig. 2, respectively. It has been reported earlier that the content of artemisinin as well as other secondary metabolites is reduced drastically over prolonged storage of A. annua leaves at room temperature.1,31 Table 1. Composition of the combined fraction containing artemisinin. Name of compound Artemisinin (1) Artemisitene (2) Dihydroartemisinic acid (3) Artemisinic acid (4) Arteannuin B (5) Coumarin (6) Solvent (n-hexane–ethyl acetate)

Concentration (mg/ml of fraction) 1.82 0.015 0.0745 0.01 0.0051 77.65:22.35 v/v



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Figure 2. Chemical structure of artemisinin (1) and impurities [artemisitene (2), dihydroartemisinic acid (3), artemisinic acid (4), arteannuin B (5) and coumarin (6)] found in the combined fraction. The combined fraction obtained from flash column chromatography in this work contain fewer impurities and also reduced artemisinin content compared to the fraction obtained previously from the same biomass and using the same procedure.26 This clearly indicates that the quality of A. annua leaves used in this work has been deteriorated due to prolonged storage. 2.2.2

Solubility measurement

The solubility of artemisinin was measured by using a classical isothermal technique. The solubility of artemisinin was measured in n-hexane–ethyl acetate mixtures of composition 75:25, 80:20, 85:15, 90:10, and 95:05 v/v at temperature 25 °C, 15 °C, and 5 °C, respectively. Each measurement was carried out in triplicates. The procedure included preparation of samples in 10 ml glass vials by suspending excess amount of artemisinin in 1 ml solvent. The sealed glass vials containing suspension and magnetic stirrer were then placed in a jacketed reactor maintained at constant temperature (accuracy ± 0.1 °C) under stirring speed of 400 rpm for 6 h to attain


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equilibrium. After the equilibration, the samples were transferred into a syringe and filtered through 0.2 µm PTFE filters to separate liquid phase from suspended solids. In case of measurements at 15 °C and 5 °C, syringes and filters pre-cooled at respective temperature were used to avoid re-dissolution of artemisinin during the filtration step. The liquid phase obtained was diluted appropriately with acetonitrile and analyzed by HPLC-CAD to determine the artemisinin content. The solid phase was dried and analyzed with Raman spectrometer to determine the polymorphic form of suspended artemisinin.29 The specific individual effect of certain impurities present in the combined fraction on solubility of artemisinin was not possible to measure due to unavailability of standard samples. Therefore, the collective effect of all impurities present in the combined fraction on the solubility of artemisinin was evaluated by comparing the artemisinin solubility in the combined fraction and in pure solvents. Three samples of 10 ml each were taken from the combined fraction, dried and re-constituted in 10 ml of n-hexane–ethyl acetate mixture of composition 75:25, 85:15 and 95:5 v/v, respectively. Another set of three samples of 20 ml each from the combined fraction was re-constituted in 10 ml of n-hexane–ethyl acetate mixture of composition 75:25, 85:15 and 95:5 v/v, respectively. The solubility of artemisinin was measured in the re-constituted fractions at temperatures 25 °C, 15 °C, and 5 °C by using isothermal technique as mentioned above in triplicates. 3. Results and Discussion 3.1 Solubility of artemisinin in n-hexane–ethyl acetate mixtures Solubility of artemisinin measured in n-hexane–ethyl acetate mixtures of varying composition at different temperatures is shown in Table 2. It is evident from Table 2 that the solubility of artemisinin decreases with increase in amount of n-hexane in the solvent mixture at a constant



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temperature. It is also clear from the data that the solubility of artemisinin decreases with decrease in temperature, however the magnitude of decrease is less significant as compared to the sharp decrease obtained by increasing amount of n-hexane in the solvent mixture. This trend clearly suggests the possibility of n-hexane being used as antisolvent to crystallize artemisinin from the combined fraction. FT-Raman spectroscopy of suspended artemisinin confirmed that there is no change in the artemisinin polymorph in the n-hexane–ethyl acetate solvent at temperatures studied in the present work. In order to understand the solute-solvent interactions, activity coefficients of artemisinin are determined from the measured solubility data by using simplified Schröder-Van Laar equation (Eqn 1). ln. =

∆ 1 1 −

(Eqn 1)

Where, is solubility in mole fraction, is activity coefficient, ∆ is enthalpy of melting, is gas constant, is melting temperature, and is temperature of solubility measurement. Enthalpy of melting (∆ = 78.4 J/g and melting temperature ( = 151.4 °C of artemisinin measured by Horosanskaia et al.29 were used. Table 2. Solubility of artemisinin measured in n-hexane–ethyl acetate mixtures at different temperatures (n = 3). n-hexane: ethyl acetate

Solubility of artemisinin (mg/ml of solution) 25 °C

15 °C

5 °C

(v/v) 75:25 80:20 85:15 90:10 95:05 100:0

16.2 ± 0.49 11.5 ± 0.53 8.30 ± 0.35 5.10 ± 0.10 3.72 ± 0.02 2.30 ± 0.14

13.7 ± 0.07 10.3 ± 0.28 6.60 ± 0.28 4.50 ± 0.42 2.60 ± 0.04 1.70 ± 0.14

11.0 ± 0.10 7.90 ± 0.35 5.80 ± 0.32 3.90 ± 0.14 2.20 ± 0.01 1.10 ± 0.42


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Table 3. Activity coefficients of artemisinin in n-hexane-ethyl acetate solvent mixtures determined from the measured solubility data. Activity coefficient

Sample 25 °C

15 °C

5 °C

n-hexane-ethyl acetate (75:25 v/v)

9.36

11.31

13.49


12.60

14.27

16.25


17.04

21.84

26.24


28.83

31.90

36.88


33.81

54.23

58.15

The calculated activity coefficients of artemisinin in n-hexane–ethyl acetate mixtures are shown in Table 3. It is evident from Table 3 that the activity coefficients of artemisinin in nhexane–ethyl acetate mixtures of varying composition at 25 °C, 15 °C and 5 °C are greater than 1, indicating the non-ideal behavior of solutions. Also, it indicates that the non-polar repulsive forces are dominant between the solute and solvent molecules. Furthermore, it is obvious that the non-polar repulsive forces are increasing further with increasing composition of n-hexane in the mixture and with decreasing temperature.



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Figure 3. Plan for crystallization of artemisinin from combined fraction devised on the basis of solubility of artemisinin. Point 1, 2, 3-4, and 5 represents artemisinin concentration in combined fraction, after evaporation of solvent from combined fraction, after addition of antisolvent, and at the end of cooling crystallization, respectively. Fig. 3 shows a possible concept for crystallization of artemisinin from combined fraction designed on the basis of solubility data of artemisinin. As shown in Fig. 3, the point 1 indicates the concentration of artemisinin in the combined fraction at room temperature. It is clear from Fig. 3 that the combined fraction is highly under saturated. Therefore, supersaturation has to be achieved, which can be done by evaporating certain amount of solvent from the combined fraction as indicated by path 1 to 2 in Fig. 3. Azeotropic composition of n-hexane–ethyl acetate mixture at 25 °C atmospheric pressure is 71 vol% n-hexane.32 Vapor-liquid equilibrium data for n-hexane–ethyl acetate mixture determined by Aspen plus indicates their azeotropic composition as 77.5 vol% n-hexane at 40 °C and 410 mbar, which is co-incidentally same as composition of


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n-hexane–ethyl acetate in the combined fraction (point 1). Therefore, evaporation of solvent from the combined fraction at 40 °C and 410 mbar is expected to follow the path 1 to 2 as shown in Fig. 3 provided that the solutes in the fraction do not shift the azeotropic composition. The solution at point 2 is saturated at 25 °C. Solubility data indicates that n-hexane can be used as antisolvent to create supersaturation and subsequently to crystallize artemisinin from the combined fraction as shown in Fig. 3 by path 2 to 3 and 3 to 4. However, taking into consideration the dilution factor due to addition of n-hexane, path from 2 to 4 should follow the dotted arrow as shown in Fig. 3. Finally, it should be possible to crystallize more artemisinin by cooling down the solution from 25 °C to 5 °C as shown by path 4 to 5 in Fig. 3 assuming that the impurities do not inhibit crystallization of artemisinin during this step also. Theoretically, the amount of artemisinin equivalent to the solubility difference between point 2 to 4 and point 4 to 5 should crystallize during antisolvent and cooling crystallization, respectively; assuming that the impurities do not inhibit crystallization of artemisinin. 3.2 Effect of impurities on solubility of artemisinin The solubility of artemisinin measured in the combined fraction, which is re-constituted into nhexane–ethyl acetate mixtures of composition 75:25, 85:15 and 95:05 v/v as mentioned in section 2.2.2, is shown in Table 4. The change in the solubility of artemisinin in re-constituted fractions relative to the artemisinin solubility in n-hexane–ethyl acetate mixtures is also shown in



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Table 4. Solubility of artemisinin in the combined fraction re-constituted in n-hexane–ethyl acetate mixtures (n = 3). The change in solubility is relative to the artemisinin solubility in n-hexane–ethyl acetate mixtures of same composition at a given temperature.

Hex:EA (v/v)

Solubility in fractiona

Solubility in fractionb

(mg/ml of solution)

(mg/ml of solution)

25 °C

15 °C

5 °C

25 °C

15 °C

Change in solubility (%) 5 °C

25 °C

15 °C

5 °C

75:25

17.2 ± 0.72

15.6 ± 0.48

13.7 ± 0.89

18.0 ± 0.07

15.6 ± 0.11

14.8 ± 0.89

a 6.37

85:15

9.80 ± 0.12

8.28 ± 0.11

7.62 ± 0.09

10.5 ± 0.14

8.70 ± 0.18

8.13 ± 0.18

18.5

26.6

25.5

32.3

31.5

40.2

95:05

4.08 ± 0.13

2.93 ± 0.36

2.72 ± 0.14

4.43 ± 0.09

3.13 ± 0.12

3.02 ± 0.02

9.73

19.3

13.0

20.5

23.8

37.6

a

b 11.3

a 14.4

b 14.4

a 24.8

b 34.6

Concentration of impurities is same as in original fraction; bConcentration of impurities is twice the concentration in original fraction.


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Table 4. It is evident from Table 4 that the solubility of artemisinin in fractions is higher than the solubility of artemisinin in n-hexane–ethyl acetate mixtures at a given temperature, indicating that the impurities present in the combined fraction increase the solubility of artemisinin. This conforms to the earlier observation that the impurities extracted by solvents such as n-hexane and petroleum ether enhance the solubility of artemisinin (co-solvency effect), thereby making them the solvents of choice in commercial extraction processes despite having low solubility of artemisinin.33,34 The solubility increase is getting more pronounced towards the lower temperature and it is also evident that the solubility increase is highest in n-hexane–ethyl acetate composition of 85:15 v/v. Moreover, the solubility of artemisinin is marginally higher in the concentrated fraction than the original fraction, although statistically not very significant. The solubility results clearly indicate that the impurities present in the combined fraction shift the artemisinin solubility curves shown in Fig. 3 upwards, which can have implications on the suggested plan for crystallization of artemisinin shown in Fig. 3. In step 1 i.e., evaporation of solvent from the combined fraction, the saturation concentration indicated by point 2 in Fig. 3 may also shift upwards and so does the artemisinin concentration indicated by point 3 and 4. Estimated activity coefficients of artemisinin in re-constituted fractions are shown in Table 5. It is clear from Table 5 that the activity coefficients of artemisinin in re-constituted fractions shows similar trend as activity coefficients of artemisinin in n-hexane-ethyl acetate mixtures. However, it is evident from Table 3 and Table 5 that the activity coefficients of artemisinin are lower in re-constituted fractions due to presence of impurities.



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Table 5. Activity coefficients of artemisinin in re-constituted combined fractions containing impurities determined from the measured solubility data. Activity coefficient

Sample 25 °C

15 °C

5 °C

a

8.31

9.36

10.74

a

Fraction (n-hexane-ethyl acetate (85:15 v/v))

14.48

18.30

18.06

Fraction (n-hexane-ethyl acetate (95:05 v/v))a

31.60

50.56

51.24

Fraction (n-hexane-ethyl acetate (75:25 v/v))b

8.13

9.80

9.82


13.38

17.30

17.57


30.45

46.72

47.56

Fraction (n-hexane-ethyl acetate (75:25 v/v))

a

Concentration of impurities same as in original fraction; bConcentration of impurities is twice the concentration in original fraction. 3.3 Crystallization of artemisinin from the chromatography fraction The crystallization of artemisinin from the combined fraction was carried out according to the plan (Fig. 3) devised on the basis of artemisinin solubility in n-hexane–ethyl acetate mixtures. The combined fraction (180 ml) obtained from flash column chromatography purification having a composition as described in Table 1 was evaporated at 40 °C, 410 mbar pressure to the final volume of 18 ml and allowed to cool down to 25 °C. Following the confirmation of no crystals present in the concentrated combined fraction, a sample was taken and appropriately diluted with ethyl acetate for HPLC analysis. The concentrated fraction was transferred to a jacketed reactor maintained at 25 °C (± 0.1 °C) under constant stirring and 60 ml of n-hexane was added to it at the rate of 10 ml/min. During the addition of n-hexane, samples were taken at regular time intervals and analyzed by HPLC-CAD to determine artemisinin concentration. At the end of the addition, the solution was maintained at constant temperature under stirring for 10 min. The solution was then cooled down to 5 °C at the cooling rate of 0.58 °C/min. Cooled solution is maintained at 5 °C for 30 min followed by filtration to collect the crystallized solid. The progress


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of antisolvent and cooling crystallization of artemisinin from the concentrated fraction along with solubility of artemisinin in n-hexane–ethyl acetate solvent mixtures and the combined fraction is shown in Fig. 4. The unsaturated combined fraction travels into the supersaturated region after evaporation of solvent as shown by path 1 to 2 in Fig. 4. As expected due to the azeoptropic composition, the solvent composition in the concentrated fraction (point 2) remains almost constant as original combined fraction after evaporation. Moreover, there were no crystals observed in the concentrated fraction (point 2) even after entering into the supersaturated region. Evaporation of solvent from the combined fraction (path 1 to 2) also increases the concentration of impurities present in it. High concentration of impurities at point 2 might increase the solubility of artemisinin as observed in the solubility measurement experiments and thereby preventing its crystallization. It is obvious from Fig. 4 that addition of n-hexane as antisolvent

Figure 4. Crystallization of artemisinin from the combined fraction and solubility of artemisinin in n-hexane–ethyl acetate mixtures (blue lines) and the combined fraction re-constituted in nhexane–ethyl acetate mixtures (red lines). Solid lines are drawn for the visual guidance.



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in the concentrated combined fraction (path 2 to 3) reduces the solubility of artemisinin and that the desupersaturation profile of artemisinin follows the solubility curve of artemisinin in the combined fraction. Desupersaturation profile of artemisinin during cooling crystallization from 25 °C to 5 °C (path 3 to 4) is also shown in Fig. 4. HPLC analysis of solution and crystals confirmed the crystallization of only artemisinin (purity 99 %). This clearly indicates that the desupersaturation profile of artemisinin follows the thermodynamic boundary set by the solubility curve and that the impurities do not influence the crystallization of artemisinin at given concentrations in both, antisolvent and cooling crystallization steps. The artemisinin concentration and n-hexane composition determined from HPLC analysis of the liquid phase at steps 1 to 4 is shown in Table 6. The theoretical yield of artemisinin for antisolvent crystallization (step 1) and cooling crystallization (step 2) calculated from the solubility in the combined fraction according to Eqn 2 and Eqn 3, respectively is also shown in Table 6 along with the experimental yield obtained. Theoretical yield in Step 1 =

)* × ,* − )- × ,- × 100 )* × ,*

(Eqn 2)

Theoretical yield in Step 2 =

)- × ,- − )0 × ,0 × 100 )- × ,-

(Eqn 3)

Where, S2, S3, and S4 represent the solubility of artemisinin in the combined fraction with nhexane-ethyl acetate composition 77.65:22.35 v/v at 25 °C as shown by point 2* in Fig. 4, the combined fraction with n-hexane-ethyl acetate composition 95:05 v/v at 25 °C, and the combined fraction with n-hexane-ethyl acetate composition 95:05 v/v at 5 °C, respectively. The solubility of artemisinin in the combined fraction with n-hexane-ethyl acetate composition 77.65:22.35 v/v at 25 °C has been approximated (16 mg/ml) from solubility curve in Fig. 4. V2, V3, and V4 represent the volume of the combined fraction before addition of anti-solvent, after addition of


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anti-solvent, and after cooling down to 5 °C, respectively. Excess molar volume of n-hexaneethyl acetate binary mixture at composition of 95:05 v/v is negligibly positive and also, the change in density of binary mixture from 25 °C to 5 °C is not significant.35 Therefore, the volume V3 and V4 is calculated as the volume V2 plus the volume of n-hexane added during the antisolvent crystallization step to make the final composition of n-hexane in the solution to 95 % by volume. Table 6. Stepwise progress of crystallization of artemisinin from combined fraction along with theoretical yield and actual yield of artemisinin.

Combined fraction (1)

77.65

Artemisinin concentration (mg/ml solution) 1.82

Evaporation (2)

77.90

17.81

-

-

Antisolvent crystallization (3)

95.15

3.50

4.8

14.84

Cooling crystallization (4)

95.05

2.07

33.30

40.85

Crystallization steps

n-hexane (vol%)

Overall yield (wt%)

Theoretical yield (wt%) -

Actual yield (wt%) -

49.63

The theoretical yield during the antisolvent crystallization step is only 4.8 wt% as the difference in solubility of artemisinin in solvent mixture with 77.65 and 95 vol% n-hexane is compensated by the dilution occurred due to n-hexane addition. However, the actual yield obtained during antisolvent crystallization is 14.84 wt% as shown in Table 6. The actual yield obtained during the experiment is different from the theoretically expected yield because the experiment did not follow the expected path as can be seen in Fig. 4. Furthermore, in cooling crystallization step the actual yield obtained is slightly higher than the theoretical yield as shown in Table 6. Thus, the overall yield of artemisinin crystallized during the process (49.63 wt%) is marginally higher than the yield obtained previously. However, this process circumvents the



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complicated antisolvent crystallization steps that required evaporation of solvent from the fraction and re-dissolution into another solvent as reported earlier.10 The results obtained in this work clearly show that the impurities present in the combined fraction at given concentration do not influence the crystallization of artemisinin. A supplement to the monograph on artemisinin in The International Pharmacopoeia describes the quality requirements for use of plant derived artemisinin in the production of antimalarial pharmaceuticals.36 It specifically raises the concerns about the presence of impurities such as artemisitene and a diastereomer of artemisinin, 9-epi-artemisinin, in the final product due to their less sensitivity to the UV detection used in most of the laboratories. The official method mentioned in the monograph for checking the purity of artemisinin uses HPLC with UV detection at 210 nm. However, in this work the purity of artemisinin obtained in the process is analyzed with HPLC-CAD and LC-MS. The purity of artemisinin obtained in this work is found to be 99 %, which conforms to the specifications mentioned in the monograph. FT-Raman analysis of the artemisinin crystals obtained in this work confirmed the polymorphic form of artemisinin as orthorhombic. 4. Conclusion Solid-liquid equilibrium data of artemisinin in n-hexane–ethyl acetate solvent mixtures has been determined and applied to design a crystallization process for recovery of artemisinin from chromatography fractions of A. annua extract. Impurities present in the combined chromatography fraction along with artemisinin were identified and quantified. The solubility of artemisinin was also measured in the fraction in order to assess the collective effect of impurities present in the fraction on solid-liquid equilibrium of artemisinin and thereby on its crystallization. It was found that the presence of impurities increased the solubility of


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artemisinin. Furthermore, the increase in solubility of artemisinin in fraction was found to be dependent on concentration of impurities. Increase in the solubility of artemisinin in presence of impurities confirmed the fact that co-solvency, a phenomenon related to the enhancement of solubility in presence of other compounds, plays an important role in extraction of artemisinin and should be taken into consideration while selecting solvents for extraction. Estimated activity coefficients of artemisinin explain the decrease in repulsive forces between solute and solvent in presence of impurities. Solubility data of artemisinin in n-hexane–ethyl acetate solvent mixtures clearly indicated the possibility of combining antisolvent and cooling crystallization in order to crystallize artemisinin from the fraction. Both, antisolvent crystallization and cooling crystallization steps followed the thermodynamic boundary of artemisinin determined from solid-liquid equilibrium data. This clearly indicated that the impurities present in the fraction at given concentration did not significantly influence the crystallization of artemisinin despite affecting the solubility of artemisinin. ASSOCIATED CONTENT Supporting Information HPLC calibration curves for artemisinin and coumarin, chromatograms of the combined chromatography fraction, mother liquor, and artemisinin obtained during crystallization, and a table containing mass balance during crystallization. AUTHOR INFORMATION Corresponding author * Chandrakant R. Malwade



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* E-mail: [email protected]; Phone: 0045 65508669 Funding sources This work was supported by The Danish Council for Strategic Research, Denmark (project No. FI 2101-08-0048). ACKNOWLEDGMENT Authors would like to thank Brian Hermansen, Hanne V. Hemmingsen and Dorthe Lillesø for their technical assistance. REFERENCES

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Crystallization of Artemisinin from Chromatography Fractions of

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