Recovery of Artemisinin from a Complex Reaction Mixture Using

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Recovery of Artemisinin from a Complex Reaction Mixture Using Continuous Chromatography and Crystallization Zoltán Horváth,† Elena Horosanskaia,†,‡ Ju Weon Lee,† Heike Lorenz,† Kerry Gilmore,§ Peter H. Seeberger,§,∥ and Andreas Seidel-Morgenstern*,†,‡ †

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany Chair of Chemical Process Engineering, Otto von Guericke University, Magdeburg, Germany § Max Planck Institute of Colloids and Interfaces, Potsdam, Germany ∥ Institute for Chemistry and Biochemistry, Free University Berlin, Berlin, Germany ‡

ABSTRACT: Artemisinin, a secondary metabolite of sweet wormwood, is the basis for the production of the most effective antimalarial drugs. Since the amount of artemisinin currently produced from plants is not sufficient to treat the worldwide malaria cases, an effective semisynthetic method was developed recently that is capable of producing artemisinin from dihydroartemisinic acid (DHAA). DHAA is a byproduct obtained during the extraction of artemisinin from plant leaves. The photocatalytic reaction to convert DHAA to artemisinin can be performed continuously in a tubular reactor using toluene as a solvent. The reactor effluent contains besides artemisinin the photocatalyst (dicyanoanthracene) and several compounds that are structurally similar to artemisinin, including unreacted DHAA starting material. To isolate artemisinin from the reaction mixture, two separation techniques were applied, crystallization and chromatography. The solid obtained by seeded cooling crystallization was highly enriched in artemisinin but contained also traces of the photocatalyst. In contrast, using a variant of continuously operated multicolumn simulated moving bed (SMB) chromatography, which splits the feed into three fractions, we were able to recover efficiently the photocatalyst in the raffinate stream. The extract stream provided already almost pure artemisinin, which could be finally further purified in a simple crystallization step. are byproducts of artemisinin extraction from A. annua,15 several low-cost semisynthetic alternatives that produce artemisinin from artemisinic acid or dihydroartemisinic acid were reported.16,17 Recently, a continuous photochemical reaction that produces artemisinin from dihydroartemisinic acid was reported.18,19 This reactor can produce artemisinin from dihydroartemisinic acid, which is available from the waste of the process of extracting artemisinin from the leaves of A. annua. Besides artemisinin, the reactor effluent contains the photocatalyst and several impurities and byproducts that are structurally related to the target molecule.19 Since the synthesis method can produce artemisinin continuously, continuous separation techniques are required for subsequent purification. Currently, continuous manufacturing that combines upstream and downstream processes is intensively investigated in the pharmaceutical industry to increase productivity and decrease costs.6,20,21 We demonstrated previously22 that continuously operated simulated moving-bed (SMB) chromatography can be directly connected to a flow reactor for the continuous production of certain target products. Also, systematic methods to design and optimize such coupled processes were introduced and validated.23 Effluents of a reaction process contain together with the target several byproducts, unconverted reactants, impurities, and eventually catalysts. Within a chromatographic elution train, typically the target component elutes somewhere in an

1. INTRODUCTION Natural products, often called secondary metabolites, are compounds that are commonly obtained from plants in low concentrations (0.01−3 wt %).1,2 Plants store these compounds as inactive components and activate them only in cases of danger (e.g., wounds, infections, natural enemies). Since early mankind’s history, natural products have found a wide usage as dyes, flavors, fragrances, stimulants, human poisons, and even as therapeutic agents. Nowadays over 10 000 plants with their secondary metabolites are known to have a medical usage, and over 90% of prevailing API’s are derived from natural products.3 The relative low availability of the natural products motivates the pharmaceutical industry to develop semisynthetic analogues.4 Hereby, proper production processes should be developed providing the target compounds with a high purity and yield.5 In particular efficient combinations of continuously operated reactors and separators possess the potential to improve process productivity and reduce production costs.6,7 There are more than 200 million malaria cases per year, and more than 650 000 people die from this disease each year.8 Currently artemisinin and its derivatives are the most effective antimalarial drugs. Due to the limited artemisinin availability in Artemisia annua (max. 1.4 wt % in dried plant material9), a stable production of artemisinin and its derivatives is required. Owing to the complexity of its molecular structure, the complete synthesis of artemisinin is not commercially viable, even though several synthetic methods have been reported.10−14 Since artemisinic acid and dihydroartemisinic acid have similar molecular structures to artemisinin and those © XXXX American Chemical Society

Received: February 18, 2015

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DOI: 10.1021/acs.oprd.5b00048 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Figure 1. Molecular structures of the target and major impurities (a: artemisinin, ARTE, b:dihydroartemisinic acid, DHAA, and c: 9,10dicyanoanthracene, DCA).

(HiperSolv CHROMANORM, VWR GmbH, Germany), ethyl acetate (LiChrosolv, Merck, Germany), and deionized water produced by a Milli-Q Advantage A10 system (Millipore Corporation, Billerica, MA, USA) were used. To dye TLC plates, a sulfuric acid (analysis grade, 95−97%, Merck, Germany) solution (10 vol. % in ethanol) was used. 2.2. Equipment. An analytical HPLC unit (Agilent 1200 Series, Agilent Technologies, Germany), consisting of a fourchannel pump, a column oven, a PDA detector, and an auto sampler, was used to quantify artemisinin with a reversed phase KINETEX RP-18 column (25.0 cm × 0.46 cm; 5 μm, Phenomenex, Germany). To develop a preparative chromatography method working with a toluene based solvent leaving the reactor (see below), in preliminary runs TLC analysis was used applying normal phase silica gel plates (aluminum oxide plate, Si60 F254, Merck, Germany), a UV lamp, and a flat bottom glass chamber (Camag, Switzerland). For preliminary preparative batch chromatography, a HPLC pump (AP 250, Armen Instrument, France), a manual injection valve (1/8″, Knauer, Germany), and a UV detector (K2501, Knauer, Germany) were used with a 50 mm I.D. column (NW50 DAC column, Merck, Germany) packed with normal phase silica gel (LUNA PREP, 10 μm, Phenomenex, Germany). For continuous chromatographic separation, a SMB unit that consists of a 48-port valve system (CSEP C9812, Knauer, Germany), four preparative pumps (K-1800, Knauer, Germany), one HPLC pump (Smartline 100, Knauer, Germany), and two UV-detectors (K-2501, Knauer, Germany), was used with four 25 mm I.D. columns (NW25 DAC column, Merck, Germany) packed with normal phase silica gel (LUNA PREP, 10 μm, Phenomenex, Germany). For crystallization, a jacketed 100 mL glass vessel equipped with a Pt-100 resistance thermometer (resolution 0.01 °C) connected to a thermostat (RP845, Lauda Proline, Germany) was used with a magnetic stirrer for agitation. During the crystallization process, the artemisinin content was measured with an attenuated total reflection Fourier transform infrared spectroscopy inline probe (ATR-FTIR, ReactIR45m, Mettler Toledo, Switzerland). A digital microscope (VHX-2000, Keyence, Germany) was used for the visualization of solid products after crystallization with 150 times magnification. To collect solid products after chromatographic separation and crystallization, a rotary evaporator (R-114 rotor and B481

intermediate position and necessitates a so-called center-cut separation. Modified SMB configurations have been introduced to achieve this task continuously including pseudo-SMB,24 integrated 8-zone SMB,25 SMB cascades,26 and 3F-SMB.27 These configurations were developed for constant solvent strengths (isocratic conditions). To perform continuous chromatographic separation in a gradient mode, a multicolumn countercurrent solvent gradient purification (MCSGP) process has been introduced.28 This process uses different solvent strengths in each zone to isolate a target component present in a complex mixture. Complementary to highly selective chromatographic separation processes, it is expedient to apply crystallization-based separation steps. Preprocessing the feed prior to chromatography simplifies the separation, while final polishing of the chromatographic product stream further increases purity. To achieve both objectives and to develop crystallization processes in a rational manner, an understanding of the specific solid− liquid equilibria (solubilities) in the system is essential. For utilization of cooling crystallization, the temperature dependences of the solubilities need to be known for the target and the key byproducts. Based on this thermodynamic information, a straightforward design of crystallization processes is possible.29,30 Here, two separation techniques, crystallization and simulated moving bed (SMB) chromatography, were applied experimentally to recover artemisinin from the reaction mixture obtained as the effluent of the photocatalytic reaction. The performances of both separation techniques applied alone and in combination were compared in terms of essential objective criteria as product purity and recovery yield.

2. EXPERIMENTAL SECTION 2.1. Materials. The reaction mixture was produced in a flow reactor.18,19 It mainly contains artemisinin, dihydroartemisinic acid, and 9,10-dicyanoanthracene. For crystallization experiment, the crude reactor effluent (total concentration: 122 g/L) was used. For preliminary batch and SMB chromatography, it was stored after the evaporation of solvent. Artemisinin standard (>97% purity) and 9,10-dicyanoanthracene, a photocatalyst standard (>98% purity), were purchased from TCI Chemicals. Dihydroartemisinic acid was purchased from Honsea Sunshine Biotech Co.,Ltd. (Guangzhou, China). Toluene (HiperSolv CHROMANORM, VWR, Germany), ethanol (Gradient grade for LC, Merck, Germany), acetonitrile B



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DCA were quantitatively analyzed to follow the progress of the different purification steps. Since the solvent toluene is not miscible with aqueous solvents and artemisinin has a limited solubility in aqueous solvents, the analytical RP-HPLC method (Table 1) is not suitable for preparative separations. For this reason, normalphase (NP) conditions with toluene as the main mobile phase were considered for the preparative chromatographic separation of artemisinin. 3.2. Solvent Screening for Crystallization. Due to the available solubility data of artemisinin in toluene and ethanol,32 the application of toluene as the reaction medium, and the possibility to apply ethanol as a strong solvent in gradient chromatography (Section 3.3), preliminary screening of the corresponding binary solvent mixtures was performed to select a suitable solvent for crystallization. Since the artemisinin solubility in pure toluene is rather high, and toluene is considered as a class 2 solvent, we decided to use a solvent mixture with toluene as minor component, and ethanol as a major component. The latter offered several advantages: ethanol acts as an antisolvent for artemisinin due to its low solubility therein and also is a harmless and cheap solvent. The solubility behavior and the accessible range of supersaturation (the width of the metastable zone) of artemisinin in toluene and ethanol as well as various mixtures of both were measured as a function of temperature. Artemisinin is highly soluble in pure toluene, and using only ethanol is not feasible according to the rather low solubility and the weak dependency of solubility on temperature, which would lead to significantly lower product amounts and yields. In Figure 3, obtained solubility

water bath, Büchi, Germany) and a vacuum-dry oven (VT 6060 M, Thermo Scientific, Germany) were used.

3. CHARACTERIZATION OF REACTOR EFFLUENT AND SELECTION OF CHROMATOGRAPHIC SYSTEMS AND SOLVENTS 3.1. HPLC Method for Analysis. In the continuous reactor, the reactant, dihydroartemisinic acid (DHAA, Figure 1, b) is converted to the target, artemisinin (ARTE, Figure 1a) by a photosynthetic mechanism with 9,10-dicyanoanthracene (DCA, Figure 1, c) as a photocatalyst.18,19 This reaction was performed in pure toluene as the solvent. According to the WHO pharmacopoeia library,31 the required minimum artemisinin purity is 97% using a proposed gradient reversed-phase (RP) HPLC assay. However, unlike the natural product, the reaction mixture contains the photocatalyst (DCA) and other reaction byproducts. Thus, adjusted conditions were used in analytical HPLC to identify artemisinin (Table 1). Figure 2 shows the comparisons of typical Table 1. Analytical HPLC conditions for artemisinin in the reaction mixture mobile phase composition flow rate column specification temperature [°C] injection volume UV wavelength

water/acetonitrile = 20:80 v/v 0.5 mL/min Phenomenex Kinetex RP-18 (25 cm × 0.46 cm, 5 μm) 20 °C 1.0 μL 210 nm

Figure 2. Analytical HPLC chromatograms of the reaction mixture, the target (ARTE), and major impurities (DHAA and DCA).

Figure 3. Solubility behavior of artemisinin as a function of temperature. Studied solvent compositions toluene/ethanol = 50:50 (■), 30:70 (●), 20:80 (▲), 0:100 (▼) (wt/wt) in the temperature range between 5 to 40 °C (black: pure artemisinin, red: with impurities, blue: 4 vol % of EtOAc in toluene).

chromatograms of the reactor effluent and the standard solutions of commercially available artemisinin, DHAA, and DCA to identify the artemisinin, DHAA, and DCA peaks. As shown in the reaction mixture chromatogram, impurities can be classified into two groups: one group contains strongly polar impurities (SPIs) including DHAA, and the other group contains weakly polar impurities (WPIs) including DCA. With this HPLC method, three major components, namely, artemisinin (the target) and two major impurities (DCA and DHAA), could be identified. Since the available standard of DHAA contained unknown impurities, only artemisinin and

curves are shown in the temperature range between 5 and 40 °C for the following solvents: toluene/ethanol = 50/50, 30/70, 20/80, 0/100 (wt/wt). For the 20:80 solvent composition, also the solubility curve of artemisinin in the presence of the impurities present in the reaction mixture was determined to evaluate possible effects on the solubility behavior. As expected, the solubility of artemisinin decreases with increasing ethanol content and decreasing temperature. According to the results of preliminarily crystallization C



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elution volume of artemisinin (VARTE/V0), and the selectivity of artemisinin to DCA (kARTE/kDCA) for various developing solvent compositions. The reduced elution volume and the selectivity were calculated from the converted retention factor (k, eq A2, Appendix A). Toluene, toluene/ethyl acetate, and toluene/isopropyl acetate mixtures show very good selectivities of artemisinin and DCA, and Rf,DHAA values are close to 0. Comparably large volumes are needed to elute artemisinin when pure toluene is used. Since the viscosity and boiling point of ethyl acetate (0.44 cP and 77.0 °C) are lower than those of isopropyl acetate (0.60 cP and 88.6 °C), toluene/ethyl acetate = 96:4 (v/v) was chosen as a mobile phase due to lower pressure drops and evaporation requirements. Figure 4b shows the TLC plate for this finally selected toluene based solvent mixture. With the selected mobile phase composition, the stronger bound impurities (including DHAA) are captured in the chromatographic column. The weaker bound impurities and the target components elute and are separated. Pure ethanol can be used to remove the impurities captured in the chromatographic column. This gradient elution method was first tested in preliminary batch column runs and then applied in a continuous multicolumn process. 3.4. Preliminary Batch Chromatography. To characterize the reactor effluent, single column batch chromatography was performed with the chosen gradient elution method in Section 3.3. As expected, with the chosen mobile and stationary phases (toluene/ethyl acetate = 96:4, v/v and LUNA PREP silica gel 10 μm, Phenomenex), the SPI did not elute. Pure ethanol could be used subsequently for the complete elution of SPIs. Figure 5 shows the chromatogram of a single column

experiments, the solubility of impurities present in the reaction mixture drastically decreased in solvents with ethanol contents above 80 wt % inducing their spontaneous nucleation. For that reason and also since the solubility curve of the reaction mixture in the 20/80 (wt/wt) of toluene/ethanol solvent was found to be close to the pure artemisinin solubility curve (Figure 3), this solvent composition was selected as a reasonable compromise to purify the reaction mixture by crystallization without attempting to further optimize the solvent composition. 3.3. TLC Screening To Identify a Suitable Preparative Chromatographic System. Also in NP chromatographic separation, the elution of the target component artemisinin occurs between of two major impurities (DCA and DHAA). However, in contrast to RP-conditions, DHAA has strong interactions with the bare silica adsorbent due to the carboxylic group. Since a stronger desorbent is required to elute the SPIs including DHAA, gradient elution is preferable for the centercut separation of artemisinin from the reaction mixture. For a fast qualitative screening of the composition of organic mobile phases, normal phase thin-layer chromatography (TLC) was applied. Several combinations of toluene containing solvent mixtures were evaluated. Figure 4a shows the determined retardation TLC factors Rf (eq A1, Appendix A), the reduced

Figure 5. Preliminary batch chromatogram for characterization of the reaction mixture.

preparative batch run. The injection volume and concentration of feed mixture were 15 mL and 81.8 g/L, respectively. The column dimension and loaded packing amount were 50 × 190 mm and 180 g (0.48 g/cm3 of the adsorbent bulk density), respectively. The mobile phase flow rate was 50 mL/min. The elution bands of WPIs, artemisinin, and SPIs were identified using accompanying TLC analysis. Three fractions (WPIs: 5− 15 min, artemisinin: 20−25 min, and SPIs: 45−56 min) were collected. All other effluents were collected together as a fourth

Figure 4. TLC screening results in various solvent compositions (a) and TLC chromatogram with the chosen solvent composition (b, toluene/ethyl acetate = 96:4, v/v). D



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waste fraction. The column effluent collected in the three fractions was evaluated by subsequent HPLC analysis using the analytical method described. The mass ratios of three fractions given above were determined after evaporation. The WPI fraction contains DCA, and the SPI fraction contains DHAA. By analyzing three characteristic product fractions, the reactor effluent contains 12 wt % of total solutes (69 wt % of artemisinin, 13 wt % of WPIs, and 18 wt % of SPIs). The weight percentage of the fourth waste fraction was less than 1.4 wt %.

4. PURIFICATION OF ARTEMISININ FROM THE REACTOR EFFLUENT After characterization of the reaction mixture, the two separation techniques described above, i.e., chromatography and crystallization, were chosen for the purification of artemisinin. Crystallization is a classical separation or purification process. The rejection of impurities by a growing crystal is principally highly specific with respect to the target molecule and an impurity to be depleted. However, the efficiency of purification by crystallization is strongly influenced by the nature of the impurities and the target molecule and also by the kinetics of the subprocesses nucleation and growth that might limit the purity achievable.33 To circumvent the stochastic character of nucleation seeded processes within metastable regions can be exploited, as done in our work. There are many chromatographic configurations capable to split feed streams continuously using in multicolumn SMB processes beyond the classical four-zone binary separator.24−28 The specificity of the pseudoternary separation problem investigated here, namely, the fact that one of the fractions (the SPI fraction) could be efficiently captured and then separately eluted, allows considering the feed just as a binary mixture of WPIs and artemisinin. Below will be described the applied three-zone open-loop SMB process with capture and regeneration steps. 4.1. Standalone Crystallization. According to the solubility data measured in Section 3.2, a crystallization process illustrated in Figure 6 was designed to purify artemisinin. The operation line of crystallization as well as the theoretical yield of artemisinin can be estimated from the solubility and nucleation border curves. Starting at 40 °C with a clear unsaturated solution (8.9 wt % of artemisinin), the system was cooled down linearly to 5 °C with a cooling rate of 6 K/h. After passing the artemisinin saturation line at 27 °C and introducing 50 mg of pure artemisinin seeds (standard material >97% purity) at 21 °C, a final concentration of 5 wt % of artemisinin was reached at 5 °C as indicated in Figure 6. At the end of the crystallization run, the suspension was stirred for 1 h at 5 °C and filtered via a 0.6 μm filter. The solid product was washed with 250 mL of cold (5 °C) ethanol in order to remove residual toluene and adhering mother liquor. The selected separation conditions guarantee the formation of the orthorhombic modification of artemisinin as the polymorph stable at ambient temperatures.34 The final separation product obtained via this crystallization process was analyzed by the analytical HPLC method described in Section 3.1. The measured chromatogram of the product is compared to that of the initial reaction mixture in Figure 7a. Except the photocatalyst DCA all impurities were removed by this single-step crystallization. The residues of DCA are clearly visible in the microscopic picture of the crystalline product (Figure 7b). As shown in Figure 6, the solubility of DCA in the

Figure 6. Crystallization procedure and trajectories together with solubility and supersolubility (nucleation border) curves of artemisinin in a solvent composition of toluene/ethanol = 20:80 (wt/wt) (upper part). The corresponding solubility curve of DCA in the solvent mixture and indication of spontaneous nucleation observed by inline process monitoring (▼, lower part). (Points: measurement data; Lines are guides to the eyes.)

Figure 7. HPLC chromatograms (a) and picture of the filtered product crystals (b) of single-step crystallization (a: reaction mixture (upper) and crystallization product (lower)).

chosen solvent mixture at 5 °C was much lower than the initial concentration of DCA, so that DCA crystallized along with the artemisinin product. The purity of artemisinin was enhanced from 69% up to 98.8%. The yield was 43.8% related to the artemisinin initially present in the synthesis solution, almost E



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chromatography. Figure 8 shows the schematic illustration of the three-zone open-loop SMB process with capture step. Similar to the process described in ref 35, this configuration can separate artemisinin and WPIs, while the SPIs are efficiently captured. When a column that was located in the zone 1 is moved to the zone 4, it is completely isolated from the threezone SMB and the SPIs captured can be removed by a strong regeneration solvent (here ethanol). The average value of the Henry constant was 7.06. By including retention delay caused by the system dead volume, the Henry constant of artemisinin was adjusted to 7.3. To identify suitable operating conditions of the three-zone openloop SMB, the retention behaviors of WPIs should be known (Appendix B). Since it is very difficult to consider all impurities and quantify their retention behavior, a representative “pseudo Henry constant” of the earlier eluting WPI fraction was presumed to be a certain fraction (99.0 99.9 62.1b 99.9 61.5

a

Average concentration of the target product in all SMB runs. bUsing the enriched SMB effluent.

the purity and yield of each purification step and of the overall process. The overall recovery yield was increased to 61.5%. Three chromatograms are compared in Figure 11a: the initial reaction mixture, the product after the first purification step with continuous chromatography, and the final crystallization product. HPLC analysis verified the final artemisinin purity to be 99.9% (Table 6). The absence of (yellow) DCA crystals was also confirmed inspecting the crystalline product under the microscope (Figure 11b).

5. CONCLUSIONS To isolate artemisinin from a complex reaction mixture produced continuously by a photocatalytic reaction carried out in a tubular reactor, two separation techniques were applied, namely, crystallization and chromatography. Most impurities except DCA were removed by single-step crystallization, while the two major impurities DCA and DHAA were removed in SMB chromatography. Both single separation techniques were not yet fully optimized, so that they did not purify alone artemisinin to meet the desired purity requirement (>99.5%). However, a chromatography-crystallization coupling just using the same operating conditions of both single separation techniques successfully achieved this goal. The coupled process can be used to efficiently purify the artemisinin present in the processed reaction mixture. The final purity and recovery yields were 99.9% and 61.5%, respectively.

■

APPENDIX A: RELATIONSHIP BETWEEN RETARDATION AND RETENTION FACTORS Thin layer chromatography (TLC) is a versatile method in chromatography frequently used for analyzing the mixtures.37 The development of TLC plates is normally done at atmospheric pressure with the mobile phase moved by capillary forces. The efficiency of rather simple TLC is lower than for column chromatography. TLC methods are often used for screening of stationary and mobile phases. The retention behavior of a solute i on a TLC plate can be characterized by a retardation factor Rf,i defined as,

Figure 11. HPLC chromatograms (a) and picture of the filtered product crystals (b) of the coupled process of SMB chromatography and crystallization (a: reaction mixture (upper), SMB chromatography product (middle), and crystallization product (lower).

other impurities. These components can be successfully removed simply by subsequent crystallization. To test this, all collected and diluted chromatography extract streams were mixed (giving a feed with a purity of artemisinin of 92%) to perform after an adjustment of the concentration a crystallization in order to isolate solid pure artemisinin. Hereby, I


Organic Process Research & Development R f, i =

■

(travelled distance of solute i from the loading spot) (travelled distance of solvent from the loading spot)

*E-mail: [email protected]. Tel.: +49-(0)391-6110 401. Fax: +49-(0)391-6110 521.

The retardation factor is equivalent to the penetrated position of solute in a chromatographic column after elution of one column volume of the mobile phase. The retardation factor obtained from TLC can be used to predict the retention factor ki of solute i in column chromatography with the following relationship,

=

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge generous financial support by the Max-Planck Society. We would like to gratefully acknowledge the help of Jacqueline Kaufmann, Dr. D. Kopetzki, AnneKathleen Kort, and Stephanie Leuchtenberg during this work.

(amount of solute in the stationary phase) (amount of solute in the mobile phase)

■

(1 − R f, i) R f, i

(A2)

■

APPENDIX B: DESIGN METHOD OF THREE-ZONE OPEN-LOOP SIMULATED MOVING BED CHROMATOGRAPHY WITH CAPTURE STEP The applied three-zone open-loop simulated moving bed (SMB) process is a modification and extension of conventional four-zone SMB chromatography as shown in Figure 8. It omits the typically applied solvent regeneration zone after the raffinate port and uses a different two-step solid phase regeneration concept in order to remove the captured fraction. Nevertheless, the established design concepts developed for the conventional SMB concept can be applied in a straightforward manner. One of the most well-known design methods for SMB chromatography, often called the Triangle method, is developed based on the equilibrium theory.36 It uses the ratio of the net liquid flow-rate to the simulated solid flow-rate as a design parameter defined as, Q j − ϵ TVC/tS (1 − ϵ T)VC/tS

(B1)

where mj is the flow-rate ratio in zone j, Qj is the volumetric flow-rate of the liquid phase in zone j, ϵT is the total void fraction of the column, VC is the empty volume of the column, and tS is the port switching interval. For binary separations with linear isotherms, the zone flow-rate ratios should be posed as below, HS < m1

(B2-1)

HW < m2 < m3 < HS

(B2-2)

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Thus, TLC can be used efficiently to screen mobile phases for column chromatography. Nevertheless, since the conditions during TLC and column chromatography are different, eq A2 cannot describe the retention behavior of solutes in the corresponding columns perfectly. Therefore, for parameter provision and process design further experiments using the columns are required.

mj =

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(A1)

ki =

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where H is the Henry constant, the subscripts S and W denote the strongly and the weakly adsorbed solutes, respectively, and the subscripts 1 to 3 denote the zone number in Figure 8. Since this method is based on the equilibrium model assuming an infinite plate number, it provides just estimates for possible operating parameters, which need to be typically refined in order to find the optimal conditions. J



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