Supporting Information

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Continuous Industrial-scale Centrifugal Partition Chromatography

with Automatic Solvent System Handling: Concept and

Instrumentation

László Lorántfy*, Dóra Rutterschmid, Róbert Örkényi*, Dávid Bakonyi, József Faragó, Gergő Dargó, Árpád Könczöl

RotaChrom Technologies LLC, 151 Fő út, H-2370 Dabas, Hungary

*Corresponding authors. E-mail address: [email protected] (L. Lorántfy);

[email protected] (R. Örkényi)

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Figure S1. Distribution of solvent systems by the number of the constituents utilized in liquid-liquid chromatographic purifications of natural products. BLSs that are containing ethyl acetate are more represented in the quaternary systems (79%) rather than the ternary systems (38%).

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The data for the following diagrams were simulated by vle-calc.com ver. 09/23/2020 and the ternary plots were created using TernaryPlot.com (Copyright © 2018, Jules Blom); for density measurements see the Experimental Section.

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Figure S9. The mixer-settler skid. Standing jacketed vessel with agitator mounted on top. Material: EN 1.4301 / AISI 304. Net volume: 250 L.

Figure S10. The buffer skid. Standing jacketed vessel. Material: EN 1.4301 / AISI 304. Net volume: 2x250 L, 60 L sample tank.

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Figure S11. The fraction collector skid. Standing vessels in a rack. Material: EN 1.4301 / AISI 304. Net volume: 9x40 L.

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Figure S13. The recycler (evaporator) skid. Wiped film evaporators. Material: EN 1.4301 / AISI 304. Jacket surface: 1.02±0.02 m2_{. Jacket medium: water, 80-95 °C. Condenser medium:}

ater, 10-40 °C.

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Figure S15. Test setup for flow visualization in the settler unit. The BLS for testing was

n-hexane/MeOH/H2O in the volumetric ratio of 5/4/1.

Figure S16. Test setup for flow visualization in the settler unit. The BLS for testing was

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Figure S17. Lab-scale CPC-UV chromatogram of a steroid API separation. COI stands for compound of interest. The sample solution’s concentration was 0.05 g mL-1 (in upper phase). To a 250 mL capacity CPC column, 40 mL of sample solution was injected. The flow rate of the elution was 15 mL min-1 in descending mode. The rotation speed of the rotor was 1600 rpm, and the UV detection was set to 206 and 243 nm, which represented by the blue and red line, accordingly. Fractions of 30 mL were collected.

Figure S18. Pilot-scale CPC-UV chromatogram of a steroid API separation. COI stands for compound of interest. The sample solution’s concentration was 0.05 g mL-1 (in upper phase). To a 2150 mL capacity CPC column, 320 mL of sample solution was injected. The flow rate of the elution was 300 mL min-1 in descending mode. The rotation speed of the rotor was 1000 rpm, and the UV detection was set to 206 and 243 nm, which represented by the green and blue line, accordingly. Fractions of 300 mL were collected.

COI

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Figure S19. HPLC-UV chromatograms and API purities of the collected fractions from the industrial-scale CPC separation. Retention time of the API was 12.02 minutes.

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Table S1. iCPC method for the model-mixture separation.

Time (s) Flow rate (L/min) Input valve Output valve Rotation speed (rpm) Detection (nm) 19 2.5 Sample Waste 700 210 315 2.5 LP Waste 700 210 292 2.5 LP Frac 1 700 210 32 2.5 LP Frac 2 700 210 32 2.5 LP Frac 3 700 210 32 2.5 LP Frac 4 700 210 32 2.5 LP Frac 5 700 210 32 2.5 LP Frac 6 700 210 32 2.5 LP Frac 7 700 210 32 2.5 LP Frac 8 700 210 292 2.5 LP Frac 9 700 210 180 2.5 UP Waste 700 210

Table S2. iCPC method for the steroid API purification.

Time (s) Flow rate (L/min) Input valve Output valve Rotation speed (rpm) Detection (nm) 48 4 Sample Waste 700 210, 243 315 3 LP Waste 700 210, 243 84 3 LP Frac 1 700 210, 243 84 3 LP Frac 2 700 210, 243 84 3 LP Frac 3 700 210, 243 84 3 LP Frac 4 700 210, 243 84 3 LP Frac 5 700 210, 243 84 3 LP Frac 6 700 210, 243 84 3 LP Frac 7 700 210, 243 84 3 UP Frac 8 700 210, 243 84 3 UP Frac 9 700 210, 243 270 4 UP Waste 700 210, 243

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Table S3. Results of the partition coefficient measurements of the compounds under studies.

Entry Compound Biphasic Liquid System (v/v/v) Partition coefficients (KD) Shake-flask methoda Calculated from CPC chromatogramb 1 4-ethylbenzaldehyde Hexane/MeOH/H2O 5/4/1 0.93 1.34 2 4-isopropylbenzaldehyde 1.24 1.78 3 Steroid API MIBK/Acetone/H2O

2/2/1 2.0 2.4-2.7

c

a_{The partition coefficient measurement protocol is described in the experimental section,}b_Partition coefficients were calculated from the CPC chromatogram as follows: KD=(VE-VM)/(VT-VM), where VE is the elution volume of the given compound, VM is the mobile phase volume in the rotor, and VT is the total volume of the rotor, c_{Measured on laboratory- and industrial-scale CPC, respectively.}