Isolera flash purification system

Apo-mimochrome VI*a (ApoMC6*a) and CoMP11-Ac were prepared as previously described.18 (link),27 (link) ApoMC6*a is the non-metallated version of CoMC6*a. Cobalt insertion into ApoMC6*a was carried out using a modified version of the acetic acid/acetate method.27 (link),55 Purified ApoMC6*a (100 mg) was dissolved in 60 : 40 AcOH : TFE (v/v) at a concentration of 20 mg mL^–1, along with a 20-fold molar excess of cobalt(ii) acetate. The reaction was performed under a nitrogen atmosphere at 50 °C for 2 hours. Metal insertion was monitored by analytical RP-HPLC (Shimadzu LC-10ADVP equipped with a SPDM10AVP diode-array detector and Vydac C18, 150 mm × 4.6 mm column) with an elution gradient of 50% to 80% acetonitrile, 0.1% trifluoroacetic acid (TFA) over 35 min with a flow rate of 1 mL min^–1, monitored by UV-vis spectroscopy (Varian Cary 50) at 410 nm. The reaction products were concentrated under vacuum, re-dissolved in 10 mL water, 0.1% TFA (v/v) and desalted by flash chromatography performed with a Biotage Isolera flash purification system, equipped with a diode-array detector. CoMC6*a was loaded on a 30 g SiO₂ C18 reversed-phase column with an elution gradient from 0 to 95% acetonitrile, 0.1% TFA over 20 min with a flow rate of 25 mL min^–1. Similar fractions were pooled and concentrated. Lyophilization yielded the final product (95% purity).

Liquid-liquid separation was conducted on a TBE-300C HSCCC (Tauto Biotechnique Company, Shanghai, China). The apparatus was consisted of three coiled columns connected in series (diameter of tube: 1.6 mm; total capacity: 300 mL), a 20 mL sample loop, and an Isolera FLASH purification system (Biotage, Uppsala, Sweden) as the pump, fraction collector, and UV monitor. The revolution speed was set at 850 rpm.
The essential oil was analyzed using an Agilent 6890N series GC equipped with a flame ionization detector (FID) and a HP-5 5% phenyl methyl siloxane capillary column (Agilent 19091J-413, 30 m × 0.32 mm i.d., film thickness 0.25 μm), and GC-MS was performed on the Agilent Technologies 7820A GC system combined with an Agilent 5977E series GC/MSD.

A TBE-300C HSCCC instrument (Tauto Biotechnique Company, Shanghai, China) equipped with an Isolera FLASH purification system (Biotage, Uppsala, Sweden) was used for separation. Before separation, 300 ml of the stationary phase was pumped into the coiled column and subsequently rotated at 400 rpm. After equilibrium was reached, 20 ml of the DCR extract solution was injected, and a series of mobile phases was delivered into the coil in the tail-to-head mode. A mixture of n-butanol (n-BuOH) and water [1:10 (v/v)] was used as the stationary phase. The injected DCR extract was dissolved in a mixture of ethyl acetate (EtOAc), n-BuOH, and water [1:1:10, (v/v/v)] to achieve a concentration of 25 mg/ml. A list of mobile phases is presented in Figure 6. Chromatograms were recorded at a wavelength of 254 nm.

The HSCCC instrument was a model TBE-1000A HSCCC (Tauto Biotechnique Company, Shanghai, China) with three multilayer coil columns (ID of the tubing: 1.8 mm, column volume: 260 mL) connected in series and a 50 mL sample loop. The β value (β = r/R, where r is the distance from the coil to the holder shaft and R is the distance between the holder axis and central axis of the centrifuge) of the multilayer coil varies from 0.60 (internal terminal) to 0.80 (external terminal). The revolution speed of the apparatus was regulated at 0–1000 rpm with an electronic speed controller. The HSCCC system was equipped with a Model Hitachi L-6200 intelligent pump (Hitachi, Tokyo, Japan) and an Isolera FLASH purification system (Biotage, Uppsala, Sweden) as UV monitor. The multilayer coil column was first entirely filled with the upper organic phase at a flow rate of 20 mL min⁻¹. The lower aqueous phase was pumped into the inlet column as the mobile phase at 5 mL min⁻¹, while the apparatus was rotated at 400 rpm. The mode for HSCCC separation was “head to tail.” After the hydrodynamic equilibrium was established, the EtOAc fraction of the MA extract (2 g in 40 mL of each phase) was injected into the separation column through the injection valve, and then each peak fraction was collected in 25 mL tubes while monitored with a UV detector at 254 nm.