3.4. Centrifugal Partition Chromatography (CPC)

The centrifugal partition chromatography (CPC) device was a SCPC-250-B Bio-Extractor (SCPE) (Armen, France). Total cell volume was 250 mL. Descending and ascending modes were selected by a four-way switching valve. SCPE was connected to a SPOTPREP II system (Armen, France), equipped with an UV detector and fraction collector (32 mL tubes) and an injection valve with 10 mL sampling loop (Note: for pulse injections 1 mL loop was used). The CPC rotor was first filled with 1.5 column volumes using the lower phase at 30 mL/min and 500 rpm rotation. Afterwards, upper phase was pumped into the system in ascending mode at a flow rates from 5–40 mL/min increasing the rotation speed up to 2000–2400 rpm. Crude B. globosa extract (300 mg) was dissolved in 10 mL of 1:1 mixture of upper and lower phase and loaded through 10 mL sample loop. Fractions (25 mL, 26 tubes) were collected, and monitored with a scan of 200–600 nm and wavelengths 280 and 320 nm. Extrusion was performed after 50 min run time with 100% stationary phase increasing the flow rate at 30 mL for 10 min. Pareto chart was used to evaluate the effect of rotation speed and flow rate upon stationary phase retention. The final operation conditions were chosen considering the retention of stationary phase, backpressure in the CPC column and time elapsed to obtain the target compounds.

The partition coefficient (KD) was determined according to Ito and coworkers [14], with slight modifications. In brief, five mg of B. globosa extracts were weighed and dissolved in 3 mL of thoroughly pre-equilibrated upper organic and lower aqueous phases. In this work, nine different solvent system were selected from the list presented in Table 3. The mixture was vigorously shaken in a 10 mL conical vials. Once settled, upper and lower phases were separated and taken to dryness. The residues were reconstituted in 1 mL of mobile phase and analyzed by HPLC following the method described above, injecting 5 µL. Based on the ratio of HPLC peak area of each target compound in lower and upper phases the KD values were calculated as follows:

Also, the partition coefficients of target compounds were calculated from the CPC chromatogram using pulse injection (1 mL) of B. globosa extract at optimal flow rate (F) according to the following Equation:

where F is the optimized flow rate used in the present study, Rt,i is the retention time of B. globosa target compound, SF is the stationary phase retention and VC is the column volume [40].

The number of theoretical plates (Ni) were calculated as follows [40]:

where Rt,i is the retention time of the target compound i and σ is the variance of the peak.

The resolution (RS) was calculated using the following equation [40]:

where Rt,j and Rt,i are the retention times for the second and the first target compounds, respectively. Peak widths at base line are denoted as wj and wi.