Hydrodynamics of PEG‐Phosphate Aqueous Two‐Phase Systems in a J‐Type Multilayer Countercurrent Chromatograph

Abstract

Recently, we have shown that countercurrent chromatography (CCC) is an effective method for the purification of plasmid DNA vaccines and gene therapy vectors.1 As a basis for further work, this paper studies the hydrodynamics of various PEG‐Salt aqueous‐aqueous two‐phase systems in a Brunel J‐type countercurrent chromatograph. The degree of stationary phase retention, S f , once a hydrodynamic equilibrium is achieved has been studied as a function of mobile phase flow rate (0.5–2.0 mL·min−1), coil rotational speed (500–850 rpm), column volume (92.3 and 167.3 mL), choice of mobile phase (PEG or phosphate), and mobile phase pumping direction (Head→Tail or Tail→Head). Three different aqueous two‐phase systems (ATPS) were studied, which consisted of PEG 300‐K2HPO4, PEG 600‐ K2HPO4, and PEG 1000‐K2HPO4, having density and viscosity ratios between 1.15–1.13 and 0.27–0.12, respectively. High S f values in comparison to previous aqueous‐aqueous studies with CCC were obtained of up to 73.7%. These high S f values were obtained when the lower aqueous phase was pumped from Tail(periphery)→Head(centre), opposite to the direction normally recommended for most organic‐aqueous systems in J‐type CCC machines.2 This is believed to be due to the high settling times and low density/high viscosity difference of aqueous‐aqueous systems compared to organic‐aqueous systems. Du3 plots of the data showed the essential linear relationship between S f and √F, provided that S f >20%. Data obtained from Du plots for each phase system could also be used to satisfactorily predict S f as a function of column rotational speed. This work gives an insight into the behaviour of aqueous phase systems in J‐type CCC machines and is useful as a basis for process design and scale‐up.