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.

Keywords:

• Countercurrent chromatography
• Hydrodynamics
• PEG‐phosphate aqueous two‐phase system

Acknowledgments

The support of the Engineering and Physical Sciences Research Council (EPSRC) Life Science Interface and Innovative Manufacturing Research Centre initiatives for the IMRC in Bioprocessing is gratefully acknowledged. The IMRC is part of The Advanced Centre of Biochemical Engineering, UCL with collaboration from a range of academic partners and biopharmaceutical and biotechnology companies. IAM would like to thank Gulf Pharmaceutical Industries (Julphar, UAE) and the Ministry of Higher Education & Scientific Research (UAE) for financial support. Prof. Ian Sutherland (Brunel University) and Dr Andrew Booth (UCL) are thanked for an initial reading of the original manuscript.