Protein separation using toroidal columns by type-J synchronous counter-current chromatography towards preparative...

Area of Science:

• Biochemistry
• Chromatography
• Separation Science

Background:

• Separating large bioactive molecules (proteins, DNA, RNA) using traditional methods risks sample loss and denaturation.
• Aqueous two-phase systems (ATPSs) and counter-current chromatography (CCC) offer a gentler alternative, reducing processing time.
• Toroidal columns are a novel configuration for preparative CCC, potentially enhancing separation efficiency.

Purpose of the Study:

• To construct and evaluate toroidal columns for preparative counter-current chromatography (CCC).
• To assess the separation capability of toroidal CCC for large biomolecules using proteins as models.
• To investigate the impact of sample loading on separation resolution and peak broadening.

Main Methods:

• Construction of toroidal columns (26-140 m length, 51-280 ml volume) for preparative CCC.
• Utilized an ATPS composed of 12.5% PEG1000 and 12.5% K2HPO4.
• Separated model proteins (myoglobin and lysozyme) using the phosphate-enriched phase as the mobile phase in Coriolis force parallel flow mode.

Main Results:

• Achieved good resolution (Rs=1.5) for myoglobin and lysozyme separation with a 53.5 m toroidal column at specific loading percentages (1.7% and 7.4% of column volume).
• Higher sample loading (13%) resulted in failed separation, indicating sensitivity to loading capacity.
• Observed peak broadening for proteins compared to dipeptides, confirming molecular weight as a factor influencing CCC peak characteristics.

Conclusions:

• Toroidal CCC columns are effective for separating large biomolecules, offering advantages over conventional methods.
• The study highlights the importance of optimizing sample loading for achieving high resolution.
• Results support the potential for scaling up toroidal CCC for industrial-scale purification of biomolecules.