Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography

Abstract

A spiral disk assembly composed of five single-channel units was designed for high-speed counter-current chromatography (HSCCC). The retention of different solvent systems ranging from moderately polar to polar organic–aqueous systems to aqueous two-phase systems (ATPs) was investigated under different elution modes. The results indicated that the spiral disk assembly can produce excellent retention of stationary phase for moderately polar organic–aqueous solvent systems, such as chloroform–methanol–water (4:3:2) and hexane–ethyl acetate–methanol–water (1:1:1:1) by pumping lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a high flow-rate (8 mL/min, S f > 70%), regardless of whether the inlet is at the inner or outer terminal of the channel. This makes it possible for fast analysis of some small molecular compounds. This has been proved in the separation of mixtures of three flavones, including isorhamnetin, kaempferol, and quercetin. The spiral disk assembly can also provide satisfactory retention for polar to ATPS such as 1-butanol–acetic acid–water (4:1:5) (<3 mL/min, S f > 70%), 12.5% poly(ethylene glycol) (PEG) 1000–12.5% K 2HPO 4–75% water (≤1 mL/min, S f > 70%) and 4% PEG 8000–5% Dextran T500–91% water (≤0.5 mL/min, S f > 50%) by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a low flow-rate, while this is not possible with the multilayer coil column. Acceptable resolutions were achieved when it was used for the separation of peptides such as Leu-Tyr and Val-Tyr, and proteins including cytochrome c and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins.