Centrifugal precipitation chromatography

Abstract

Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. The countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.

Application of centrifugal precipitation chromatography and high‐speed counter‐current chromatography equipped with a spiral tubing support rotor for the isolation and partial characterization of carotenoid cleavage‐like enzymes in Enteromorpha compressa (L.) Nees

“…The residue obtained by filtration (Durapore 0.45 mm, Millipore, Bedford, MA, USA) was homogenized in 250 mL chilled buffer (50 mM Tris-HCl, 125 mM KCl, 5 mM MgCl 2 , adjusted with HCl to pH 6.8) by a Psycotron homogenizer (Microtec, Chiba, Japan) at 30 000 rpm for 1 min. Further steps were carried out as described earlier [14]. Briefly, the samples were centrifuged and the soluble proteins in the supernatant were precipitated with 95% acetone.…”

“…Centrifugal precipitation chromatography separates molecules according to their solubilities in ammonium sulfate or other precipitants, such as organic solvents [14]. Previously, this instrument was successfully applied for the isolation of enzymes from Camellia sinensis with the preservation of the biological activity [15].…”

Novel Design for Centrifugal Counter-Current Chromatography: VI. Ellipsoid Column

“…2. A pair of flow tubes is put together and passed through the center of the central shaft downward and the hollow horizontal shaft of a miter gear, then led upward into the vertical hollow tube support, and finally exits the centrifuge from the center of the upper plate where they are tightly held with a pair of clamps [19] .…”

Studies on the effect of column angle in figure-8 centrifugal counter-current chromatography

“…Each terminal of the column is connected to PTFE flow tube (0.46 mm I.D., Zues Industrial Products) with a set of tubing connectors (Upchurch Scientific, Palm Spring, CA, USA). A pair of flow tubes is put together and passed through the center of the central shaft downward and the hollow horizontal shaft of a miter gear, then led upward into the vertical hollow tube support, and finally exits the centrifuge from the center of the upper plate where it is tightly held with a pair of clamps [16]. The total space in the feed and return tubing (dead volume) is approximately 0.5 cm 3 .…”