Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.

Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.

Precipitation of a 6.9 kb pSVb plasmid DNA using three different alcohols, i.e., methanol, ethanol, and 2-propanol, as precipitating agents was performed. Precipitate composition was analyzed, and it was found that the quantity of supercoiled plasmid DNA recovered varied according to type and concentration of alcohol. Precipitation with 2-propanol concentration greater than 70% (v/v) resulted in mostly open circular plasmid DNA, which could be converted back to supercoiled by diluting 2-propanol concentration in the process. It appeared in this study that 2-propanol at high concentration could affect supercoiling of the plasmid, and lead to the temporary supercoiled-open circular transition without covalent breaking of the strands. Supercoiled DNA quantity slightly ORDER REPRINTS decreased in precipitation with methanol at high concentration, but remained quite constant in precipitation using ethanol.

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Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel. Solutes of interest undergo repetitive precipitation-dissolution, and fractionate at different locations, and finally elute out from the channel according to their solubility in the precipitant solution. In the practical apparatus, two channels are formed by inserting a piece of dialysis membrane between a pair of disks with mutually mirror-imaged spiral grooves. A sample is injected into one channel and the precipitant solution is fed into another one. When the precipitant travels across the membrane to the sample channel, its concentration gradient is formed. Gradient profile of precipitant inside the sample channel is crucial to the success of CPC; therefore, it is worthwhile to investigate the formation of the precipitant concentration gradient. In this paper, an established mathematical model used to explain the steady-state gradient formation of ammonium sulfate, (NH 4 ) 2 SO 4 , in CPC was adopted for studying the gradient formation of cationic surfactant cetyltrimethylammonium bromide (CTAB). Its concentration, velocity and pressure profile obtained from the model are discussed.