Separation of prostaglandins using the new horizontal flow-through coil planet centrifuge

Prostaglandins and their metabolites have been separated using the new horizontal flow-through coil planet centrifuge to perform countercurrent chromatography. The system is a continuous-flow system which provides separations similar to column and thin-layer chromatography without the use of solid supports. Either phase of a two-phase solvent system can be used to elute compounds with the low pressure produced by a metering pump. Separations were produced in PTFE tubing, 0.55 mm i.d. (wound in 0.68 cm helical diameter) with a total capacity of 24 ml. A solvent system of chloroform/acetic acid/water (2 : 2 : 1, v/v) was used at flow rates of 2.4 and 6 ml/h and at a rotation of 500 rev./min. Microgram quantities of prostaglandins E₂, F_2α, A₂, and B₂ and thromboxane B₂ can be separated. This method of can be scaled up to 260-ml columns and thromoboxane B₂ can be separated. This method can be scaled up to 260-ml columns with comparable results.     

Countercurrent gradient chromatography: A continuous focusing technique

The continuous separation of proteins was performed in a countercurrent gradient chromatography (CGC) system. A magnetically stabilized fluidized bed (MSFB) was used to establish true countercurrent contact of a solid resin with a liquid buffer. STable pH gradients were formed in the system in less than 10 min and remained stable throughout the course of the separation experiment (>2 h). The shape of the pH gradient, which ultimately controls the resolution and purity of the separation, can be controlled by making simple adjustments in the interstitial velocities of the liquid and solid phases. We have performed the separation of myoglobin and human serum albumin (HSA) using this device and achieved concentration factors of 1.75 for myoglobin and 1.2 for HSA. A mathematical model that has no adjustable parameters has been developed that predicts the focusing behaviour and capabilities of the CGC system. Using the model, we have estimated the optimum phase velocities, particle diameters, and equilibrium parameters necessary for achieving high purity and high concentrations. (c) 1995 John Wiley & Sons, Inc.