RESUMEN

Counter-current chromatography is a form of liquid-liquid chromatography which uses low-speed centrifugation to hold one phase of an immiscible liquid pair stationary while the other is eluted through it. Two types of countercurrent chromatography are described: one suitable for preparative/analytical separation with aqueous-organic phase systems and the other for analytic fractionations using aqueous-aqueous phase systems. Applications of both processes are described, ranging from the purification of antibiotics, pesticides, and peptides to the fractionation of whole cells.

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Separation of subcellular organelles by two-phase partition is thought to reflect differential partition of the organelles between the two phases or between one of the phases and the interface. Studies by Fisher and colleagues [Fisher & Walter (1984) Biochim. Biophys. Acta 801, 106-110] suggest that cell separation by phase partition is a dynamic process in which the partition changes with time. This is mainly due to association of the cells with sedimenting droplets of one phase in the bulk of the other. Rat liver organelle partition was studied to determine whether the same dynamic behaviour is observed. Partition was clearly time-dependent during 24 h at unit gravity, and was also affected by altering the volume ratio of the two phases and the duration of phase mixing. These results indicate that, as with cells, the partition of organelles between phases is a dynamic process, and is consistent with the demonstration that organelles adhere to the phase droplet surfaces. Optimization of the volume ratio between phases may lead to significant processing economies. Organelle sedimentation in the upper phase was significantly faster than in the isoosmotic sucrose. Theoretical modelling of apparent organelle sizes indicates that aggregation occurs in the poly(ethylene glycol)-rich upper phase. This phenomenon is likely to limit the use of this technique in organelle separations unless means can be found to decrease aggregation.

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The principal organelles of rat liver homogenates were fractionated by two-phase partition chromatography using toroidal-coil centrifugation with a mixture of dextran T 500 and poly(ethylene glycol) 6000 in 0.26 M-sucrose containing 10 mM-sodium phosphate/phosphoric acid buffer, pH 7.4. The effects of varying the following parameters on organelle elution profiles, as reflected by their marker-enzyme activities, were studied: centrifuge speed; the composition and relative proportion of dextran-rich and poly(ethylene glycol)-rich phases in the eluent; flow rate; sample volume; homogenate concentration; helix diameter; tubing bore and the number of loops in the coil. Optimal resolution of the organelles was achieved with a toroidal coil of internal diameter 1.07 mm with a 4.55 mm helix diameter on a 0.42 m-diameter rotor running at 1000 rev./min. The eluent was prepared by combining, in a ratio of 93:7 (v/v), the poly(ethylene glycol)-rich upper phase and dextran-rich lower phase obtained from a phase mixture containing 3.3% (w/w) dextran and 5.4% (w/w) poly(ethylene glycol). The flow rate of the eluent was 14ml/h. Optimal conditions for separation of the organelles were evaluated. Resolution of plasma membrane and lysosomes was achieved. Separation of endoplasmic reticulum, which showed marked heterogeneity, from plasma membrane was also demonstrated. DNA and marker enzymes for peroxisomes, mitochondria and cytosol showed distinct elution profiles.

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Isoelectric soya-protein precipitate densities were measured for mean particle sizes ranging from 3.4-65 microm by gradient centrifugation, centrifugation in water-immiscible solvents, tracerdilution, gravity sedimentation of isolated particles. Coulter counter volume determination, and a comparison of Coulter counter and centrifugal sedimentation size distributions. The immiscible system and tracer dilution methods were both found to be unreliable due to experimental uncertainties. The Coulter counter volume measurement indicated the existence of a density-size relationship with the aggregate density decreasing as the size increased. Comparison with sedimentation measurements showed that the Coulter counter measures 80% of the total aggregate volume for 6-microm particles. The relation between aggregate density (rho(a), kg m (-3)) and size (d, microm) was measured for isoelectric soya protein and casein precipitated by ammonium sulfate, using a comparison of the Coulter counter size distribution and centrifugal sedimentation. The functions were described for soya by [rho(alpha) - 1001 = 246d-0.408 and for casein by rho(alpha) - 1136 = 31d-0.441]. The gradient centrifugation method measured the buoyant density of hydrated protein precipitate which was independent of size, and is consistent with an aggregate structure consisting of primary particles. However, the aggregate structure was not described for all sizes by the theoretical cubic packing of hard-sphere primary particles, nor by the successive random addition of primary particles. The density-size functions indicated up to a fivefold difference in Stokes settling velocities compared to those calculated assuming a constant density difference.