phoresis as an industrial separation tool [59]. Under most circumstances in
particle separations, conditions can be arranged so that the sedimentation of
individual particles, say cells, can be minimized, but not always [51]. Finally, the
diffusion-driven formation of droplets containing high concentrations of
particles or solutes [60] results in ‘droplet sedimentation’, and at very high
concentrations of particles in density-gradient electrophoresis [52] but not in
low-gravity electrophoresis [54].
Another type of mixing problem encountered in free electrophoresis,
although less critical compared to ohmic heating, is the mixing caused by gas
release at the electrodes. This problem is addressed by employing non-gassing
electrodes [61] or membrane-separated electrodes [62]. This experience points
to the possibility of using non-gassing Pd electrodes (as in the present work)
rather than the more complicated membrane based system of Tulp et al. [63].
A score of methods has been developed to effect free electrophoresis [41].
These methods can be broadly divided into static and flowing methods, neither
of which has satisfactory capacity for application as a manufacturing tool. Batch
and continuous methods have also been developed. In almost all cases maximum sample input rates have been of the order of a few milliliters per hour. In
one important case, Tulp et al. [63] designed a re-orienting free electrophoresis
device consisting of a flat disk-shaped container with thin sample bands and a
short migration distance. The top and bottom electrode fluids served as
coolant, the total height of the separation column was 1–2 cm, and its diameter
was greater than 15 cm. The distance between unrelated separands was about
1–2 mm, and this distance was increased during fractionation after electrophoresis by re-orienting the disk.
Based on research experience and needs identified for free electrophoresis in
various fields of bioprocessing and analysis such as virology, endocrinology,
enzymology, hematology, and mammalian cell culture [64–69], in the present
study an attempt is made to overcome the major bottlenecks of free electrophoresis.
2.2.2
Multistage Electrophoretic Method
The multistage electrophoretic method has been developed by combining free
electrophoresis and multistage extraction and is explored as an improved
alternate method for the purification and concentration of cells or macromolecules. The present discussion is restricted to cells and particles. The present
design is based on a derivative of the thin-layer multistage extractor design of
Albertsson [70] and Treffrey et al. [71] and called ADvanced SEParation apparatus (ADSEP), designed by SHOT, Inc. [42].
To test this method extraction of fixed human red blood cells and latex
suspended in 0.01 mol l –1 phosphate buffer was performed at different electric
field strengths such as 0.05 V m –1 or 0.1V m –1 . A simple mathematical model
was developed (discussed in Sect. 2.2.3) to describe the mass and heat transfer
during the electrophoretic separation process in the countercurrent extractor
employed. The experimental results agree reasonably well with those predicted
158
K.S.M.S. Raghavarao et al.
particle separations, conditions can be arranged so that the sedimentation of
individual particles, say cells, can be minimized, but not always [51]. Finally, the
diffusion-driven formation of droplets containing high concentrations of
particles or solutes [60] results in ‘droplet sedimentation’, and at very high
concentrations of particles in density-gradient electrophoresis [52] but not in
low-gravity electrophoresis [54].
Another type of mixing problem encountered in free electrophoresis,
although less critical compared to ohmic heating, is the mixing caused by gas
release at the electrodes. This problem is addressed by employing non-gassing
electrodes [61] or membrane-separated electrodes [62]. This experience points
to the possibility of using non-gassing Pd electrodes (as in the present work)
rather than the more complicated membrane based system of Tulp et al. [63].
A score of methods has been developed to effect free electrophoresis [41].
These methods can be broadly divided into static and flowing methods, neither
of which has satisfactory capacity for application as a manufacturing tool. Batch
and continuous methods have also been developed. In almost all cases maximum sample input rates have been of the order of a few milliliters per hour. In
one important case, Tulp et al. [63] designed a re-orienting free electrophoresis
device consisting of a flat disk-shaped container with thin sample bands and a
short migration distance. The top and bottom electrode fluids served as
coolant, the total height of the separation column was 1–2 cm, and its diameter
was greater than 15 cm. The distance between unrelated separands was about
1–2 mm, and this distance was increased during fractionation after electrophoresis by re-orienting the disk.
Based on research experience and needs identified for free electrophoresis in
various fields of bioprocessing and analysis such as virology, endocrinology,
enzymology, hematology, and mammalian cell culture [64–69], in the present
study an attempt is made to overcome the major bottlenecks of free electrophoresis.
2.2.2
Multistage Electrophoretic Method
The multistage electrophoretic method has been developed by combining free
electrophoresis and multistage extraction and is explored as an improved
alternate method for the purification and concentration of cells or macromolecules. The present discussion is restricted to cells and particles. The present
design is based on a derivative of the thin-layer multistage extractor design of
Albertsson [70] and Treffrey et al. [71] and called ADvanced SEParation apparatus (ADSEP), designed by SHOT, Inc. [42].
To test this method extraction of fixed human red blood cells and latex
suspended in 0.01 mol l –1 phosphate buffer was performed at different electric
field strengths such as 0.05 V m –1 or 0.1V m –1 . A simple mathematical model
was developed (discussed in Sect. 2.2.3) to describe the mass and heat transfer
during the electrophoretic separation process in the countercurrent extractor
employed. The experimental results agree reasonably well with those predicted
158
K.S.M.S. Raghavarao et al.