electrophoretic mobility. The assembly of the plates containing the chambers is
shown in Fig. 7a. Identically designed plates assure uniform loading and sealing
when the plates are clamped together. The experimental samples are loaded and
withdrawn through the fill ports located on the side of each plate as shown in
Fig. 7b. ADSEP is driven by an independent power supply (Lamda, Model No.
LP-532-FM) for rotating the plates to bring the chambers into interfacial contact
with each other. This ADSEP was modified to use as ELECSEP by replacing the
chamber bottoms with metal cover plates. Electrodes are kept over these cover
plates with gaskets in between them.
Preliminary electrophoretic transfer experiments were carried out [72] with
two types of particles, fixed human red blood cells and latex particles (average
diameter is 3.5 mm and 2.3 mm, respectively). The particles were counted by
hemacytameter with a minimum of three counts per fraction. Average values of
duplicate experiments were reported.
Fixed human red blood cells, in the concentration range of 90 – 245 ¥
10 4 ml –1 , were placed in suspension in 0.01 mol l –1 phosphate buffer (pH 8.0) in
the lower cavity of stage 1 in a total volume of about 0.4 ml. A field of known
intensity (0.05 Vm –1 or 0.01Vm –1 ) was applied for a specified time period (30 s
or 60 s) then a fresh top chamber containing buffer only was aligned with the
bottom chamber of stage 1. This process was repeated until several transfers
had been completed. Cells were then removed from the top cavities, and the
fraction of the original population transferred at each step was obtained by
counting the suspended cells with a hemacytameter.
Precautions are required when using sliding chambers. Swapping of the
liquids between the chamber liquid surfaces occurs during the time period
while the chambers approach each other for the extraction transfer step and
depart after the transfer. The swapping of liquid and enhanced mass transfer
associated with such hydrodynamic flow for similar equipment was reported
[73]. The schematic diagram indicating the swapping of liquid and flow pattern
during the alignment and separation of the chamber is shown in Fig. 8 [73]. In
order to assess the magnitude of cell migration due to this phenomenon, a few
control experiments were performed without the application of an electric
field. Results are shown in Table 2.
Considerable numbers of cells are transferred due to the hydrodynamic flow
during transfer steps, especially in the initial steps. This problem is alleviated by
giving sufficient settling time for the cells to settle toward the bottom of the
lower cavity and by considerably reducing the speed at which the cavities are
aligned during the transfer steps.
160
K.S.M.S. Raghavarao et al.
Table 2. CCD without application of electric field
S No.
Initial
Transfer
Transfer
Transfer
Residual
cells
#1
#2
#3
cells
1
273
178
29
5
52
2
328
200
32
4
46
shown in Fig. 7a. Identically designed plates assure uniform loading and sealing
when the plates are clamped together. The experimental samples are loaded and
withdrawn through the fill ports located on the side of each plate as shown in
Fig. 7b. ADSEP is driven by an independent power supply (Lamda, Model No.
LP-532-FM) for rotating the plates to bring the chambers into interfacial contact
with each other. This ADSEP was modified to use as ELECSEP by replacing the
chamber bottoms with metal cover plates. Electrodes are kept over these cover
plates with gaskets in between them.
Preliminary electrophoretic transfer experiments were carried out [72] with
two types of particles, fixed human red blood cells and latex particles (average
diameter is 3.5 mm and 2.3 mm, respectively). The particles were counted by
hemacytameter with a minimum of three counts per fraction. Average values of
duplicate experiments were reported.
Fixed human red blood cells, in the concentration range of 90 – 245 ¥
10 4 ml –1 , were placed in suspension in 0.01 mol l –1 phosphate buffer (pH 8.0) in
the lower cavity of stage 1 in a total volume of about 0.4 ml. A field of known
intensity (0.05 Vm –1 or 0.01Vm –1 ) was applied for a specified time period (30 s
or 60 s) then a fresh top chamber containing buffer only was aligned with the
bottom chamber of stage 1. This process was repeated until several transfers
had been completed. Cells were then removed from the top cavities, and the
fraction of the original population transferred at each step was obtained by
counting the suspended cells with a hemacytameter.
Precautions are required when using sliding chambers. Swapping of the
liquids between the chamber liquid surfaces occurs during the time period
while the chambers approach each other for the extraction transfer step and
depart after the transfer. The swapping of liquid and enhanced mass transfer
associated with such hydrodynamic flow for similar equipment was reported
[73]. The schematic diagram indicating the swapping of liquid and flow pattern
during the alignment and separation of the chamber is shown in Fig. 8 [73]. In
order to assess the magnitude of cell migration due to this phenomenon, a few
control experiments were performed without the application of an electric
field. Results are shown in Table 2.
Considerable numbers of cells are transferred due to the hydrodynamic flow
during transfer steps, especially in the initial steps. This problem is alleviated by
giving sufficient settling time for the cells to settle toward the bottom of the
lower cavity and by considerably reducing the speed at which the cavities are
aligned during the transfer steps.
160
K.S.M.S. Raghavarao et al.
Table 2. CCD without application of electric field
S No.
Initial
Transfer
Transfer
Transfer
Residual
cells
#1
#2
#3
cells
1
273
178
29
5
52
2
328
200
32
4
46