Chapter 15
The Continuous Rotating Annular Electrophoresis Column
Downstream Processing and Bioseparation Downloaded from pubs.acs.org by UNIV LAVAL on 07/12/16. For personal use only.
A Novel Approach to Large-Scale Electrophoresis Randall A. Yoshisato , Ravindra Datta, Janusz P. Gorowicz , Robert A. Beardsley, and Gregory R. Carmichael 1
2
Department of Chemical and Materials Engineering, University of Iowa, Iowa City, IA 52242 The continuous rotating annular electrophoresis (CRAE) column design is capable of processing relatively large flowrates and can be scaled-up for industrial use. Previous modelling studies indicate that this column design offers considerable flexibility in meeting process objectives through the decoupling of several important design parameters. The column geometry, voltage gradient, residence time, angular velocity, and packing material/size can be adjusted in order to achieve the desired separation while controlling the peak temperature rise in the bed. However, actual specification of the operating parameters requires careful consideration of buoyancy effects, dispersion, and electrophoretic mobilities in order to achieve optimum results. A laboratory scale CRAE column has been constructed to verify these findings. This paper summarizes the work that has been done so far in developing the CRAE column.
Electrophoresis is one of the most sensitive methods for the separation and purification of charged chemicals available. Most species acquire a charge in a polar or ionic solution through ionization or ion adsorption. These species can be separated from one another based on the relative differences between their electrophoretic migration velocities. Proteins, ions, colloids, cellular materials, organelles and whole cells (1-5) have been separated by electrophoresis on an analytical scale. This ability to separate a wide range of compounds with high selectivity suggests that large-scale electrophoretic methods may be a useful adjunct to current techniques used in downstream bioprocessing (6-8). M a n y novel electrophoretic devices and techniques have been proposed for continuous electrophoretic separations such as the velocitystabilized Biostream/Harwell device (9-11), the recycle continuous-flow electrophoresis device (12-14), Bier's isoelectric focusing technique (15), Current address: Dow Chemical U.S.A., 2800 Mitchell Drive, P.O. Box 9002, Walnut Creek, C A 94598-0902 Current address: Johnson Controls, Automation Systems Group, Saline, MI 48176
1
2
0097-6156/90/0419-0285$06.00/0 © 1990 American Chemical Society
Downstream Processing and Bioseparation Downloaded from pubs.acs.org by UNIV LAVAL on 07/12/16. For personal use only.
286
DOWNSTREAM
PROCESSING AND BIOSEPARATION
Gidding's electrical field-flow fractionation technique (16), and the rotating annular electrochromatograph (17-18). The rotating annular electrochromatography column developed by Scott (17) has bulk f l o w i n the axial direction. This device is actually a rotating continuous Chromatograph, w i t h a radial electric field p r o v i d i n g a radial electrophoretic separation, similar to the Biostream/Harwell device. Despite these advances and the success of electrophoresis i n analytical applications, the scale-up of electrophoresis for industrial separations has been hampered variously by l o w throughput, substantial Joule heating, significant dispersion phenomena, and the inability to handle m u l t i component separations. The recently developed C R A E column is a design that utilizes an axial electric field i n an annular column (19-21). U n l i k e other electrophoretic separators, the C R A E c o l u m n operates w i t h the electric field imposed i n the same direction as the elutant flow. The bed is rotated slowly about its axis such that each component leaves the column at a different angular position. Products form helical bands as they traverse from the stationary feed point, d o w n the column to stationary product collection points at the bottom of the column, as shown i n Figure 1. Similar rotating annular separators have been developed previously for use i n gas and liquid chromatography (21-24); however, the C R A E column is the first attempt to utilize this principle i n electrophoresis. A key advantage of this configuration for electrophoresis is that it decouples the directions of separation (angular), electric field gradient (axial) and heat removal (radial), thus offering greater design flexibility.
BASIC ELECTROPHORESIS THEORY For a charged species i carried by elutant flow and under the influence of an electric field, the net species velocity, , is the sum of the convective and electrophoretic migration velocities, (1) The convective velocity is the bulk average velocity of the elutant given by = hr t where t is the mean residence time of elutant. migration velocity for species i can be written as Wi = - Ui4^-
dz
The
(2) electrophoretic
(3)
where Ε is the electric potential and uj is the electrophoretic mobility of species i w h i c h can be positive, negative, or zero depending upon whether
Downstream Processing and Bioseparation Downloaded from pubs.acs.org by UNIV LAVAL on 07/12/16. For personal use only.
YOSHISATO ET AL. Continuous Rotating Annuhr Electrophoresis Column 287
Figure 1. C R A E Helical Product Bands
288
DOWNSTREAM
PROCESSING AND BIOSEPARATION
the species has a net charge that is positive, negative, or zero, respectively. For a spherical particle, the electrophoretic mobility is related to its total net charge, particle radius, and fluid viscosity by Q U ; =-
6πr μ
()
Downstream Processing and Bioseparation Downloaded from pubs.acs.org by UNIV LAVAL on 07/12/16. For personal use only.
p
4
However, the electrophoretic mobility calculated using Eq. (4) is usually not accurate a n d , i n general, the electrophoretic mobility must be measured experimentally. A n alternate expression for Uj i n terms of the zeta potential is given by Henry
Ui = - ^ - f ( 4 > ) 6πμ
(5)
where φ is the ratio of particle radius and double layer thickness, and ί(φ) varies between 1.0 for small φ and 1.5 for large φ. The mean residence time for species i i n the column is given by _
L
_
- -
L
+ Wj
'
If the species residence times are sufficiently different, the various components w i l l be well-resolved. The resolution between two exiting bands is defined by R
s
= ^ 4σ
2
(7)
assuming that the baseline band width is given by four times the standard deviation.
MATHEMATICAL MODEL FOR THE CRAE COLUMN A comprehensive mathematical model has been formulated for the C R A E column w h i c h considers temperature and velocity gradients, dispersion, electroosmosis a n d adsorption (20). For the sake of completeness, the governing equations are summarized below. Conservation of momentum is expressed by
(8)
15. YOSHISATO ET AL.
Continuous Rotating Annular Electrophoresis Column 289
with boundary conditions Ρ =P Ρ =P
at ζ at ζ at r atr
0
L
v = v 7
P
=0 =L =η =r
(9) (10) (11) (12)
n
Downstream Processing and Bioseparation Downloaded from pubs.acs.org by UNIV LAVAL on 07/12/16. For personal use only.
A slip boundary condition is assumed to exist at the walls due electroosmotic flow given by the Helmholtz-Smoluchowski equation
v
eo
where the electroosmotic mobility u
u
G O
= -
e o
u
e o d z
to
(
1
3
)
is given by εζ 4πμ
=
, (14)
v
The conservation of energy is expressed by the heat equation 3T
1 3 / dT\
(15)
with boundary conditions T =T 3T ^ =0 T =T T =T 0
0
0
at
z=0
(16)
at at at
z =L r = rj r=r
(17) (18) (19)
0
The conservation of species i i n a packed C R A E column is given by 3Q ,„ 3ni _ 3Q IÎÛ 3 Q ^ ff^ Q ε ω» — — + (1- ε ) ω ——- + ε — - = — ^ — r + K g 30 30 3z r dz 2
Β
1
x
Β
T
Β
η
2
d Q
2
e
1
2
(20)
290
DOWNSTREAM
PROCESSING
AND BIOSEPARATION
The second term on the left accounts for possible adsorption of species i onto the surface of the packing material. For a single feed inlet, the boundary conditions are Q =0 Q = Qf Q =0
Downstream Processing and Bioseparation Downloaded from pubs.acs.org by UNIV LAVAL on 07/12/16. For personal use only.
Ci = 0
For all ζ
at at
θ=0 0 < θ < Qf
z
