Comparison of Methods of Preparative Cell Electrophoresis - ACS

Chapter 15

Comparison of Methods of Preparative Cell Electrophoresis

Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: June 10, 1991 | doi: 10.1021/bk-1991-0464.ch015

Paul Todd Center for Chemical Technology, National Institute of Standards and Technology, 325 Broadway 831.02, Boulder, CO 80303-3328

In the electrophoretic separation of macromolecules, resolution, purity and yield establish the merits of a separation method. In the separation of living cells by electrophoresis, additional significant criteria include viability, capacity, convenience and cost, as the method must also be acceptable to cell biologists, nondestructive to living cells and in free solution. These demands lead to the following quantitative characteristics of methods of cell electrophoresis: separation (resolution), purity, capacity, product viability, convenience and capital cost. Thirteen different methods of electrophoretic cell separation were compared on the basis of these six criteria using a relative scale of 0 to 4. The methods compared were free zone electrophoresis, density gradient electrophoresis, ascending vertical electrophoresis, re-orienting density gradient electrophoresis, free-flow electrophoresis, agarose sol electrophoresis, low-gravity electrophoresis, continuous flow low-gravity electrophoresis, rotating annular electrophoresis, density-gradient isoelectric focusing, free flow isoelectric focusing, stable flow free boundary electrophoresis, and continuous magnetic belt electrophoresis. In each case the sum of the six scores provides an overall "figure of merit"; however, no method rates a perfect score of 24. While quantitative trade-offs, such as resolution for capacity and vice versa, impose technical limitations on each method, lack of convenience is one of the most significant factors in the failure of cell electrophoresis to become a widely popular method.

C e l l e l e c t r o p h o r e s i s , l i k e the e l e c t r o p h o r e s i s o f macromolecules, a h i g h - r e s o l u t i o n s e p a r a t i o n method t h a t i s d i f f i c u l t t o s c a l e up.

This chapter not subject to U.S. copyright Published 1991 American Chemical Society

In Cell Separation Science and Technology; Kompala, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

15.

TODD

Preparative Cell Electrophoresis: Comparison of Methods

217

S u b p o p u l a t i o n s o f c e l l s f o r w h i c h no a f f i n i t y l i g a n d h a s b e e n d e v e l o p e d and f o r w h i c h t h e r e i s no d i s t i n c t s i z e o r d e n s i t y range a r e o f t e n s e p a r a b l e on t h e b a s i s o f t h e i r e l e c t r o p h o r e t i c m o b i l i t y , w h i c h may i n t u r n be r e l a t e d t o t h e i r f u n c t i o n , whether f o r t u i t o u s l y o r o t h e r w i s e . Electrophoresis


E l e c t r o p h o r e s i s i s the m o t i o n o f p a r t i c l e s ( m o l e c u l e s , s m a l l p a r t i c l e s and whole b i o l o g i c a l c e l l s ) i n an e l e c t r i c f i e l d and i s one o f s e v e r a l e l e c t r o k i n e t i c transport processes. The v e l o c i t y o f a p a r t i c l e p e r u n i t a p p l i e d e l e c t r i c f i e l d i s i t s e l e c t r o p h o r e t i c m o b i l i t y , μ; t h i s i s a c h a r a c t e r i s t i c o f i n d i v i d u a l p a r t i c l e s and c a n be u s e d as a b a s i s o f s e p a r a t i o n and p u r i f i c a t i o n . T h i s s e p a r a t i o n method i s a r a t e ( o r transport) process. The four principal e l e c t r o k i n e t i c processes of i n t e r e s t are e l e c t r o p h o r e s i s (motion o f a p a r t i c l e i n an e l e c t r i c f i e l d ) , s t r e a m i n g p o t e n t i a l ( t h e c r e a t i o n o f a p o t e n t i a l by f l u i d f l o w ) , sedimentation p o t e n t i a l ( t h e c r e a t i o n o f a p o t e n t i a l by p a r t i c l e m o t i o n ) , and electroosmosis ( t h e i n d u c t i o n o f f l o w a t a c h a r g e d s u r f a c e by an electric field). These phenomena always o c c u r , and t h e i r r e l a t i v e magnitudes d e t e r m i n e the p r a c t i c a l i t y o f an e l e c t r o p h o r e t i c s e p a r a t i o n o r an e l e c t r o p h o r e t i c measurement. I t i s generally desirable, f o r example, t o m i n i m i z e m o t i o n due t o e l e c t r o o s m o s i s i n practical applications. A b r i e f d i s c u s s i o n o f the general e l e c t r o k i n e t i c relationships follows. The s u r f a c e c h a r g e d e n s i t y o f suspended p a r t i c l e s p r e v e n t s t h e i r c o a g u l a t i o n and l e a d s t o s t a b i l i t y o f l y o p h o b i c colloids. This s t a b i l i t y d e t e r m i n e s t h e s u c c e s s e s o f p a i n t s and c o a t i n g s , p u l p and p a p e r , sewage and f e r m e n t a t i o n , and numerous o t h e r m a t e r i a l s and processes. The s u r f a c e c h a r g e a l s o l e a d s t o m o t i o n when such p a r t i c l e s a r e suspended i n an e l e c t r i c f i e l d . The p a r t i c l e s u r f a c e has an e l e c t r o k i n e t i c ("zeta") p o t e n t i a l , ζ, p r o p o r t i o n a l ( i n a g i v e n i o n i c e n v i r o n m e n t ) t o σ , i t s s u r f a c e c h a r g e d e n s i t y - a few mV a t the hydrodynamic s u r f a c e o f s t a b l e , n o n c o n d u c t i n g p a r t i c l e s , i n c l u d i n g b i o l o g i c a l c e l l s , i n aqueous s u s p e n s i o n . I f t h e s o l u t i o n has a b s o l u t e d i e l e c t r i c c o n s t a n t €, e l e c t r o p h o r e t i c v e l o c i t y i s β

3η f o r s m a l l p a r t i c l e s , such as m o l e c u l e s , whose r a d i u s o f c u r v a t u r e i s s i m i l a r t o t h a t o f a d i s s o l v e d i o n (Debye-Huckel p a r t i c l e s ) , and v=Ç*E

(2)

η f o r l a r g e ("von Smoluchowski") p a r t i c l e s , such as c e l l s and o r g a n e l l e s . These r e l a t i o n s h i p s a r e e x p r e s s e d i n r a t i o n a l i z e d MKS ( S I ) u n i t s , as c l a r i f i e d , f o r example, by H u n t e r ( 1 ) , where η i s t h e v i s c o s i t y o f the b u l k medium, and e i s t h e d i e l e c t r i c c o n s t a n t . At typical ionic


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s t r e n g t h s (0.01 t o 0.2 e q u i v . / L ) p a r t i c l e s i n the nanometer s i z e range u s u a l l y have m o b i l i t i e s (v/E) d i f f e r e n t from t h o s e specified by e q u a t i o n s (1) and (2) ( 2 ) .


Preparative Electrophoresis A n a l y t i c a l e l e c t r o p h o r e s i s i n a g e l matrix i s v e r y popular, because c o n v e c t i o n i s s u p p r e s s e d ; however, h i g h sample l o a d s c a n n o t be u s e d owing t o the l i m i t e d volume o f g e l t h a t can be c o o l e d s u f f i c i e n t l y t o p r o v i d e a u n i f o r m e l e c t r i c f i e l d , and c e l l s do n o t m i g r a t e i n g e l s . C a p i l l a r y zone e l e c t r o p h o r e s i s , a p o w e r f u l , h i g h - r e s o l u t i o n a n a l y t i c a l t o o l ( 3 ) , depends on p r o c e s s e s a t the m i c r o m e t e r s c a l e and i s n o t a p p l i c a b l e to p r e p a r a t i v e c e l l e l e c t r o p h o r e s i s . Therefore p r e p a r a t i v e e l e c t r o p h o r e s i s must be p e r f o r m e d i n f r e e f l u i d , so g r e a t c a r e must be t a k e n t o p r e v e n t t h e r m a l and s o l u t a l c o n v e c t i o n and c e l l s e d i m e n t a t i o n . The two most frequently used free-fluid methods are zone e l e c t r o p h o r e s i s i n a d e n s i t y g r a d i e n t and f r e e - f l o w ( o r continuous f l o w ) e l e c t r o p h o r e s i s (FFE o r C F E ) . O t h e r , l e s s p o p u l a r , methods w i l l n o t be d i s c u s s e d i n d e t a i l . These a r e d e s c r i b e d i n p a r t i a l r e v i e w s by I v o r y (4) and by Mosher e t a l . ( 5 ) . P h y s i c a l C o n s t r a i n t s Imposed by L i v i n g

Cells

Buffers. T y p i c a l c a t e g o r i e s o f b u f f e r s u s e d w i t h each o f the methods p o s s e s s a wide range o f ionic strengths, partly to a v o i d the a p p l i c a t i o n o f h i g h c u r r e n t s i n p r e p a r a t i v e separations., w h i c h r e q u i r e l o w - c o n d u c t i v i t y b u f f e r s t o a v o i d c o n v e c t i v e m i x i n g due to Joule heating. A t v e r y low i o n i c s t r e n g t h , phosphate b u f f e r s a r e f o u n d h a r m f u l t o c e l l s , and t h i s i s the p r i n c i p a l r e a s o n t h a t t r i e t h a n o l a m i n e was introduced as a general carrier buffer for free-flow e l e c t r o p h o r e s i s by Z e i l l e r and Hannig ( 6 ) . When c e l l s a r e p r e s e n t n e u t r a l s o l u t e s s u c h as s u c r o s e o r m a n n i t o l must be added f o r o s m o t i c balance. These b u f f e r s have undergone t e s t i n g i n a n a l y t i c a l and preparative c e l l electrophoresis. Temperature. Temperature c o n t r o l i s g e n e r a l l y imposed on a l l f r e e e l e c t r o p h o r e s i s systems as a means o f s u p p r e s s i n g t h e r m a l c o n v e c t i o n and avoiding v i s c o s i t y gradients. Biological separands impose additional constraints on thermoregulation of electrophoretic separators. W i t h a few n o t a b l e e x c e p t i o n s , l i v i n g c e l l s do not t o l e r a t e t e m p e r a t u r e s above 40°C. A d d i t i o n a l l y , mammalian c e l l s a r e damaged by b e i n g a l l o w e d t o m e t a b o l i z e i n n o n - n u t r i e n t medium, so i t i s necessary t o reduce m e t a b o l i c r a t e by reducing temperature, t y p i c a l l y t o 4 t o 10°C. P r o t e i n s a r e more t h e r m o t o l e r a n t , b u t a t 37°C the enzymes o f homeothermic organisms and many b a c t e r i a ( i n c l u d i n g E_j_ c o l i ) a r e a c t i v e , and unwanted h y d r o l y s i s r e a c t i o n s o c c u r i n samples containing mixtures of p r o t e i n s . Electric field. I t i s p o s s i b l e t o move membrane p r o t e i n s i n c e l l s i m m o b i l i z e d i n an e l e c t r i c f i e l d (7) and, i n s u f f i c i e n t l y s t r o n g f i e l d s (> 3 kV/cm) t o p r o d u c e d i e l e c t r i c breakdown o f the plasma membrane i n a few / i s , r e s u l t i n g i n a p o t e n t i a l f o r c e l l - c e l l f u s i o n o r p o r e s t h r o u g h w h i c h macromolecules can r e a d i l y p a s s ( 8 ) . The s t r e n g t h o f such f i e l d s , 1 t o 15 kV/cm, c o n s i d e r a b l y exceeds the range o f f i e l d s t r e n g t h used f o r e l e c t r o p h o r e s i s .


15.


TODD

219

R o l e o f c e l l s i z e and d e n s i t y . Over a wide range o f r a d i i t h e m o b i l i t y o f a g i v e n c e l l t y p e i s independent o f c e l l s i z e , as p r e d i c t e d b y e l e c t r o k i n e t i c t h e o r y ( E q u a t i o n (2) , a b o v e ) . I s o l a t e d e l e c t r o p h o r e t i c f r a c t i o n s c o l l e c t e d a f t e r d e n s i t y g r a d i e n t e l e c t r o p h o r e s i s have been a n a l y z e d i n d i v i d u a l l y w i t h a p a r t i c l e s i z e d i s t r i b u t i o n a n a l y z e r , and no c o n s i s t e n t r e l a t i o n s h i p between s i z e and e l e c t r o p h o r e t i c f r a c t i o n was f o u n d ( 9 ) . T h i s f i n d i n g i m p l i e s t h a t , a l t h o u g h t h e g r a v i t y and electrokinetic vectors are a n t i p a r a l l e l (or p a r a l l e l ) i n density gradient electrophoresis, s i z e plays very l i t t l e r o l e i n determining the e l e c t r o p h o r e t i c m i g r a t i o n o f c u l t u r e d c e l l s . As e a r l i e r c h a p t e r s have i m p l i e d , d e n s i t y p l a y s a n i m p o r t a n t r o l e i n c e l l s e d i m e n t a t i o n , and when ρ - p i s e x c e s s i v e (>0.03 g/cm ) f r e e e l e c t r o p h o r e s i s i n a d e n s i t y g r a d i e n t i s dominated b y s e d i m e n t a t i o n , w h i l e s e p a r a t i o n b y f r e e - f l o w e l e c t r o p h o r e s i s i s l e s s so (10, 1 1 ) . 3


0

Droplet

Sedimentation 6

The d i f f u s i o n c o e f f i c i e n t s , D, o f s m a l l m o l e c u l e s a r e i n t h e range 10~ -10" cm /s, o f m a c r o m o l e c u l e s , 10~ -10~ ; and o f whole c e l l s and p a r t i c l e s , 10~ -10~ . I f a s m a l l zone, o r d r o p l e t , o f r a d i u s R c o n t a i n s η p a r t i c l e s o f r a d i u s a i n s i d e , whose d i f f u s i v i t y i s much l e s s t h a n t h a t o f s o l u t e s o u t s i d e , t h e n r a p i d d i f f u s i o n o f s o l u t e s i n and slow d i f f u s i o n o f p a r t i c l e s o u t o f t h e d r o p l e t ( w i t h c o n s e r v a t i o n o f mass) l e a d s t o a l o c a l l y i n c r e a s e d d e n s i t y o f t h e d r o p l e t : 5

2

7

12

6

9

I f p > p t h e n t h e d r o p l e t f a l l s down; i f ρ < p i t i s buoyed upward (12). D r o p l e t s e d i m e n t a t i o n ( o r buoyancy) i s a s p e c i a l c a s e o f convection, and i t s occurrence i s governed by the f o l l o w i n g s t a b i l i t y rule D

Q

Ό

D(d() /dz) 0

+D (dp/dz) s

Q

>0

W

in which D and D are dif f u s i v i t i e s o f the c e l l and s o l u t e , respectively. T y p i c a l l y άρ/άζ w i l l be somewhere n e g a t i v e , and dp /dz 0 except i n density gradient electrophoresis. I n column e l e c t r o p h o r e s i s d r o p l e t s e d i m e n t a t i o n c a n n o t be p r e v e n t e d e x c e p t a t low c e l l d e n s i t i e s (13), so a s u i t a b l e way t o s c a l e column e l e c t r o p h o r e s i s i s t o i n c r e a s e column c r o s s - s e c t i o n a l a r e a ( 1 4 ) . Under c o n d i t i o n s o f d r o p l e t s e d i m e n t a t i o n p a r t i c l e s s t i l l behave i n d i v i d u a l l y u n l e s s t h e i o n i c environment a l s o p e r m i t s a g g r e g a t i o n (15, 1 6 ) . I n t h e c a s e o f e r y t h r o c y t e s t h e r e i s s u f f i c i e n t e l e c t r o s t a t i c r e p u l s i o n among c e l l s t o p e r m i t t h e maintenance o f s t a b l e d i s p e r s i o n s up t o a t l e a s t 3 χ 1 0 cells/mL (1Z,18)· Droplet sedimentation i s an avoidable initial c o n d i t i o n i n zone p r o c e s s e s , b u t i t c a n be c r e a t e d d u r i n g p r o c e s s i n g when s e p a r a n d s become h i g h l y c o n c e n t r a t e d as i n i s o e l e c t r i c f o c u s i n g . The absence o f d r o p l e t s e d i m e n t a t i o n i n low g r a v i t y i s one o f t h e most s i g n i f i c a n t a t t r a c t i o n s o f low-gravity separation science. s

0

8


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E v a l u a t i o n o f S e p a r a t i o n Methods:


Resolution Two d e f i n i t i o n s o f r e s o l u t i o n a r e u s e d i n t h i s r e v i e w ( s e e C h a p t e r 1) . One f o l l o w s t h e paradigm o f m u l t i - s t a g e e x t r a c t i o n o r a d s o r p t i o n and i d e n t i f i e s t h e p r a c t i c a l l i m i t o f t h e t o t a l number o f f r a c t i o n s t h a t can be c o l l e c t e d from a g i v e n s e p a r a t i o n d e v i c e , NSU ("number o f s e p a r a t i o n u n i t s " ) , which i s u s u a l l y s p e c i f i e d i n equipment d e s i g n . The i n v e r s e v i e w i s a l s o u s e f u l . I n s e d i m e n t a t i o n , i n w h i c h r e s o l u t i o n depends on t h e s t a n d a r d d e v i a t i o n o f t h e d i s t a n c e s e d i m e n t e d and e l e c t r o p h o r e s i s , i n which i t depends on t h e s t a n d a r d d e v i a t i o n o f m o b i l i t y , and i n f l o w s o r t i n g , where i t depends on t h e s t a n d a r d d e v i a t i o n o f o p t i c a l f l u o r e s c e n c e , f o r example — a l l c a s e s i n w h i c h the moments o f t h e a p p r o p r i a t e measurement a r e known — t h e c o e f f i c i e n t o f v a r i a t i o n ("CV") o r " r e l a t i v e s t a n d a r d d e v i a t i o n , " i n any c a s e t h e r a t i o o f s t a n d a r d d e v i a t i o n t o t h e mean i s u s e d . CV i s n o t a s i m p l e r e c i p r o c a l o f NSU, as t h e measured e l e c t r o p h o r e t i c s t a n d a r d d e v i a t i o n a c c o u n t s f o r p h y s i c a l d i s t o r t i o n o f s e p a r a n d zones d u r i n g m i g r a t i o n and c o l l e c t i o n , i n t r i n s i c b i o l o g i c a l h e t e r o g e n e i t y and c o u n t i n g s t a t i s t i c s i n a d d i t i o n t o t h e f i n i t e s i z e o f c o l l e c t e d f r a c t i o n s . So, as a r u l e , CV > 1/NSU.

5

()

Thus b o t h v a l u e s w i l l be i n c l u d e d , as t h e y e x p r e s s b o t h t h e g e o m e t r i c a l and p r a c t i c a l r e s o l u t i o n o f each i n s t r u m e n t . Purity I n t h e c a s e o f c e l l s e p a r a t i o n p u r i t y i s d e f i n e d as t h e p r o p o r t i o n o f c e l l s i n a separated f r a c t i o n t h a t a r e o f the d e s i r e d type. In m u l t i f r a c t i o n "peaks," s i n g l e f r a c t i o n s c a n be i d e n t i f i e d w i t h g r e a t e r t h a n 99% p u r i t y ; however, i n d i v i d u a l f r a c t i o n s may have low volume ( a n d hence low c e l l numbers) , and a whole "peak," when p o o l e d , may have much lower p u r i t y . P u r i t y and y i e l d a r e thus always r e l a t e d . The o v e r l a p o f peaks i s more s e v e r e i n some s e p a r a t i o n systems t h a n i n o t h e r s , and even narrow peaks c a n be h e a v i l y c o n t a m i n a t e d w i t h c e l l s from a d j a c e n t fractions. T h e r e f o r e i t i s m e a n i n g f u l t o u s e t h e maximum r e p o r t e d p u r i t y as an i n d i c a t o r f o r each method. Viability I t i s u s u a l l y p o s s i b l e t o c o n t r i v e s e p a r a t i o n c o n d i t i o n s t h a t do n o t k i l l living cells. I n some c a s e s , i n g e n u i t y i s r e q u i r e d t o m i n i m i z e shear f o r c e s , e l i m i n a t e t o x i c chemicals ( i n c l u d i n g c e r t a i n a f f i n i t y ligands) , incorporate p h y s i o l o g i c a l l y acceptable b u f f e r ions, maintain o s m o l a r i t y , c o n t r o l t h e temperature, etc. In n e a r l y a l l cases, abnormal s a l t c o n c e n t r a t i o n s a r e r e q u i r e d , so t h e system t h a t c a n u s e the h i g h e s t s a l t c o n c e n t r a t i o n w i l l p r o d u c e t h e h i g h e s t v i a b i l i t y . T h i s , i n t u r n , i s t h e system w i t h t h e most e f f e c t i v e h e a t r e j e c t i o n . I t i s l i k e l y , a l t h o u g h n o t a r g u e d i n t h i s r e v i e w , t h a t minimum R a y l e i g h number, Ra, w i l l r e s u l t i n maximum v i a b i l i t y .


15.

TODD


Capacity C a p a c i t y ( a l s o known as " t h r o u g h p u t " ) i s measured as mass o f f e e d s t o c k p r o c e s s e d p e r h o u r o r mass o f p r o d u c t s e p a r a t e d p e r hour, d e p e n d i n g on the o b j e c t i v e . T h i s f i g u r e a p p l i e s whether p r o c e s s i n g i s c o n t i n u o u s or batchwise. I n the case o f c e l l separation, e s p e c i a l l y by e l e c t r o p h o r e s i s , t y p i c a l u n i t s o f c a p a c i t y would be m i l l i o n s o f c e l l s p e r h o u r . Because, under most e l e c t r o p h o r e s i s c o n d i t i o n s , t h e maximum f e e d c o n c e n t r a t i o n i s 1 0 c e l l s / m L ( d e p e n d i n g s l i g h t l y on c e l l s i z e and d e n s i t y ) , t h e n c a p a c i t y i s d i r e c t l y r e l a t e d t o t h e volume o f f e e d t h a t c a n be p r o c e s s e d p e r hour. 7


Convenience I n t h e l a b o r - i n t e n s i v e a r e n a o f b i o m e d i c a l r e s e a r c h c o n v e n i e n c e may come i n one o f two forms: a s i m p l e p r o c e s s t h a t r e q u i r e s v e r y l i t t l e e n g i n e e r i n g s k i l l o r p h y s i c a l m a n i p u l a t i o n o r a complex p r o c e s s t h a t has been h i g h l y automated. B i o m e d i c a l r e s e a r c h e r s do n o t w i s h t o dedicate excessive amounts o f manpower to separation process development o r maintenance. I n c e l l e l e c t r o p h o r e s i s t h e f o r m a t i o n o f a d e n s i t y g r a d i e n t o r t h e m i x i n g o f a m u l t i t u d e o f b u f f e r s may be c o n s i d e r e d an i n c o n v e n i e n c e ; t h e need t o t r a v e l t o a u n i q u e f a c i l i t y i s a l s o a d e t e r r e n t to m u l t i p l e experiments. Cost The cost of a separation process includes capital equipment (apparatus), r e a g e n t s and l a b o r . The economics o f i n v e s t i n g i n s e p a r a t i o n t e c h n o l o g y a r e h i g h l y dependent upon g o a l s . A frequently r e p e a t e d p r o c e s s , f o r example, i s b e t t e r done by an automated system and i s c a p i t a l - i n t e n s i v e . A r a r e l y p e r f o r m e d , i n t e l l e c t u a l l y demanding p r o c e d u r e would be l a b o r - i n t e n s i v e . And a p r o c e s s t h a t r e q u i r e s l a r g e q u a n t i t i e s o f e x p e n s i v e a f f i n i t y r e a g e n t s (such as a n t i b o d i e s ) would n o t be u s e d t o s e p a r a t e l a r g e q u a n t i t i e s o f s e p a r a n d w i t h o u t p r o v i s i o n s for r e c y c l i n g ligands or reducing t h e i r cost. Thus t h e " c o s t " o f a c e l l e l e c t r o p h o r e s i s method i s t h e sum o f c a p i t a l , l a b o r and s u p p l i e s . Over t h e f u l l range o f methods c o n s i d e r e d t o t a l c o s t ranges o v e r 4 o r d e r s o f magnitude. I n t h i s review only c a p i t a l c o s t i s evaluated. Methods o f P r e p a r a t i v e

Cell

Electrophoresis

T h e r e a r e two v e r y b r o a d c a t e g o r i e s o f c e l l e l e c t r o p h o r e s i s methods. The f o l l o w i n g d i s c u s s i o n d i v i d e s a l l methods i n t o s t a t i c column methods, w h i c h a r e d i s c u s s e d f i r s t , and f l o w i n g methods. S t a t i c column methods a r e b a t c h p r o c e s s e s w h i l e f l o w methods a r e c o n t i n u o u s . Each method i s i n t r o d u c e d by a q u a n t i t a t i v e d e s c r i p t i o n and t h e n e v a l u a t e d according t o the 6 c r i t e r i a . 1.

F r e e Zone E l e c t r o p h o r e s i s (FZE)

H o r i z o n t a l column e l e c t r o p h o r e s i s has been p e r f o r m e d i n r o t a t i n g tubes o f 1-3 mm i n n e r d i a m e t e r ( 1 9 ) . The s m a l l d i a m e t e r m i n i m i z e s c o n v e c t i o n d i s t a n c e and f a c i l i t a t e s h e a t r e j e c t i o n w h i l e r o t a t i o n c o u n t e r a c t s a l l t h r e e g r a v i t y - d e p e n d e n t p r o c e s s e s : c o n v e c t i o n , zone s e d i m e n t a t i o n and


221

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p a r t i c l e sedimentation. The c o n c e p t i s i l l u s t r a t e d i n F i g u r e 1, w h i c h shows t h a t the t o t a l v e r t i c a l v e l o c i t y v e c t o r o s c i l l a t e s as a sample zone moves i n the e l e c t r i c f i e l d , so t h a t s p i r a l m o t i o n r e s u l t s w i t h a r a d i u s v e c t o r t h a t c a n be d e r i v e d from e q u a t i o n s o f m o t i o n i n w h i c h the sample zone i s t r e a t e d as a s o l i d p a r t i c l e . I n a c t u a l i t y , the sample zone i s more l i k e a s e d i m e n t i n g d r o p l e t , w h i c h c a n be t r e a t e d as a p a r t i c l e w i t h d e n s i t y ρ (see " D r o p l e t S e d i m e n t a t i o n , " equation ( 3 ) , a b o v e ) . Due t o g r a v i t y and c e n t r i f u g a l a c c e l e r a t i o n , the c e n t e r o f the s p i r a l i n F i g u r e 1 ( c o o r d i n a t e s k , l ) i s n o t the c e n t e r o f the tube, and the v e r t i c a l c i r c l e , i n χ and y, d e s c r i b e d by the sample zone is Ό

2

2

2

6

(x-k) + ( y - i ) = r e x p (2γ t)

i n w h i c h a zone i s s t a b i l i z e d i n s u s p e n s i o n when k +1 and 7 , the r a t e c o n s t a n t f o r c e n t r i f u g a l motion, a r e m i n i m i z e d . Hjertén s o l v e d the e q u a t i o n s o f m o t i o n f o r t h e s e v a l u e s and f o u n d t h a t i f the Hjertén number, r e c e n t l y d e f i n e d (20) as


2

2

i s < 1.0, t h e n a c c e p t a b l e c o n d i t i o n s f o r s t a b i l i t y o f the sample zone exist. I n a t y p i c a l s e p a r a t i o n r , the r e s i d e n c e time i s between 1000 and 10000 s e c , and 7 i s between 10~ and 10" s" . 5

3

1

I n an open system, i n which f l u i d f l o w i n the d i r e c t i o n o f the a p p l i e d e l e c t r i c f i e l d i s p o s s i b l e , the n e t n e g a t i v e c h a r g e on the tube w a l l r e s u l t s i n p l u g - t y p e e l e c t r o o s m o t i c f l o w (EEO) t h a t enhances t o t a l f l u i d t r a n s p o r t a c c o r d i n g t o the H e l m h o l t z r e l a t i o n s h i p (21) i n t e g r a t e d o v e r the d o u b l e l a y e r a t the chamber w a l l v=t*E.

(8)

η I n a c l o s e d system, such as i s u s e d i n a l m o s t a l l p r a c t i c a l c a s e s , f l o w a t the w a l l s must be b a l a n c e d by r e t u r n f l o w i n the o p p o s i t e d i r e c t i o n a l o n g the c e n t e r o f the c y l i n d r i c a l tube, so t h a t the f l o w p r o f i l e i s a p a r a b o l a (22, 23, 24) 2

2

v=-&E(r /R -l/2)

9

(>

Thus, i f the e l e c t r o o s m o t i c m o b i l i t y μ^ ( c o e f f i c i e n t o f Ε i n e q u a t i o n ( 8 ) ) i s h i g h ( o f the o r d e r 10~ cm /V-s), t h e n sample zones w i l l be d i s t o r t e d i n t o p a r a b o l a s as shown i n the s i m u l a t i o n o f F i g u r e 2. An a d d i t i v e p a r a b o l i c p a t t e r n i s superimposed i f t h e r e i s a r a d i a l temperature g r a d i e n t t h a t causes a v i s c o s i t y g r a d i e n t (19). High t e m p e r a t u r e i n the c e n t e r o f the chamber r e s u l t s i n low v i s c o s i t y and h i g h e r v e l o c i t y due t o b o t h EEO r e t u r n f l o w and f a s t e r m i g r a t i o n o f separands. Increased temperature also increases conductivity. E x c e s s i v e power i n p u t under t y p i c a l o p e r a t i n g c o n d i t i o n s c a n r e s u l t i n 4

2


15. TODD


THE FREE-ZONE ELECTROPHORESIS PRINCIPLE


+ F i g u r e 1. F r e e zone e l e c t r o p h o r e s i s . T r a j e c t o r y f o l l o w e d b y a p a r t i c l e i n FZE i n a r o t a t i n g tube. I n the presence o f g r a v i t y the c e n t e r o f t h e s p i r a l i s below t h e c e n t e r o f r o t a t i o n (19).

(Reproduced with permission from reference 20. Copyright 1990 American Institute of Aeronautics and Astronautics.)

8 9 10 DISTANCE MIGRATED.CM

12

F i g u r e 2. C o n t o u r l i n e s o f sample bands p r e d i c t e d i n s i m u l a t i o n o f human ( r i g h t ) and r a b b i t ( l e f t ) e r y t h r o c y t e m i g r a t i o n , f r o m l e f t t o r i g h t , i n a c y l i n d r i c a l column i n low g r a v i t y , u s i n g s i m u l a t i o n method o f V a n d e r h o f f and M i c a l e (1979; M i c a l e e t a l , 1976). Assumptions were r - 1.0 h , μ « -0.06, /i(human) - 2.05, / i ( r a b b i t ) - -1.05 χ 10" cm /V-s, Ε - 18.6 V/cm, sample w i d t h - 0.75 χ chamber d i a m e t e r . C o u r t e s y o f F. J . M i c a l e . β ο

4

2

(Reproduced with permission from reference 20. Copyright 1990 American Institute of Aeronautics and Astronautics.)


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25% h i g h e r s e p a r a n d v e l o c i t y i n the c e n t e r o f the tube even i n the absence o f EEO ( 1 9 ) . I n s m a l l - b o r e tubes w i t h c l o s e d ends, s c r u p u l o u s c a r e must be t a k e n t o s u p p r e s s EEO by u s i n g a n e a r l y n e u t r a l , h i g h v i s c o s i t y c o a t i n g ( 1 9 ) , and r a p i d h e a t r e j e c t i o n and/or low c u r r e n t d e n s i t y i s necessary to minimize r a d i a l thermal g r a d i e n t s . A l l o f the above problems were c o n s i d e r e d i n the e n g i n e e r i n g o f a f r e e zone e l e c t r o p h o r e s i s system w i t h a 20 cm l o n g χ 3 mm d i a m e t e r r o t a t i n g tube w i t h no EEO (19, 25). A p r a c t i c a l minimum c o l l e c t i o n volume o f 0.05 mL from a t o t a l o f 1.5 mL g i v e s NSU - 30, and the r e s u l t s o f o p t i c a l s c a n n i n g o f the e l e c t r o p h o r e s i s tube d u r i n g a r u n shown i n F i g u r e 3 i n d i c a t e s CV - 5%. Owing t o the s h o r t n e s s o f a s i n g l e b a t c h p u r i f i c a t i o n (20 min) and the e f f i c i e n t h e a t t r a n s f e r , w h i c h a l l o w s e x p e r i m e n t s i n 0.15 M ( i s o t o n i c ) s a l t , t h i s system has no e f f e c t s on c e l l v i a b i l i t y beyond t h o s e c a u s e d by s p e n d i n g the same amount o f time i n s u s p e n s i o n , e x c e p t p o s s i b l y i n t h o s e c a s e s where a g g r e g a t i o n o r o t h e r c e l l - c e l l i n t e r a c t i o n s o c c u r (25, 15). The e l e c t r i c f i e l d i s o r t h o g o n a l t o the g r a v i t y v e c t o r , and r o t a t i o n compensates f o r c o n v e c t i o n , so d r o p l e t s e d i m e n t a t i o n c a n o c c u r f r e e l y , and the maximum c e l l l o a d i s e s t i m a t e d a t around 1 0 c e l l s i n a s t a r t i n g zone o f 0.1 mL ( v e r y d e n s e l y p a c k e d ) . A 30-min p r o c e s s i n g time g i v e s c a p a c i t y = 2 χ 1 0 c e l l s / h . The main s o u r c e s o f i n c o n v e n i e n c e a r e the p r e p a r a t i o n o f the e l e c t r o p h o r e s i s tube w i t h low EEO and the m e t i c u l o u s t a s k o f l o a d i n g the sample. O n l y 1 o r 2 such u n i t s a r e i n o p e r a t i o n i n t h e world. The c o s t o f i n s t r u m e n t p r o d u c t i o n , o n e - a t - a - t i m e , s h o u l d be $30000 - $50000. 8

8

2.

D e n s i t y G r a d i e n t E l e c t r o p h o r e s i s (DGE)

D e n s i t y g r a d i e n t e l e c t r o p h o r e s i s has been u s e d f o r p r o t e i n and v i r u s s e p a r a t i o n (26) and has been shown t o s e p a r a t e c e l l s on the b a s i s o f e l e c t r o p h o r e t i c m o b i l i t y as measured by m i c r o s c o p i c e l e c t r o p h o r e s i s (27, 28) . Methods and a p p l i c a t i o n s o f t h i s t e c h n i q u e have been r e v i e w e d (29). Downward e l e c t r o p h o r e s i s i n a c o m m e r c i a l l y a v a i l a b l e device u t i l i z i n g a Ficoll g r a d i e n t was first used to separate immunological cell t y p e s (30, 31» 22.)· Similar gradient-buffer c o n d i t i o n s a r e employed i n the B o l t z - T o d d a p p a r a t u s (13, 23) i n w h i c h e l e c t r o p h o r e s i s o f c e l l s and o t h e r separands i s u s u a l l y p e r f o r m e d i n the upward d i r e c t i o n ( F i g u r e 4 ) . The use o f a d e n s i t y g r a d i e n t counteracts (within c a l c u l a b l e l i m i t s ) a l l three e f f e c t s of g r a v i t y : p a r t i c l e s e d i m e n t a t i o n , c o n v e c t i o n and zone, o r d r o p l e t , s e d i m e n t a t i o n . The m i g r a t i o n r a t e s o f c e l l s i n a d e n s i t y g r a d i e n t a r e , however, q u a n t i t a t i v e l y d i f f e r e n t from t h o s e o b s e r v e d under t y p i c a l a n a l y t i c a l e l e c t r o p h o r e s i s c o n d i t i o n s (6, 24), b e c a u s e the f o l l o w i n g p h y s i c a l p r o p e r t i e s a r e n o t depend on p o s i t i o n i n the d e n s i t y g r a d i e n t : (1) The s e d i m e n t a t i o n component o f the v e l o c i t y v e c t o r changes as the d e n s i t y o f the s u s p e n d i n g f l u i d d e c r e a s e s . (2) The v i s c o s i t y o f the s u s p e n d i n g F i c o l l s o l u t i o n d e c r e a s e s r a p i d l y w i t h i n c r e a s i n g h e i g h t (33) . (3) The c o n d u c t i v i t y o f the s u s p e n d i n g e l e c t r o l y t e i n c r e a s e s , c o n s i s t e n t w i t h the v i s c o s i t y r e d u c t i o n . (4) C a r b o h y d r a t e polymers, i n c l u d i n g F i c o l l , t y p i c a l l y u s e d as s o l u t e s t o form d e n s i t y g r a d i e n t s i n c r e a s e the z e t a p o t e n t i a l o f c e l l s (34, 35). (5) C e l l s a p p l i e d t o the d e n s i t y g r a d i e n t i n h i g h c o n c e n t r a t i o n s undergo d r o p l e t s e d i m e n t a t i o n (12, 13) t h e r e b y increasing the magnitude o f the s e d i m e n t a t i o n component o f the


15.

TODD


EXP 1041, t = 83min 1.0 CM Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: June 10, 1991 | doi: 10.1021/bk-1991-0464.ch015

I

H I 11 I M M 11 I I I I I I I I M I

J I I 0 15 21 MIGRATION DISTANCE, CM F i g u r e 3. E l e c t r o p h o r e t i c s e p a r a t i o n o f c h i c k e n a n d r a b b i t e r y t h r o c y t e s ( f i x e d w i t h g l u t a r a l d e h y d e ) b y FZE. Sampling r e g i o n s 1, 2 a n d 3 were u s e d t o d e t e r m i n e p u r i t y , and d a t a were u s e d t o d e t e r m i n e CV.


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F i g u r e 4. Diagram o f a DGE column l a b e l e d t o i n d i c a t e the l o c a t i o n o f v a r i o u s s o l u t i o n s a t the s t a r t : A. Top b u f f e r , B. M i g r a t i o n zone w i t h o r w i t h o u t F i c o l l g r a d i e n t c o n t a i n i n g g e l l i n g m a t e r i a l , C. Sample t o be s e p a r a t e d , D. Dense s o l u t i o n t o s u p p o r t s o l u t i o n s i n the column, E. Top s o l u t i o n w h i c h p r e v e n t s e l e c t r o d e s o l u t i o n s from e n t e r i n g the column, F. E l e c t r o d e s o l u t i o n i n which i s immersed a b r i g h t p l a t i n u m w i r e e l e c t r o d e , G. P o l y a c r y l a m i d e g e l p l u g t o s u p p o r t h y d r o s t a t i c p r e s s u r e o f the column, H. T h e r m o s t a t e d j a c k e t . Typically Β c o n t a i n e d a g r a d i e n t o f 1.7 - 6.2% F i c o l l and an i n v e r s e g r a d i e n t o f 6.5 - 5.8% s u c r o s e w i t h o r w i t h o u t a g a r o s e , D c o n t a i n e d 15% F i c o l l , and F was s a t u r a t e d N a C l ( 1 3 ) .


15. TODD


migration v e l o c i t y i n e q u a t i o n ( 3 ) , above.

equation

(10),

below,

by

replacing

it

A l l o f t h e s e v a r i a b l e s have been s t u d i e d (10, 33, 35, 36, 37). a d d i t i o n c e l l p o p u l a t i o n heterogeneity w i t h respect to both d e n s i t y s i z e c o n t r i b u t e s t o the d i s t r i b u t i o n o f m i g r a t i o n r a t e s ( 1 0 ) .

227 with

In and

The i n s t a n t a n e o u s m i g r a t i o n v e l o c i t y o f a p a r t i c l e (assumed n e g a t i v e l y c h a r g e d ) u n d e r g o i n g upward e l e c t r o p h o r e s i s in a vertical density g r a d i e n t may be d e s c r i b e d by


dx/dt=»

(x) E(X)

-2a g(p-pjx)

) / 9 η (x)

2

10

where, i n t y p i c a l s i t u a t i o n s o f i n t e r e s t , i n e r t i a l and d i f f u s i o n terms are neglected. I n e q u a t i o n (12), χ i s d e f i n e d as v e r t i c a l d i s t a n c e from the s t a r t i n g zone, μ i s the a n o d i c e l e c t r o p h o r e t i c m o b i l i t y , Ε i s the e l e c t r i c f i e l d s t r e n g t h , u s u a l l y d e t e r m i n e d from E(x)=l/(Ak(x))

n

i n w h i c h I i s the c o n s t a n t a p p l i e d c u r r e n t , k i s the c o n d u c t i v i t y o f the g r a d i e n t s o l u t i o n , and A i s the c r o s s - s e c t i o n a l a r e a o f the g r a d i e n t column. The s e c o n d term o f e q u a t i o n (10) r e p r e s e n t s the Navier-Stokes sedimentation v e l o c i t y f o r spheres o f d e n s i t y ρ and r a d i u s a under the i n f l u e n c e o f the g r a v i t a t i o n a l a c c e l e r a t i o n g. The x-dependent v a r i a b l e s a r e e x p l i c i t l y i n d i c a t e d i n e q u a t i o n s (10) and (11). S t a n d a r d e l e c t r o p h o r e t i c m o b i l i t i e s , μ , a r e commonly e x p r e s s e d a t the v i s c o s i t y o f pure water a t 25°C, η . Thus β

Β

μ(χ)=μ,η,/η(χ).

12

< >

Explicit e x p r e s s i o n s have been e s t a b l i s h e d for the x-dependent v a r i a b l e s t o a l l o w i n t e g r a t i o n o f e q u a t i o n (10) t o y i e l d the d i s t a n c e o f m i g r a t i o n up the column as a f u n c t i o n o f time o f a p p l i c a t i o n o f the electric field. These e m p i r i c a l r e l a t i o n s h i p s c o n s i s t o f t r e a t i n g d e n s i t y and m o b i l i t y as l i n e a r f u n c t i o n s o f C ( x ) , the polymer ( o r d e n s i t y g r a d i e n t s o l u t e ) c o n c e n t r a t i o n , c o n d u c t i v i t y as a q u a d r a t i c f u n c t i o n o f C ( x ) , and v i s c o s i t y as an e x p o n e n t i a l function. S u b s t i t u t i o n o f t h e s e f u n c t i o n s i n t o e q u a t i o n (10) g i v e s an e x p l i c i t r e l a t i o n s h i p between v e l o c i t y and m i g r a t i o n d i s t a n c e . The time t ( x ) t o m i g r a t e a d i s t a n c e χ may be f o u n d by n u m e r i c a l i n t e g r a t i o n o r a n a l y t i c a l l y i n terms o f e x p o n e n t i a l i n t e g r a l s ( 3 7 ) :

f

0(x)exp(ax) ^

t(x)=

J

91

0.7

4

SGE

50

100

1.0

5

DGI

600

10000

>6

90

90

50

7

LGC

20

10

90

98.9

9

CFI

48

7+

99+

10

RAE

19

>5.5

11

LGF

197

12

STF

12

13

CBE

20

4-5 5

10

LOW

30-50

HIGH

2.0-2.5

"

HIGH HIGH

1.5-2.0

"

HIGH HIGH

2.0-2.5

MOD-- MOD

2.5-3.0

MED

LOW

4.5-5.5

LOW

ZERO

>300

10

MOD

LOW

20-60

10

LOW

LOW

20-60

7

M

"

?

?

10000 " MOD

55

99

>10

>8

>90

100

V.LOW ZERO >10000

5

MOD

MOD

6-20

50

MOD

LOW

50-100


15.

TODD


249


Table II. Comparison of methods of cell electrophoresis usingfiguresof merit NO. METH. RESOL. VIABIL. PURITY CAPACITY CONVENIENCE

COST

TOTAL

1

FZE

3+

4

4

3

1

2

17+

2

DGE

4

4

4

2

3

4

21

3

AVE

1

3

3

3

4

4

18

4

SGE

3

3

3

3

4

4

20

5

DGI

4

1

4

2

2

4

17

6

RGE

3

4

3

4

3

3

20

7

LGC

4

2

3

4

0

0

13

8

FFE

3

4

2

3

2

2

16

9

CFI

?

1

3

3

2

2

11+

10

RAE

1

1

?

4

2

1

9+

11

LGF

3+

2

4

4

0

0

13+

12

STF

2

4

2

3

2

4

17

13

CBE

3

4

3

3

2

2

17


250

CELL

SEPARATION

SCIENCE AND

TECHNOLOGY

i n s t i t u t i o n a l machine shop f o r $5000-12000 i n c l u d i n g t h e p r i c e o f a power s u p p l y , so t h e c a p i t a l c o s t and c o n v e n i e n c e a r e comparable t o t h o s e o f RGE.


13.

Endless Continuous

B e l t E l e c t r o p h o r e s i s (EBE)

In a process called "Endless Belt Continuous Flow Deviation E l e c t r o p h o r e s i s " K o l i n (97, 98) d e s i g n e d a h o r i z o n t a l a n n u l a r v e s s e l s u r r o u n d i n g a b a r magnet. The h o r i z o n t a l passage o f a c u r r e n t t h r o u g h the b u f f e r i n t h e annulus p e r p e n d i c u l a r t o t h e r a d i a l f i e l d l i n e s o f the magnet c r e a t e s a s t e a d y f l o w o f t h e b u f f e r a r o u n d t h e annulus due to t h e L o r e n t z f o r c e . T h i s f l o w mimics t h e r o t a t i o n o f the h o r i z o n t a l tube i n FZE (see above) t h e r e b y c o u n t e r a c t i n g b o t h s e d i m e n t a t i o n and the b u i l d - u p o f c o n v e c t i v e v o r t i c e s . The a n n u l a r space i s t y p i c a l l y 1.5 mm t h i c k . The sample i s i n t r o d u c e d c o n t i n u o u s l y a t the a p p r o p r i a t e end o f t h e a n n u l u s , and separands m i g r a t e i n h e l i c a l p a t h s t h e p i t c h o f w h i c h i s d i r e c t l y p r o p o r t i o n a l t o μ . A f t e r a n adequate number o f h e l i c a l t u r n s (about 5) a r e a c c o m p l i s h e d , separands a r e removed from the annulus a t t h e o p p o s i t e end through a l i n e a r a r r a y o f i n d i v i d u a l p o r t s , t y p i c a l l y 20, so NSU - 20, and minimum CV - 5%. A field s t r e n g t h up t o 150 V/cm i s p o s s i b l e , so t h a t m i g r a t i o n i s r a p i d . Zone sedimentation i s avoided by the b u f f e r c i r c u l a t i o n , and o v e r a l l c a p a c i t y i s c l a i m e d t o be 5 χ 1 0 c e l l s / h ( 9 8 ) . B u f f e r s o f a r o u n d 0.6 mS/cm c o n d u c t i v i t y a r e used, and v i a b i l i t i e s a r o u n d 85% c a n be expected. Photographs i n d i c a t e p r a c t i c a l l y no o v e r l a p between bands o f separands d u r i n g m i g r a t i o n , so p u r i t y s h o u l d be a r o u n d 99%. β

8

Summary : T a b l e I l i s t s the 13 methods o f c e l l e l e c t r o p h o r e s i s c o n s i d e r e d i n t h i s study and t h e a b s o l u t e v a l u e s o f t h e 8 p a r a m e t e r s c h o s e n f o r e v a l u a t i o n . Table I I l i s t s the corresponding f i g u r e s o f merit f o r the d i f f e r e n t methods. These f i g u r e s were d e r i v e d from an a p p r o x i m a t e l y l i n e a r i n t e r p r e t a t i o n o f the data i n Table I. O v e r a l l , a simple, accessible, inexpensive method, i f i t can provide v i a b l e cell p o p u l a t i o n s i s t h e r e v e r s i b l e g e l method, s i n c e r e l a t e d t e c h n i q u e s a r e p r a c t i c e d i n many l a b o r a t o r i e s d a i l y . On t h e o t h e r hand, f r e e f l o w e l e c t r o p h o r e s i s i s t h e most w i d e l y p r a c t i c e d c o n t i n u o u s method. I t s capacity and advanced stage of development, including c o m m e r c i a l i z a t i o n , rank i t h i g h among p r o c e s s e s t h a t a r e u s e f u l i n l a b o r a t o r i e s t h a t wish to separate c e l l s d a i l y . Acknowledgments : T h i s work was s u p p o r t e d by t h e N a t i o n a l A e r o n a u t i c s and Space A d m i n i s t r a t i o n ( C o n t r a c t s NAS9-15583, NAGW-694, and NAS9-17431), U. S. P u b l i c H e a l t h S e r v i c e ( G r a n t s R01 CA-12589 and N01 CB-43984) and t h e N a t i o n a l I n s t i t u t e o f Standards and T e c h n o l o g y . E x p e r i m e n t a l and t h e o r e t i c a l work by L i n d s a y D. Plank, M. E l a i n e Kunze, Kenneth D. C o l e and R o b i n S t e w a r t and t h e guidance o f F. J . M i c a l e and S c o t t R. Rudge a r e g r a t e f u l l y acknowledged. Senior design students o f the Colorado S c h o o l o f Mines a r e acknowledged f o r t h e i r c o n t r i b u t i o n t o t h e s t u d y of r e - o r i e n t i n g d e n s i t y g r a d i e n t e l e c t r o p h o r e s i s .


15.

TODD

Preparative Cell Electrophoresis: Comparison of Methods 251

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6. 7. 8. 9. 10. 11.

12. 13.

14. 15. 16. 17. 18. 19. 20.

21.

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15.

TODD


46. Troitsky, G. V.; Azhitsky, G. Yu. Isoelectric focussing of proteins in natural and artificial pH gradients. Kiev Nauka Dumka: Kiev, 1984. 47. Boltz, R. C., Jr.; Miller, T. Y.; Todd, P.; Kukulinsky, Ν. E. In Electrophoresis '78: Catsimpoolas, Ν., Ed.; Elsevier/NorthHolland Biomedical Press: Amsterdam, 1978; pp 345-355. 48. Boltz, R. C., Jr.; Todd, P.; Hammerstedt, R. H.; Hymer, W. C.; Thompson, C. J . ; Docherty, J. J. In Cell Separation Methods; Bloemendal, H., Ed.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1977; pp 145-155. 49. Sherbet, G. V. The Biophysical Characterisation of the Cell Surface; Academic Press: London, 1978. 50. McGuire, J. K.; Miller, T. Y.; Tipps, R. W.; Snyder, R. S.; Righetti, P. G. J. Chromatog. 1980, 194, 323-333. 51. Leise, E.; LeSane, F. Prep. Biochem. 1974, 4, 395-410. 52. Sherbet, G. V.; Lakshmi, M. S.; Rao, Κ. V. Exp. Cell Res. 1972, 70, 113-123. 53. Hammerstedt, R. H.; Keith, A.D.; Boltz, R. C., Jr.; Todd, P. Arch. Biochem. Biophys. 1979, 194, 565-580. 54. Zarkower, D.; Plank, L. D.; Kunze, M. E.; Keith, Α.; Todd, P; Hymer, W. C. Cell Biophys. 1984, 6, 53-66. 55. Thompson, C. J . ; Docherty, J. J . ; Boltz, R. C., Jr.; Gaines, R. Α.; Todd, P. J. Gen. Virol. 1978, 39, 449-461. 56. Cole, K. D., Dutta, Β. K. and Todd, P. Non-amphoteric isoelectric focusing III. A borate-polyol density gradient for rapid isoelectric focusing of proteins. In preparation, 1990. 57. Snyder, R. S. Electrophoresis demonstration on Apollo 16. National Aeronautics and Space Administration Report NASA TMX-64724; National Aeronautics and Space Admin., Huntsville, AL, 1972. 58. Snyder, R. S.; Bier, M.; Griffin, R. N.; Johnson, A. J . ; Leidheiser, H.; Micale, F. J . ; Ross, S.; van Oss, C. J. Sep. Purif. Meth. 1973, 2, 258-282. 59. McKannan, A. C.; Krupnick, E. C.; Griffin, R. N.; McCreight, L. R. National Aeronautics and Space Administration Report NASA TMX-64611; National Aeronautics and Space Admin., Washington, DC, 1971. 60. Micale, F. J . ; Vanderhoff, J. W.; Snyder, R. S. Sep. Purif. Meth. 1976, 5, 361-383. 61. Vanderhoff, J. W.; Micale, F. J. In Electrokinetic Separation Methods: Righetti, P. G.; Van Oss C. J . ; Vanderhoff, J. W., Eds.; Elsevier/North-Holland: Amsterdam, 1979; pp 81-93. 62. Vanderhoff, J. W.; van Oss, C. J. In Electrokinetic Separation Methods: Righetti, P. G.; van Oss, C. J . ; Vanderhoff, J. W., Eds.; Elsevier/North-Holland: Amsterdam, 1979; pp 257-274. 63. Allen, R. E.; Rhodes, P. H.; Snyder, R. S.; Barlow, G. H.; Bier, M.; Bigazzi, P. E.; van Oss, C. J . ; Knox, R. J . ; Seaman, G. V. F.; Micale, F. J . ; Vanderhoff, J. F. Sep. Purif. Meth. 1977, 6, 1-59.


253


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SEPARATION

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64. Snyder, R. S.; Rhodes, P. H.; Miller, T. Y.; Micale, F. J.; Mann, R. V.; Seaman, G. V. F. Sep. Sci. Technol. 1986, 21, 157-185. 65. Morrison, D. R.; Lewis, M. L.; In 33rd International Astronautical Federation Congress: 1983, Paper No. 82-152. 66. Sarnoff, Β. E.; Kunze, M. E.; Todd, P. Adv. Astronaut. Sci. 1983, 53, 139-148. 67. Patterson, W. J. Development of polymeric coatings for control of electro-osmotic flow in ASTP MA-011 electrophoresis technology experiment. NASA TMX-73311, National Aeronautics and Space Admin., Huntsville, AL, 1976. 68. Heard, D. H.; Seaman, G. V. F. Biochim. Biophys. Acta 1961, 53, 366-372. 69. Barlow, G. H.; Lazer, S. L.; Rueter, Α.; Allen, R. In Bioprocessing in Space: NASA TM X-58191; Morrison, D. R., Ed.; L. B. Johnson Space Center: Houston, TX, January 1977; pp 125-132. 70. McGuire, J. K.; Snyder, R. S. In Electrophoresis '81, Allen, R. C.; Arnaud, P., Eds.; Walter deGruyter & Co.: Berlin, 1981; pp 947-960. 71. Hannig, K. In Modern Separation Methods of Macromolecules and Particles; Gerritsen, T., Ed.; Wiley Interscience: New York, NY, 1969; pp 45-69. 72. Saville, D. A. Physicochem. Hydrodyn. 1977 2, 893. 73. Biscans, B.; Alinat, P.; Bertrand, J.; Sanchez, V. Electrophoresis 1988, 9, 84-89. 74. McCreight, L. R. In Bioprocessing in Space: Morrison, D. R., Ed.; NASA TMX-58191, Lyndon B. Johnson Space Center: Houston, TX, January 1977, pp 143-158. 75. Strickler, Α.; Sacks, T. Prep. Biochem. 1973, 3, 269-277. 76. Snyder, R. S.; Rhodes, P. H. In Frontiers in Bioprocessing; Sikdar, S. K.; Bier, M.; Todd, P., Eds.; CRC Press: Boca Raton, FL, 1989; pp 245-258. 77. Miller, T. Y.; Williams, G. P.; Snyder, R. S. Electrophoresis 1985, 6, 377. 78. Rhodes, P. H.; Snyder, R. S.; Roberts, G. O. J. Colloid Interface Sci. 1989, 129, 78. 79. Taylor, G. I. Proc. Roy. Soc. 1966, A291, 159-167. 80. Rhodes, P. H. In Electrophoresis '81; Allen, R. C.; Arnaud, P., Eds.; W. deGruyter & Co.: Berlin, 1981; pp 919-932. 81. Vanderhoff, J. W.; Micale, F. J.; Krumrine, P. H. In Electrokinetic Separation Methods; Righetti, P.G.; van Oss, C. J.; Vanderhoff, J. W., Eds.; Elsevier/North-Holland: Amsterdam, 1979; pp 121-141. 82. Ostrach, S. J. Chromatog. 1977, 140, 187. 83. Deiber, J. Α.; D. A. Saville. In Materials Processing in the Reduced Gravity Environment of Space; Rindone, G. Ε., Ed.; North-Holland: New York, NY, 1982; pp 217-224. 84. Rhodes, P. H.; Snyder, R. S. In Materials Processing in the Reduced Gravity Environment of Space; Rindone, G. Ε., Ed.; North-Holland, New York, NY, 1982; pp 217-224. 85. Bauer, J.; Hannig, K. In Electrophoresis '86: Dunn, M. J., Ed.; VCH Verlagsgesellschaft: Berlin, 1986; pp 13-24.



15.

TODD


86. Just, W. W.; Werner, G. In Cell Separation Methods; Bloemendal, H., Ed.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1977; pp 131-142. 87. Just, W. W.; Werner, G. In Electrokinetic Separation Methods; Righetti, P. G.; Van Oss, C. J.; Vanderhoff, J. W., Eds.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1979; pp 143-167. 88. Haydon, D. Α.; Seaman, G. V. F. Arch. Biochem. Biophys. 1967, 122, 126. 89. Levesque, M. J.; Nerem, R. M. ASME J. Biomech. Engin. 1985, 176, 341-347. Stathopoulos, Ν. Α.; Heliums, J. D. Biotechnol. Bioengin. 1985, 27, 1021-1026. 90. Hannig, K.; Wirth, H. Prog. Astronaut. Aeronaut. 1977, 52, 411-422. 91. Hannig K.; Bauer, J. Adv. Space Res. 1989, 9 (11), 91-96. 92. Mel, H. C. J. Theoret. Biol. 1964, 6, 159-180. 93. Tippetts, R. D.; Mel, H. C.; Nichols, Α. V. In Chemical Engineering in Medicine and Biology. Plenum Press, NY, 1967; pp 505-539. 94. Paulus, J. M.; Mel, H. C. Exper. Cell Res. 1967, 48, 27-38. 95. Mel, H. C. Chem. Engin. Science 1964, 19, 847-851. 96. Mel, H. C. In Myeloproliferative Disorders of Animals and Man: Clark, W. J.; Howard, Ε. B.; Hackett P.L., Eds.; U.S. Atomic Energy Commission: Washington, DC, 1970; pp 665-686. 97. Kolin, A. J. Chromatogr. 1967, 26, 164-183. 98. Kolin, A. In Electrokinetic Separation Methods: Righetti, P. G; Van Oss, C. J.; Vanderhoff, J., Eds.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1979; pp 169-220. RECEIVED March 15, 1991


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Comparison of Methods of Preparative Cell Electrophoresis - ACS

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