Chapter 12 Continuous Flow Deviation Electrophoresis*

Chapter 12

Continuous flow deviation electrophoresis*

.

A KOLIN

CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theoretical aspects of continuous flow deviation electrophoresis . . . . . . . . . . . . . . . . . . . Velocity distribution in fluid band . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distortion of the streak cross-section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resolving power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electroosmotic streaming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abolition of streak-profile distortion by electroosmosis . . . . . . . . . . . . . . . . . . . . . . Modes of implementation of continuous flow deviation electrophoresis . . . . . . . . . . . . . . . Flat fluid band electrophoresis (“free-flow” electrophoresis) . . . . . . . . . . . . . . . . . . . . . The preparative instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modification of the fluid curtain apparatus for analytical work . . . . . . . . . . . . . . . . . . Endless fluid belt electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Objectives of the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The circular endless belt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Principle of suppression of thermal convection . . . . . . . . . . . . . . . . . . . . . . . . . Achievement of electromagnetic circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . A simple apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of magnetohydrodynamic driving force . . . . . . . . . . . . . . . . . . . . . . . . . Some illustrations of instrument performance . . . . . . . . . . . . . . . . . . . . . . . . . . Some unique properties of the endless belt system . . . . . . . . . . . . . . . . . . . . . . . Modes of operation of the endless fluid belt system . . . . . . . . . . . . . . . . . . . . . . The noncircular endless belt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transition to the vertical “racetrack” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The separation space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Serpentine fluid belt electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Examples of preparative separations by continuous flow deviation electrophoresis . . . . . . . Measurement of electrophoretic mobility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Symbols and units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

253 255 255 257 258 259 260 261 261 261 264 267 267 267 267 269 269 270 272 273 275 276 276 277 278 279 282 291 295 295 296 296

INTRODUCTION

In this chapter the frequently used term “free-flow’’ electrophoresis will not be used. as it is inappropriate . An example of fluid motion to which the term “free flow” could be

* The author of this chapter preferred not to use the recommended SI units.For the convenience of readers. a table of symbols may be found on p . 296 .

254

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

applied is the efflux of water from a tap. It flows without solid confines and in this sense it is “free flow”. In the electrophoretic methods that will be considered here, a continuous fluid flow is confined between walls in channels of different shapes and we shall classify the methods according to the shape of the confining channel. If the term “free flow” was meant to indicate an absence of solid obstacles that would retard the flow by viscous drag, it would be equally untenable because the walls of the channel exert such a retarding force which results in the laminar flow profile. The subject of this chapter is electrophoresis in electrically conductive fluid ribbons flowing between two solid walls, which may be flat or curved. An electric field is maintained at right angles to the direction of flow. The charged particles to be subjected to electrophoresis are injected into the fluid stream as a fine streak. Owing to superposition of the transverse electrophoretic velocity upon the fluid flow velocity, the direction of the streak is deviated by an angle ad, which depends on the electrophoretic mobility of the particle, the electric field intensity and the velocity of fluid flow. Fig. 12.1a1 shows the simplest configuration of this type. The fluid flows downwards with a velocity u between the plates P, and Pz (at first we shall make the unrealistic assumption of a uniform velocity distribution). E is the transverse electric field and is the angular deviation of the electrophoretically slower negative particles from the vertical dotted line, which is the path of electrically neutral particles; a2 is the angle of deviation of an electrophoretically faster component in the particle mixture emerging from the injector, IN. This figure illustrates the essence of deviation electrophoresis, which is the subject of this chapter.

IN

U

”f

2

a2

b

C

Fig. 12.1. Fluid paths used in deviation electrophoresis. Perspective view of the flatcurtain flow. P I , P,: plates between which the fluid flow is sandwiched. MI,M,: membranes forming the lateral confines of the curtain flow. IH: influx h+oIes for buffer. EH: exit holes for buffer. IN: injection capillary for sample. 2: fluid velocity vector. E: electric field vector. a,,a*:angles of deviation of two electrophoretic components. (a,) Lateral view of curtain. Symbols as in a,. (b) Serpentine flow path. S,, S,: solid serpentine surfaces between which the serpentine flow is sandwiched. M I: lateral membrane (m, on the opposite side of walls S,, S, is not shown). 2: fluid velocity vector. (c) Endless fluid belt. S , , S,: solid surfaces confiing the fluid belt. 3 fluid velocity vector. (There are no lateral membranes confining the flow.)

THEORETICAL ASPECTS

255

Fig. 12.1a2 is a side view of the configuration shown in Fig. 12.1al. The electric field vector is to be imagined as pointing away from the reader at right angles to the page. This is the scheme of what is referred to in the literature as “free-flow electrophoresis” [2]. Another configuration that has been used [3] employs a serpentine ribbon of fluid between suitably shaped solid confmes, as shown in Fig. 12.lb. Finally, the fluid band may have the shape of an endless belt of an arbitrary shape. Fig. 1 2 . 1 shows ~ a shape [4] that is used in a more recent version of the endless belt electrophoresis system [5]. We can thus see that the continuous fluid flow may be unidirectional, tortuous or cyclic. In one of the figures illustrating such motions (Fig. 12.1b and 1 2 . 1 ~ the ) electric field is perpendicular to the page and the sample to be subjected to electrophoretic analysis is injected in the direction of the velocity ti or vf. The resolving power of this electrophoretic scheme for the separation of molecular mixtures is considerably lower than that of methods in which chromatographic and/or molecular sieving effects are combined with electrophoresis. However, the absence of a porous matrix makes continuous flow deviation electrophoresis ideally suitable for the separation of mixtures of particles, such as cells and cell organelles. In molecular separations, the disadvantage of a lower resolving power is compensated for by the advantageous preparative capabilities of continuous collection of separated fractions.

THEORETICAL ASPECTS OF CONTINUOUS FLOW DEVIATION ELECTROPHORESIS The following theory is common to all modes of continuous flow deviation electrophoresis, namely to flat curtain (“free-flow”), serpentine flow and endless belt electrophoresis. It was developed in connection with endless belt electrophoresis [6,7], with reference t o its applicability to fluid curtain electrophoresis, to which it was eventually applied [2,8]. In fact, the treatment of curved fluid paths uses the approximation of a fluid band width of negligible thickness compared with the radius of curvature and treats the fluid ribbon as being flat. Velocity distribution in fluid band We shall now consider, as a prototype of fluid motion in a deviation electrophoresis apparatus, the flow between two parallel plates. Fig. 12.2 illustrates fluid circulation in an endless fluid belt around a noncircular core C. This fluid belt has two flat sections adjacent to the front and back vertical surfaces of the core C. As a result of viscous interaction with the walls, the velocity profile in the fluid curtain is given by [9] (1 2.1 a) (1 2.1 b)

Eqn. 12.la follows from two integrations of the differential equation [lo]

256

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Fig. 12.2. Perspective view of the endless fluid %It (partial view, showing the front portion of the fluid belt) [5]. C: Inner core; M: outer mantle. u : fluid velocity; vo: fluid velocity a t centre of fluid belt. X, Y,Z coordinate axes centred at mid-point o:fluid belt. Z: distance o t a point from centre of belt. f,: force density of force propelling the fluid;& magnetic field vector; J : current density vector. h : thickness of endless belt; v: lateral flow velocity vector; F, R: front and rear surfaces confining the fluid belt, respectively; L , : section on level 1; L,: section on level 2. C,: original circular cross-section of injected sample streak;C,, C,: shapes of distorted sample streak downstream of C , . D: distance between levels C, and C,. a: angle of deviation of electrophoretic component. 1-1: points along line A-B, I*?*: final locations of points originating from line A B . A*B*: final position of points on line A B in ideal case.

(12.2) and consideration of the velocity u vanishing at the walls;f, is the vertical force per unit volume which maintains the flow of the fluid belt. Eqn. 12.lb is the familiar velocity distribution of laminar flow. It is immediately clear that only the centrally located particles injected from a well centred injector IN in Fig. 12.la

THEORETICAL ASPECTS

257

will move downwards with the maximal curtain velocity uo. The particles closer to the walls of the curtain will move more slowly. This would have an obvious effect upon the deviation of a charged particle in the transverse electric field E . It is also clear that the injector must be centred exactly in the fluid curtain and must be parallel to the flow. In the worst case, the injector would be perpendicular to the walls of the curtain confining the fluid band injecting its output in the direction of one of the walls. This would increase the excentric spread of the particles, making their velocity profile more inhomogeneous. Because of the flatness of the velocity distribution curve near the centre (Fig. 12.2), a good approximation to a uniform vertical particle velocity within the streak emanating from a well aligned injector can be achieved by using an injector with a small diameter compared with the thickness, h , of the fluid band (see also subsequent discussion of resolving power). Distortion of the streak cross-section Fig. 12.2 shows two perpendicular sections, L1 and L2, through the fluid band. Let us imagine that a species of negatively charged electromigrating particles is introduced on the upper level L1,not through a tubular injector, but rather through a fine slit AB extending almost from wall to wall (we avoid the immediate vicinity of the wall where the downward flow velocity u is zero); 1, m, n, 0 , p, q and r are representative particles (o being centrally located) at different distances z from the centre of the fluid band. The electrophoretic velocity of the particles, u, = - pE,is the same for all values of z . If the same were true of the vertical particle velocity, u , within the fluid band, the above row of particles would maintain its linear configuration on its way down while moving to the left due to electrophoresis. It would eventually arrive at the downstream level L in a linear arrangement between points A* and B* .We could imagine a collection system, COL, consisting of fine exit slits parallel to A*B* intercepting the arriving particles. The particle that we are considering would thus exit through slit A*B* . A particle species moving more rapidly in the same direction would have entered a slit to the left of A* B* . Let us now remember that the downward velocity, u , is not uniform but rather is represented by the parabolic velocity profile shown in Fig. 12.2. As a result, the centrally located particle o would move downwards faster than all of its neighbours and would thus require the shortest time to reach the level L2. Its displacement to the left will therefore be smaller than for other particles located on the line AB owing to the shorter electromigration time. The closer a particle is located to one of the walls confining the fluid flow, the slower will be the downward velocity u and the longer will the particle migrate t o the left on its way to the level L.As a result, particles 1, m, n, 0,p, q and r will no longer lie on a straight line by the time they have reached the level b.Their arrangement will have been distorted as indicated by the points 1*, m*, n*, o*, p*, q* and r* on level L2. These particles will no longer pass through the single linear slit A*B* as they did for a uniform distribution of the downward velocity u , but will now invade many neighbouring escape slits. The effect of this distortion of the slit profile on the resolving power in preparative separations is obvious. In practice, the injector is not shaped like a slit, but is rather a thin cylindrical tube. The larger is the desired throughput in preparative work, the larger will be the diameter

258

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

of the injected particle streak. Such a circular initial particle distribution is depicted as circle, C1, on level L1. We can imagine the particles within C1 to be arranged in lines parallel t o AB. These linear particle arrays will suffer the same distortion as was illustrated for the particle row AB. Thus, the initially circular cross-section C1 of the particle streak will have been distorted into the shape C1, vaguely resembling a crescent by the time the particles have reached the level L,. Particles issuing from position C1 with a higher electrophoretic mobility will form a “crescent”, C , , to the left of C2. If the collector at level L, consists of a row of adjacent circular openings, the particle streak of originally circular cross-section could no longer pass through one entrance circle. The “horns” may invade adjacent collector openings, thus decreasing the preparative resolving power. The simplest means of reducing this streak-broadening effect is to reduce the diameter of the circle C1 [6]. The particles will then lie close to the apex of the parabola of the velocity profile where the velocity varies relatively little with the distance z from the velocity maximum vo. One thus has to make a compromise between high resolution and a high throughput in preparative work, sacrificing high resolution in order to increase the yield. The above effect of impairment of resolving power is due to a mismatch between the parabolic velocity distribution in the flowing fluid belt and the uniform velocity distribution in the transverse particle motion due to electrophoresis. We have considered in a previous example the case where both the vertical and the horizontal velocity distributions were uniform. There was no distortion of the streak profile. Similarly, it is easy to show [7] that there would be no streak profile distortion if the vertical and horizontal velocity distributions were both parabolic. In general, both of these velocity distributions may be arbitrary. There will be no streak distortion if the vertical and horizontal velocity distributions are described by the same function [5, 71. Resolving power When, in Fig. 12.2 at the level b,we view the separated streak originating from C1 in the direction of the Z-axis, the “horns” of the crescent Cz may obstruct from vision the apex of the “crescent” C, . The electrophoretic fractions, although distinctly separated, would appear in a side view to form a single broad streak. We shall consider two electrophoretic components as resolved when the angle of deviation of the apex of the “crescent” (trailing edge) of the faster component ( C , in Fig. 12.2) is larger than the angle of deviation of the “horns” (leading edges) of the slower component (C, in Fig. 12.2). On the basis of the above criterion of resolution [7] and a definition of resolving power, R , as the ratio between the mean mobility, p, of two electrophoretic components and the difference, A p , between their mobilities [7]:

we can arrive at an expression for the resolving power in terms of the instrumental parameters d and h of the continuous flow electrophoresis apparatus [7]:

259

THEORETICAL ASPECTS

(12.4)

This expression shows that very high resolution can be obtained by making the streak diameter, d , very small in comparison with the thickness, h , of the fluid curtain. For instance, in order to resolve two components that differ by 1% in their mobilities ( A p = 10-2p)rwe could choose an injector of inner diameter 0.15 mm with a fluid curtain 1.5 mm thick. Electroosmotic streaming In all of the systems of implementation of continuous flow deviation electrophoresis, there is an unintended complex fluid motion, i.e., the electroosmotic flow, which has usually been considered as a source of a serious disturbance that would impair resolution. However, it was shown in 1960 [ 6 ] that one can actually take advantage of this effect in order to improve the electrophoretic resolving power by abolishing the streak broadening (“crescent” effect) caused by mismatching of the electrophoretic and hydrodynamic velocity profiles. As a result of the ionic double layer at the walls confining the fluid ribbon, electric forces set in motion the electrolyte adjacent to the cell walls. A flow in the opposite direction results in the central region between the fluid “curtain” walls to ensure zero net fluid transfer in a closed system. Such electroosmotic streaming between two parallel plates PI,P2 is shown in Fig. 12.3. The dashed lines depict the electroosmotic convection and the solid line shows the resulting parabolic velocity distribution [5]. This flow is unlike the parabolic flow maintained at right angles to the electric field

I

0

Fig. 12.3. Electroosmo_tic convection: infinitely extended parallel plates separated by distance h which confine the fluid 151. J electric current density. Z,, 2,: Srnoluchowski zones of zero velocity. The closed dashed lines depict qualitat$ely electroosmotic convection. The electroosmotic velocity distribution is shown by the parabola. v,: central velocity vector. v,: velocity at the walls (v, = - 2 ~ ~ ) .

260

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

(velocity ;in Fig. 12.2), where the velocity vanishes at the wall. The velocity is actually highest at the walls, v,, and is opposite to the central velocity, vo. Smoluchowski [l 11 showed that there are two parallel zones of zero velocity located at a distance of 0.21h from the walls of the cell. These zones are labeled Z, and Z2 in Fig. 12.3. The velocity distribution for the electroosmotic flow follows from considerations [121 similar to those which led to eqn. 12.1. The horizontal force density,fy ,which is responsible for the electroosmotic streaming is given by t

62V

fY

=7)s

(12.5)

Its integration yields V =

"[

:I'

- z+27)

[ I;]

+Az+-

+B

(12.6)

The condition that the velocity becomes % at the cell walls yields the integration constants A and B. We thus obtain the velocity distribution for the electroosmotic flow [6, 121: (12.7) Abolition of streak-profile distortion by electroosmosis The horizontal velocity, v", of a charged particle in the electrophoresis apparatus will be a resultant of its electrophoretic velocity, v, = - $3, and the electroosmotic streaming velocity, given by eqn. 12.7: (12.8) We have introduced in this equation the symbol W for the electroosmotic mobility, i.e., for the electroosmotic velocity at the wall at unit electric field strength of E = 1 V/cm, and thus the electroosmotic velocity is v, = EW. The slope of the path of a particle will be given by the ratio of its horizontal velocity, v * , to its vertical velocity, u . We thus obtain from eqn. 12.la for u and eqn. 12.8 for v* (12.9a)

Thus, (1 2.9b)

This is the essence of an equation derived in 1960 [ 6 ] ,which showed that the angle a becomes independent of the coordinate z (i.e., from the distance of the particle from the wall) when the second term in eqn. 1 2 9 b vanishes. It was pointed out that this would happen only for one set of particles whose electrophoretic velocity was equal to the

MODES OF IMPLEMENTATION

26 1

electroosmotic wall velocity. For a streak of such particles, no matter how thick (it could be as thick as the fluid curtain), all particle trajectories would be parallel and the crosssection of the streak would remain constant without broadening. This would be most favourable for high-yield preparative electrophoresis. The disadvantage is that it is not simple to modify the wall zeta potential in order to make the second term in eqn. 12.9b vanish. A clever method for modification of the wall zeta potential in a flat continuous flow electrophoresis cell was suggested in 1973 by Strickler and Sacks [8]. However, this method, even when maximally successful, could achieve optimal collection for only one electrophoretic component of a given mixture.

MODES OF IMPLEMENTATION OF CONTINUOUS FLOW DEVIATION ELECTROPHORESIS FLAT FLUID BAND ELECTROPHORESIS (“FREE-FLOW’ ELECTROPHORESIS) The origins of flat fluid band continuous flow electrophoretic separators go back to prototypes of deviation electrophoresis which utilized a fluid flow through a porous medium such as a bed of sand or glass beads [13] or a filter-paper curtain [14]. The porous matrix was effective in suppressing thermal convection but proved troublesome in separations of particulate component mixtures, such as cells. This led to the development of fluid curtain electrophoresis apparatus in whch the porous matrix was omitted [ 15, 161. These early instruments consisted of parallel dielectric plates between which was sandwiched a thin layer (typically 0.5 mm in thickness) of a flowing buffer solution. The plates had a slight inclination against the horizontal plane which helped to suppress thermal convection. However, sedimentation of particles and of the injected streak of molecular solutes towards the lower plate led to a modification in which the plates were mounted vertically [ 171. An elaborate thermoelectric cooling system with Peltier elements was used to minimize thermal convection. A substantially vertical fluid path was later also adapted to the endless belt electrophoresis system [4] for the same reasons.

The preparative instrument Hannig’s first preparative vertical plate instrument [ 171 was designed as follows (Fig. 12.4). Fig. 12.4a shows the front view and Fig. 12.4b a section along the axis A-B indicated in Fig. 12.4a; 3 and 4 are glass plates of unequal thickness between which the fluid curtain is sandwiched (see legend for dimensions and other specifications). The buffer flow is delivered at a rate of 0.2-50 ml/h through six openings (8) from a peristaltic pump which acts upon six silicone rubber tubes. The buffer of the fluid curtain leaves at the bottom through 50 silicone rubber tubes (9) (0.5 mm I.D.) which are driven by a 50channel peristaltic pump. The output of these exits tubes is channelled to a fraction collector. The rate of buffer delivery through the channels (8) must, of course, be equal to the rate of buffer drainage through the channels (9). The achievement of this critical

262

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

A

2

9

a)

1

B

Fig. 12.4. Scheme of fluid curtain apparatus [ 171. (a) Front view; (b) section along A-B. 1 : Copper plate (48 X 48 cm); 2: Peltier thermoelectric cooling elements (Philips valve; Type PT 20/20); 3: glass plate (50 X 50 cm, 3 mm thick); 4: glass plate (50 X 50 cm 6 m m thick); 5: spacers (0.5 mm thick); 6: insulating mantle; 7: electrode chambers; 8: buffer inlets; 9: collector tubes; 10: water level tube; 1 1 : resistance thermometer; P and 12: sample inlets; 1 3 and 14: inlet and outlet for cooling water; 15: ion-exchange membrane.

adjustment for equality of buffer in- and out-flow through the tubes 8 and 9 which are operated b y two different peristaltic pumps [17] is achieved as follows. A capillary tube (1 0) shaped as shown in Fig. 12.4 communicates with the fluid curtain and is filled with buffer up to a point in its lowest horizontal section. When the rate of buffer delivery exceeds the rate of buffer drainage, the meniscus in the horizontal section of tube 10 moves to the right;in the converse case it moves to the left. One of the two peristaltic pumps can now be adjusted so that the meniscus in 10 does not move, which indicates equality of in- and out-flow of buffer. The location of the fluid curtain is indicated in Fig. 12.4b by arrows (top and bottom) which point in the direction of flow and indicate where the buffer enters the space between the glass plates (3 and 4) and where it leaves this separation chamber. Spacers (5) (0.5 mm thick) ensure parallelism of the glass plates and fix their distance apart. The sample mixture t o be separated is injected into the fluid curtain through channels (12), of which the channel P is shown in use. As the sample enters at right angles to the

FLAT FLUID BAND ELECTROPHORESIS

263

plane of the curtain, there is the following problem. Optimal pIacement of the sample streak would have been at the centre of the curtain at the maximum of the parabolic velocity profile. Even if the injector tube (IN) were to terminate in the mid-plane of the curtain, the momentum of the injected fluid would tend to carry it towards the glass plate facing it, i.e., away from the optimal mid-plane. It is thus clear that the rate of sample injection could affect the centering of the sample streak within the curtain. This problem can be avoided by orienting the injector parallel to the curtain flow [4]. The cooling is accomplished by 28 thermo-electric Peltier elements ( 2 ) ,the cool junctions of which face the copper plate (1) and whose hot junctions are cooled by water entering at 13 and leaving at 14. The copper plate serves to achieve a more uniform temperature distribution along the glass plate (3) that is being cooled by 28 discrete cooling elements spaced along the 50 x 50 cm surface area of the curtain surface. Uniform thermal contact between the copper plate (1) and glass plate (3) is secured by a thin layer of silicone oil. As only the rear glass plate is cooled, the temperature distribution normal to the glass plates cannot be symmetrical. There is also a vertical temperature gradient because the cool buffer entering the curtain at the t o p is heated by the electrophoretic current as it moves toward the collection tubes (9), which it reaches at a higher temperature. An electrical resistance thermometer permits measurement of the curtain temperature at location 1 1. Laterally adjacent to the 0.5 mm thick fluid curtain are the two electrode chambers(7), which contain the positive and negative electrodes shown in Fig. 12.4a. These chambers are perfused by a buffer with an electrical conductivity higher than that of the intermediate buffer (3-fold or higher) in order to reduce heating in the electrode chambersaswellas losses in available voltage, The ionic strength of the electrode compartment buffer is about 0.03-0.05 and the electrical conductivity is approximately 0.1-0.25 (ohm-cm)-’ [ 171. The electrode compartments (7) are separated from the fluid curtain by ionexchange membranes (1 5), which seal them hydraulically from the fluid in the curtain while permitting an electrical current t o flow between them. An anionexchange membrane is used on the cathode side and a cation-exchange membrane on the anode side. These membranes have the advantage of a low electrical resistance (and hence lower heating effects) compared with neutral membranes, such as acetylcellulose. The asymmetry in the properties of the cathodic and anodic ionexchange membranes results in changes in electrolyte concentration within the separation curtain with an enrichment of anion concentration near one membrane and cation concentration near the other [ 171. The resulting gradients in pH and electrical conductivity are illustrated in Fig. 12.5. It can be seen that the uniform range o f these parameters extends only about from fraction No. 8 to fraction No. 40, i.e., over approximately two thirds of the separation space. The data plotted in Fig. 12.5 were obtained with a typical buffer solution traversing the curtain and exiting from the terminals (9) in Fig. 12.4a. The plotted values of pH and conductivity are those of the collected fractions in the numbered collection tubes. These membrane-induced shifts in pH and electrical conductivity of the buffer in the separation space are one of the main distinguishing essential features between fluid curtain (“free-flow”) and endless belt electrophoresis. The former system requires the membranes because the curtain flow must be maintained across a rectangular channel by means of a pressure gradient, which could not be generated without the channel’s side

264

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Fig. 12.5. Conductivity and pH distribution in the collected fractions after electrophoresis in the fluid curtain apparatus [ 171. Acetate buffer, pH 4.8; conductivity K = 2.6 10-3(ohm * ern)-'. Solid line, pH distribution, and dashed line, conductivity distribution in collected fractions.

-

walls (membranes). To transmit the transverse electrical current, these side walls must be electrically conductive. The mechanism of fluid propulsion in endless belt electrophoresis, on the other hand, requires no pressure gradient and is purely magneto-hydrodynamic. A force is exerted upon each fluid element of the endless fluid belt (which corresponds t o the buffer curtain of the former system) by the interaction of the electrophoretic current with a transverse magnetic field. As a result, the lateral boundaries of the endless fluid belt need not be closed and the membranes can be (and actually are) omitted altogether [18, 191. This omission results in elimination of above-mentioned temporal and spatial shifts in pH and electrical conductivity within the separation space. Fig. 12.6 shows a partial view of the instrument. The dimensions are apparent from comparison with the separation curtain whose glass plates (1) are about 50 x 50cm. On the right-hand side of the fraction collector (6) is the electrophoresis d.c. power supply (21, and on the left the d.c. power supply for the Peltier elements. The buffer supply vessels (4)are located in the cooling chamber ( 5 ) and the mixture to be analysed is located at 7 and delivered by a pump (8). This instrument is particularly suitable for preparative cell separations.

Modification of the fluid curtain apparatus for analytical work Recently, the preparative instrument described above has been modified [20] so as to incorporate some of the performance features of the endless belt apparatus [ 4 , 7 , 181. The length of the curtain is approximately equal to the circumference of the current endless belt apparatus, the cooling of the curtain is no longer accomplished by Peltier elements but rather, as in the endless belt apparatus, by cooling water circulating through cooling jackets adjacent to the curtain, The streaks can be seen and photographed or photoelectrically recorded through quartz windows and the exiting buffer can be fed back so as t o reenter the curtain at the t o p to achieve a buffer circulation as in the endless belt apparatus.

FLAT FLUID BAND ELECTROPHORESIS

265

Fig. 12.6. View of the main part of the vertical fluid curtain apparatus [ 171. 1 : Separation chamber; 2: d.c. power supply; 3: d.c. power supply for Peltier cooling elements;4: buffer supply vessels; 5: cooling chambers;6 : fraction collector; 7 : sample reservoir; 8: pump for sample supply.

We shall describe this instrument with comparative references to its flat-curtain predecessor and to the endless belt apparatus. Fig. 12.7 shows an exploded view of the separation chamber. It consists of two frames (1 and 2) that can be clasped and locked together, the fluid curtain being sandwiched in between them. Glass plates (3) form the walls of the fluid curtain. Quartz windows (5) permit the transillumination of the buffer curtain with W light for streak detection. A wavelength of 225 nm (range of the peptide absorption band) is used for detection of protein streaks. Cooling jackets (4) are provided inside the frames (1 and 2), and are supplied with cooling fluid through nipples (1 5 ) . Platinum electrodes (7) are located in electrode chambers (6) through which the electrode buffer solution is circulated via nipples (1 6). Ionexchange membranes (8) are placed on the sides of the curtain with their planes parallel to it and are sealed against leakage by sealing strips (9 and 10). A spacing step (1 1) fixes the distance between the chamber walls (3), i.e., the thickness of the buffer curtain. The buffer solution enters the curtain at 12 and exits at 15. Thus, unlike the previous preparative instrument, this one

266

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

@j

i

c 1

I

4 , i -

11

Fig. 12.7. Exploded view of analytical fluid curtain separation chamber 1201. I , 2: Frames; 3: chamber walls; 4: cooling jacket; 5: quartz window; 6: electrode chambers; 7 : Pt electrodes: 8: ionexchange membranes; 9, 10: sealing gaskets; 1 1 : spacing step; 12, 13: inlet and outlet of curtain buffer; 14: sample inlet; 15: inlets for cooling fluid; 16: inlet for electrode chamber buffer,

uses an upward flow of curtain buffer. The sample to be subjected to electrophoretic analysis through an inlet (14). The width of the windows, which limits the electrophoretic migration distance, is 3 cm, compared with 50 cm in the preparative curtain and 6 cm in the current endless belt apparatus. The height of the buffer curtain which determines the residence time of the sample in the curtain at a given rate of buffer flow, is 18 cm, compared with 50 cm in the preparative instrument and about 20 cm circumference of the endless belt instrument. Actually, I f turns is the minimal path length in the endless belt, which corresponds to a migration path of about 30 cm. Like the preparative instrument, this analytical device uses a much narrower thickness of the buffer curtain (0.35 mm) as compared to the endless belt (1.5 mm). Although this has the advantage of better cooling and greater stabilization against thermal convection, this narrowness of buffer gap has the disadvantage of a steeper velocity gradient in the flowing curtain for a given maximal buffer velocity in the buffer curtain. The result is a greater tendency for streak broadening (“crescent” effect) to occur, which impairs the sharpness of visual resolution. To counter this, exceedingly thin (0.05 mm) well centred streaks of sample must be used. The voltage of 140V/cm applied

ENDLESS FLUID BELT ELECTROPHORESIS

267

across the narrow width of the cell (3 cm) accounts for the rapid separations that can be achieved with this instrument (see Fig. 12.23). The volume of sample injected may be as small as 0.1-0.3 p1 (comparable with 0.1 pl reported for the endless belt apparatus [7]). ENDLESS FLUID BELT ELECTROPHORESIS Objectives of the system The objective of this method [4-7, 211 is to utilize a combination of electric, magnetic and gravitational fields to achieve a stable system of deviation electrophoresis. The constancy of the gravitational field can be matched, for all practical purposes, by the constancy of the field of a permanent magnet. The constancy of the electric field, however, requires a current- or voltage-stabilized power supply like any other electrophoretic method. The gravitational and magnetic fields serve to propel the buffer solution without any discontinuities in operation such as those which can be minimized, but not entirely eliminated, in a peristaltic pump. While maintenance of the gravitational and magnetic field within a buffer solution produce no changes in it, the electric field can only be maintained with a concomitant electric current which heats the solution. This heat escapes by conduction via the solid boundaries of the flowing ribbon of buffer. As a result, the buffer temperature will be lowest at these boundaries and highest at the centre of the buffer curtain. The result will be thermal convection [21], such as the circulation pattern shown in Fig. 12.8. It is obvious that such convection could re-mix or at least blurr a streak separation pattern. Hence its effective suppression is the main objective in any similar separation method. Endless fluid belt electrophoresis achieves the suppression of thermal convection vortices by fluid circulation in a vertical plane about a horizontal axis as described below [6,21]. The circular endless belt Principle of suppression of thermal convection Fig. 12.9 illustrates the principle on which we shall base suppression of thermal convection in the buffer curtain. Fig. 12.9a shows a section through two concentric horizontal circular cylinders. Let us assume that the annular space between them is filled with a liquid and that we maintain the inner cylinder at a higher temperature than the outer cylinder. The vector g gives the direction of the gravitational field. As a result of the temperature gradient between the two cylinders, vortex formation will occur, such as the solid vortex shown on the right-hand side of the annulus. The specifically lighter warm fluid rises near the warm inner cylinder and the specifically heavier fluid, which is cooled by the outer cylinder, descends so that a clockwise circulation of fluid results. Let us now assume that we can manage to rotate the fluid in the annulus in the direction of the upper curved arrow so as to place this vortex in the diametrically opposed location indicated by the dashed circulation path. Owing to inertia, the fluid near the

268

‘i

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

a

a

b

b C

Fig. 12.8. Thermal convection pattern in a rectangular channel cooled at the ve$cal walls W, and w b (51. The temperature is highest at the centre (To) and lowest at the walls (TWJg: gravitationalfield vector.

Fig. 12.9. Thermal convection patterns in channels of d!ferent shapes extending perpendicular to the page [S]. The inner wall is warmer than the outer wall. g: gtavitational field vector. Solid closed line: original thermal convection vortex;dotted closed l i e : inverted vortex after relocation by circulation of the fluid in the channel. (a) Circular annular channel; (b) non-circular quasi-annular channel; (c) endless fluid belt “annulus”.

inner cylinder will move downwards and the fluid near the outer cylinder upwards, as indicated by the arrows. These directions of motion are, however, contrary to the directions of the forces now acting upon the fluid elements. The descending fluid near the inner cylinder, being warmer and specifically lighter than the outer fluid masses, will suffer a net upward force, whereas the ascending cooler, and thus specifically heavier, fluid near the outer cylinder will experience a net downward force. Thus, transplantation of the vortex in this new location exposes it to forces that retard its rotational motion. Eventually the vortical motion will come to a standstill and subsequently resume, but now in a counter-clockwise sense because the warm fluid near the inner cylinder will be rising and the cooler outer fluid will be sinking near the outer cylinder [2 11. As a certain amount of time is required in order to give a vortex its rotational energy, a slow, circular flow of fluid in the annulus in the direction of the upper curved arrow will expose nascent vortices to forces that will periodically tend to reverse, and thus stop, the vortical motion. The circulation path of the fluid need not be circular, as in Fig. 12.9a, but could have the shape of an arbitrary closed path as in Fig. 1 2 9 b and c [21]. It is indeed possible to suppress thermal convection by such imposed circulation of fluid about a horizontal axis so effectively that high electric field intensities can be maintained in the buffer belt (of the order of 100 V/cm) without detriment to the sharpness of the streak separation pattern in deviation electrophoresis [6,2 I]. It now remains t o be shown how one can maintain such a constant circulation of the circular buffer belt in the annulus.

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269

Achievement of electromagnetic circulation Let us assume that the horizontal annular space between the concentric cylinders shown in Fig. 12.9a is filled with a buffer solution and that it represents the electrophoretic separation column. The electrophoretic current will be perpendicular to the page. If we now introduce a cylindrically shaped magnetic pole, say a North pole, into the inner cylinder, its radial magnetic field will be perpendicular to the current. As a result, the same electromagnetic forces that drive an electric motor will be exerted upon the fluid elements in the annulus at right angles to the magnetic field and the electric current and the annular buffer column will be set in rotational motion with clock-like constancy [ 6 ] . If the current vector points away from the reader, the circulation about a magnetic North pole will be clockwise but could be reversed by reversing either the current or the magnetic field. Fig. 12-10 shows how such a radial magnetic field can be generated in practice. It is encountered close t o the surface of a soft-iron cylinder, m, between two cylindrical co-axial bar magnets, NS,facing each other with their like poles. A simple apparatus

Fig. 12.1 1 shows the configuration in the simplest type of instrument in which endless belt electrophoresis can be conducted [ 6 ] .The magnets, NS,with the intermediate softiron core, m (in which a “window”, W,is milled to permit passage of light through the core region), are placed inside an inner transparent plastic (lucite) cylinder, C1. The outer, larger cylinder, C2,leaves a gap of 1.5 mm between the two concentric cylinders, which is the annulus in which electrophoresis is to be conducted. The cylinders C, and C2 are cemented t o the electrode compartments, ECI and EC2, in such a way that the annulus serves as a communication channel between them. The electrodes El and E2 permit the passage of the electrophoretic current through the annulus, thus generating in it an electric field. As soon as the current is turned on, the buffer in the annulus begins to revolve about a horizontal axis. The mixture t o be analysed electrophoretically is in the reservoir R. It is conveyed into the separation space via a fine injection capillary, IN (made of electrically nonconductive material), which places it centrally between the cylinders C1 and C2. Neutral injected particles are entrained in the revolving buffer and are accumulated in a stationary orbit (assuming absence of electroosmosis and other lateral streaming). Negative particles are transported electrophoretically towards the anode while orbiting counter-clockwise, as seen in Fig. 12.8. The combination of these two motions is a left-handed helix indicated as the particle path in Fig. 12.1 1. Positive particles will move towards the cathode in a right-handed helical path. The solid and dashed helical paths in Fig. 12.8 illustrate the helical streaks corresponding to two electrophoretic components of different mobilities. These streaks can be intercepted at the end of their paths, as described below, and conveyed to a fraction collector. This figure indicates another characteristic feature of endless belt electrophoresis. If the separation between two components is x after one revolution, it will be nx after n revolutions as the separation will be increasing by equal increments with each revolution. There is a hazard, however, which is analogous to superposition of grating spectra of

270

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Fig. 12.10. Magnetic field distribution about two cylindrical bar magnets, NS, with an intermediate soft-iron cylinder, m [ 71.

different orders encountered in optics. After a certain number of revolutions, the faster component of the nth revolution may merge with the slower component of the (n + 1)th turn. This can be easily avoided by superimposing a lateral flow of buffer in the direction of the electromigration. In the arrangement shown in Fig. 12.8 this will amount to adding buffer to compartment EC2 while removing it at the same rate from EC,.

Effect of magnetohydrodynamic driving force Instead of considering a curved buffer belt, we shall make an approximation in which the distance, h , between the cylindrical walls confining the buffer belt will be considered to be infmitely small in comparison with their radii of curvature. This amounts to a

ENDLESS FLUID BELT ELECTROPHORESIS

27 1

Fig. 12.1 1. Circular endless belt electrophoretic separator IS].NS: north and south poles of bar magnets; m: intermediate soft-iron cylinder; W: window in m; C,, C,: plastic cylinders, which enclose buffer-filed annulus; EC,, EC, : electrode compartments communicating through the annulus; IN: injector; R: sample reservoir.

consideration of flow between flat plates. A magnetic field of intensity B is maintained at right angles to these walls and a current of density ?flows through the buffer parallel to them. Under these conditions, each fluid element experiences an electromagnetic force per unit volume, say in the direction of the x-axis:

f7, =

lo-"Jxzi]

(1 2.10)

where ?is in A/cmz, B in gauss and f, in dynes/cm3. The result o f this force is a viscous flow of the buffer. The velocity distribution can be found from the differential equation for the force per unit volule [ 2 2 ] :

272

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

(12.11) where q is the fluid viscosity in poise. Remembering that the velocity vanishes at the walls and integrating twice, we obtain [23] (12.1a)

and, for the central maximal velocity, uo, (12.12) From eqns. 12.10 and 10.12 we obtain an expression for the central maximalvelocity, u o , in the fluid belt: (12.1 3) We can now calculate the streaming velocity for a typical set of parameters: h = 0.1 5 c m , i = 2 0 0 m A , J = 0 . 6 1 A/cmZ;B =340gaussand (at 16"C)q= 1.llpoise.Eqn. 12.13 then yields the value uo = 0.53 cm/s. The circumference of a typical instrument such as that shown in Fig. 12.1 1 is about L = 22 cm. From this value and uo,we can calculate the time, t o ,a centrally located particle takes to complete one revolution:

L

t o = - = 41.5s UO

(12.14)

Some illutrations o f instrument p e r f o m n c e Fig. 12.12 illustrates a helical streak obtained with erythrocytes in an instrument filled with physiological saline [7]. The cells enter through an injector IN at the bottom center and perform 15 turns of undiminishing sharpness, which can be seen by dark-field illumination through a window, W, in the central core, m, in Fig. 12.1 1. Fig. 12.13 shows electrophoretic separation between two yeasts: Saccharomyces cerevisiue and Rhodotomla [7]. A streak of the mixture enters from injector, IN, and splits in two as the particles spiral in a helical path around the core to the left. The consecutive turns are designated by a, b, c and d. The descending streaks in front of the iron core can be seen most clearly, but some of the ascending streaks moving behind the core upwards and to the left can still be distinguished. The separating particles are seen to gain equal increments in separation with each consecutive turn. The double streaks are spread far apart from each other by superposition of lateral streaming from right t o left. A clearly visible separation has been obtained after one turn (or in about 30s). Fig. 12.14 shows a separation [7] of a mixture of small molecular components (dyes). The point of injection of the dye mixture is marked by an arrow. The two slower components are not resolved after one turn, but are clearly separated after two turns (in about

ENDLESS FLUID BELT ELECTROPHORESIS

273

Fig. 12.1 2. A 15-turn helix of erythrocytes in circular endless belt revealed by light scattering [ 71. IN: injector; W : window in central core cylinder.

1 min). Finally, after four turns the dye streaks reach the collector, C, from the openings of which plastic tubes convey the separated components to a fraction collector.

Some unique properties of the endless belt system Of the unique properties of the endless belt electrophoretic system, at this point we shall mention two that are both a consequence of the utilization of the same current for electrophoresis and for the propulsion of the buffer perpendicular to the electrophoretic path. The first is the stability of the streak deviation against variation in the voltage applied to the electrophoretic cell. Fig. 12.15a shows four helical turns of the dye Evans Blue obtained at a cell voltage of 75 V. Fig. 12.15b shows four helical turns of Evans Blue after the cell voltage has been increased to 150 V. The reason why the pitch of the helix (i.e., the electromigration distance traversed by a dye ion during one revolution) has not changed is as follows. Although doubling of the voltage did double the electrophoretic velocity, it also doubled the rate of revolution of the buffer, thus reducing the revolution time by half and with it, of course, the pitch of the helix [ 6 ] . The second unique and essential property of this system is based on the fact that the propulsion of the buffer does not require a pressure gradient. As an electromagnetic force is exerted upon each buffer element, it is not necessary to enclose the channel through which the buffer flows completely. The two surfaces between which the buffer is

274

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Fig. 12.1 3. Separation in circular endless belt of two micro-organisms (Saccharomyces cereuisiae and Rhodotomla) revealed by light scattering 171. a, b, c and d represent consecutive helical turns. IN: injector. W: window in iron core.

Fig. 12.14, Separation and collection of dyes in circular endless belt [7].The sample enters at arrow. Five helical turns of separating dye components can be seen (from right to left: Evans Blue, “Brush” green recording ink and Rose Bengal). At the end of their paths, the helical turns are seen entering the collector.

ENDLESS FLUID BELT ELECTROPHORESIS

275

Fig. 12.15. Effect of cell voltage o n helical pitch in endless belt electrophoresis 161. (a) Helix of Evans Blue obtained at a voltage of 75 V across t h e cell and a current of 25 mA. (b) Ifelix of Evans Blue after incrcasing the voltage t o 150 V. There is n o perceptible change in helical pitch.

sandwiched are sufficient and the sides of the flowing belt can be left open to allow it to communicate with the electrode or buffer compartments without separation by membranes. The omission of membranes [18, 191 eliminates the drifts in pH and electrical conductivity in the separation space which they cause [ 171 in fluid curtain electrophoresis. The absolute smoothness of buffer propulsion, which cannot be achieved by a peristaltic pump, is also unique. Finally, the helical path and the cyclic nature of the buffer movement which imparts, without discontinuities, equal increments in separation to the components under analysis are unusual features characteristic of this system.

Modes of operation of the endless fluid belt system The endless fluid belt electrophoresis apparatus can be operated in several different modes 171.

Single-order collection This is the simplest normal mode of operation. The mixture to be separated is introduced via the injector and one then has to decide after how many helical turns the separated fractions are to be allowed to enter the collector. The output of the helical turn in which two or more fractions of interest are far enough apart to enter separate entry ports of the collector (which are about 1 mm apart) can be guided into the collector. Such guidance can be accomplished simply by modifying the imposed lateral flow. Split-order collection Sometimes it is advantageous not to collect all of the separated components after the same number of turns. For instance, suppose that we have a collector with 20 entrance holes and three fractions which, after four turns, have separated so that the two slowest fractions, A and B, enter after four turns the adjacent collector holes 1 and 2, while the fastest component, C, enters hole 5 . One could adjust the lateral flow so that the slowest component just misses the collector and proceeds to make a fifth turn, while the component of intermediate mobility is collected in collector tube 1. The slowest

276

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

component will then, after an additional turn, enter the collector ahead of the fastest component and may, for example, enter collector hole 12. Such a manoeuvre would have been particularly advantageous if components A and B had been too close after four turns to be collected in two separate tubes. An extra turn for both of these components could have separated them sufficiently for separate collection after five turns, while the fastest component would have been collected after four turns.

Orbital accumulation The imposition of an axial flow makes it possible to make the sum of the axial flow and electrophoretic velocities of a particle zero relative to the apparatus for a given particle species. In this instance, such particles would accumulate in a stationary orbit. Particles present at too low a concentration to be observed in the normal mode of operation could thus be detected by orbital enrichment after a sufficient number of buffer revolutions. Axial flow could then be used to shift the storage orbit so as to intercept it by the collector.

Zonal separation Microanalysis of suspension or solution volumes of the order of a few tenths of a microlitre can be accomplished by “orbiting zone” electrophoresis. One injects a fine streak (about 0.1 mm or less in diameter) about 1 cm in length, which forms a tiny zone moving on a helical path, Axial flow can be adjusted so that the helical pitch of the slowest component is zero, i.e., it moves in a stationary circular orbit, The other components of the mixture form other tiny orbiting zones that progress in the direction of the electric field. After the separation of the components has reached the desired value, axial flow is used to guide the separated fractions into the collector. The noncircular endless belt

Tkansition to the vertical “racetrack” The circular endless belt apparatus has the advantage of simplicity of construction, but this virtue is overshadowed by a drawback illustrated in Fig. 12.1 6. Particles entering the centre of the annulus via the injector, IN, will sediment if their density has not been adjusted so as to be equal to that of the buffer. This adjustment would be laborious and of limited usefulness, as equilibrium can thus be achieved for only one component of the mixture. Fig. 12.16A shows how the combination of circulation with sedimentation results in an eccentric particle orbit. If the disparity in density is great enough, the particles may precipitate on the walls of the annulus. A similar behaviour wiIl also be exhibited by a streak of molecular solutes emanating from the injector which will act like a large sedimenting object if it is denser than the surrounding buffer. This problem is solved effectively by distorting the circular annulus of Fig, 12.16A into the vertical “racetrack” [ 4 , 5 , 2 1 ] shown in Fig. 12.16B. The soft-iron core now has an elongated form with high vertical flat surfaces, C1 and Cz.The magnetic field is perpendicular to the surfaces of C1 in the immediate vicinity of the core, m, and the electrophoretic current, flowing at right angles to the page, is perpendicular to this

ENDLESS FLUID BELT ELECTROPHORESIS

277

Fig. 12.16. Particle circulation paths in annuli of different shapes [4]. m: Iron core inside the annulus; IN: injector;C, ,C, : walls confining the annulus; COL: collector; t : collector tubing;CC: collector compensator; T: test-tube.

magnetic field. We thus obtain, as with the circular annulus, a tangential electromagnetic force that moves the buffer as indicated by the arrows. A dense particle entering from the injector will not deviate from the central path until it reaches the short horizontal section where the flow turns around. There, the particle may sediment below the mid-line and continue its upward path to the left of the left vertical mid-line, This slight deviation is, however, corrected when the particle reaches the upper turning point of the flow, where sedimentation returns it to the mid-line, With this shape of the circulation path, one can use particles and solutions whose densities differ considerably from that of the surrounding buffer. Fig. 12.16C shows interception of a descending streak by the collector, COL. The fluid escaping from the collector can be replenished at an equal rate by injection via a compensator, CC.

The separation space The effective separation space of the non-circular endless belt apparatus is shown schematically in perspective in Fig. 12.17. The electrode and buffer chambers have been omitted. The central soft-iron core, C (corresponding to m in Fig. 12.16), is hollow and is cooled by a cooling liquid conveyed via the tubes CP. A quartz window, W,permits observation by visible and ultraviolet light (L in Fig. 12.17) transmitted normal to the core. Only the North poles, N, of the four magnets used are shown. The core, C,is surrounded with a plastic mantle, MA, in which hollow chambers (not shown) parallel to

278

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

CP

Fig. 12.17. Electrophoretic separation space of non-circular endless fluid belt apparatus [ S ] . N: North Boles of magnets (South poles are not shown); C: hollow cooled iron core; CP: tubes carrying cooling fluid to and from C; W: window in C; MA: mantle surrounding annulus (i.e.,&he fluid belt); IN: injector; Cap: ca@llary; COL: collector; t: c+ollector tubing; &? electric field vector;l: electric current density vector; B : magnetic field vector;F: electromagnetic force vector; 1*, 2*: first and second ascending streaks (behind the core C); 1, 2, 3: first, second and third descending streaks (in front of the core C); L: light beam passing at the bottom of the annulus.

the front and back surfaces of the core serve as outer cooling chambers by carrying a cooling solution. Thus, the fluid belt which is sandwiched between the core, C, and mantle, MA, is cooled from both the inside and outside. The sample to be analysed enters the annulus centrally from the injector, IN, and moves towards the intercepting collector, COL, in a non-circular helical path. The first, second and third descending streaks are labelled 1 , 2 and 3, respectively, and the corresponding ascending streaks (behind the core, C) I* and 2* (there is no third ascending streak). The dimensions of the core are roughly 10 cm in height, 6cm in width and about 1 cm in thickness.

The apparatus Fig. 12.1 8 shows the separation cell in a state of partial disassembly [5]. The central separation chamber includes the outer mantle, MA, and the outer cooling chambers, CC (incorporated in the mantle); the route of the cooling water, CW, through the mantle is shown. The tubes CP convey the cooling water to the iron core, C. WO is a window opening and COL (with attached plastic tubes, CT) is the collector which plugs into the gasketed opening, CO. The quartz window, W,, also plugs into a gasketed opening, WO, in the mantle. IN is the injector channel and Cap the injector capillary that conveys the sample solution to the separator (it corresponds to IN in Fig. 12.17).

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279

The left-hand side o f the drawing shows the sequence of chambers in the assembled instrument. In direct contact with the buffer in the annulus are the buffer chambers, BC, which possess three openings: OT, through which the core can be introduced, and O1 and 02, which create hydraulic and electrical communication with the adjacent electrode chambers, EC. Instead of membranes, these openings are closed by perforated plates, P. The perforations are in the lower portions of the plates and serve the following purpose. Bergrahm 1241 has shown that a centrifugal flow of buffer away from a separation column towards the electrodes can effectively prevent migration of ions of electrolysis products from the electrodes into the separation space. This flow must be sufficiently rapid, w h c h is achieved by the small perforations in the plates, P, which offer a small cross-section t o the flow. Bergrahm’s idea was adapted by Luner [19] t o endless belt electrophoresis. The buffer solution for the centrifugal buffer flow is supplied by the Mariotte bottle, MB, shown in Fig. 12.19. It enters the buffer chambers, BC, via the tubes, DT, from the distributor, D. After passing through the perforations in plates P, the buffer moves upwards past the electrodes, E, carrying the evolved gases t o the “balconies”, B, where the flow slows owing t o the large channel cross-section so as t o allow enough time for the gas bubbles t o escape before the fluid descends through the drain tubes, D, in order t o leave the cell via channels. d , and nipples, N. The flow scheme of the instrument is shown in Fig. 12.19. The Mariotte bottle, MB, is a source of buffer at a constant pressure head, which delivers a constant buffer influx into the cell via PVC tubes, DT. I n addition t o creating the centrifugal buffer flow, these tubes can also be used t o create a lateral buffer flow through the annulus. By transferring an appropriate number of tubes from the right to the left buffer chamber, one can change the direction o f the lateral buffer flow or alter its magnitude. The “see-saw”, SS, provides a fine control for the lateral buffer flow by changing the relative level of the end points of the drainage tubes, dt. The Mariotte bottle also feeds the collector-compensator, CC, via a variable-flow resistance, RV. The sample is injected from a syringe, SY, which is driven by a motor, MD, and drive, DR, via tubing, CT, into the injector capillary, Cap. As an example of the preparative throughput of the instrument, a normal run in cell separations will process 0.5 mI o f a cell suspension of 5 * 10’-10*cells/ml in one operation. Such a separation run may last from 30 t o 45 min. Serpentine fluid belt electrophoresis An alternative way of stabilizing a fluid curtain against thermal convection by inversion of nascent vortices is shown in Fig. 12.20. The fluid enters the serpentine flow channel in tube A at IN from a well centred injector and the vortices shown in tube A are inverted after transfer into tube B and inversions are repeated as the fluid moves from one vertical section into another. A similar sequence of inversions with consequent stabilization can also be obtained b y rotating the configuration of Fig. 12.20 through 90” [3,21]. Fig. 12.2 1A shows a serpentine cell and Fig. 12.2 1B and C show a dye separation illustrating the mode of action of such a serpentine separation cell [3]. This system has the advantage o f compacting a long separation path into a small space, but it has two serious disadvantages: (1) the serpentine channel is difficult t o make and (2) like the flat curtain

280

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

ENDLESS FLUID BELT ELECTROPHORESIS

28 1

4 Fig. 12.19. Endless fluid belt apparatus- flow system [S]. SCH: separation chambers; W: window; Cap: injection capillary; COL: collector; CT: collector tubing; CC: collector-compensator nipple; TT: test-tubes; BC: buffer compartments; EC: electrode compartments; E l , E,: electrodes; N: nipples of rigid drainage tubes (not shown); dt: narrow plastic drainage tubes; SS: “see-saw”; P: perforated plates; MB: Mariotte bottle; MT: tube of Mariotte bottle; CS: clean-out syringe; D: distributor; DT: distributor tubes; CT:coiled tubing linking capillary to sample syringe; SY: sample syringe; MD: motor drive; DR: screw and plate which drive the plunger of syringe SY; RV: regulator valve for control of collector compensator inflow.

Fig. 12.18. Endless fluid belt apparatus IS]. The magnets have been removed; they are shown in relation to the iron core C in Fig. 12.17. WO: Window opening; MA: mantle surrounding iron core; S: syringe needle for removal of bubbles from top of annulus; IN: injector mount; Cap: injector capillary; CC: cooling chambers in mantle; CO: gasketed opening for insertion of collector; COL: collector; CT: collector tubing; Wm: gasketed pug-in quartz window fitting into opening WO; CW: cooling water tubes; the arrows indicate the path of cooling water through these tubes and cooling chambers CC. The construction IS the same on thc back side of the mantle, which is not shown. BC: buffer chambers; OT,, OT,: openings for passage of magnet tunnel TU shown in Fig. 12.18B; the opening OT, is sealed by the plate TP of the tunnel of Fig. 12.18B; 0 , - 0 , : openings in the outer walls of the buffer chambers against which the four perforated gasketed plates P are pressed; CP: cooling pipe conveying coolant to core C; V: vent for removal of air bubbles from core C; E: electrodes; EC: electrode compartments; EP: electrode connection posts; B: four balconies; D: drainage tubes; d: continuation of drainage tube; N: terminal nipple of drainage tube for detachment of tubes, dt, shown in Fig. 12.19; the balconies are shown in place o n the left side of the cell and removed on the right; G : gasket; CB: anticonvection baffles; MG: magnet.

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CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Fig. 12.20. Serpentine flow channel 1211. The buffer is transferred from left to right as it meanders through the channel. Engendered vortices are inverted relative to the gravitational field vector $as they are transferred from one vertical channel t o the adjacent one.

apparatus (“free-flow’’ electrophoresis), the serpentine flow channel must be closed off laterally by membranes with the consequent above-mentioned shifts in pH and conductivity in the separation space. These shifts in pH and conductivity may not be objectionable, however, if the system were used for continuous flow isoelectric focusing where a pH gradient must be maintained in the cell and the long migration path (in a small space) will provide the required long residence time of ampholytes to be separated in the electromigration column [25]. Examples of preparative separations by continuous flow deviation electrophoresis In order to illustrate the capabilities and effectiveness of the methods of deviation electrophoresis, we shall present some illustrative examples of separations. We shall not tabulate here the variety of buffers that have been used, as the buffer composition can be found in the literature cited. The buffers used in molecular separations differ from those employed in separations of cells notably in the use of solutes, such as sucrose, which must be used in the latter application at appropriate concentrations to protect the cells from osmotic damage. Other considerations in designing buffer solutions for cell separations are optimization of the viability of the cells and avoidance of cell aggregation. A detailed consideration of buffers suitable for cell separations by deviation electrophoresis has been published [26].

Molecular mixtures As the resolving power of deviation electrophoresis is inferior to that of methods in which chromatographic and filtration effects are combined with electrophoresis, relatively few uses have been made of it for molecular analysis. Nevertheless, the ease of continuous collection of separated fractions may sometimes outweigh the drawback of lower resolution. Fig. 12.14 presents an example of the separation and collection of lowmolecular-weight solutes (dyes) by circular endless belt electrophoresis [7]. Whereas separations in the endless belt are usually exhibited as photographs of separated streaks,

ENDLESS FLUID BELT ELECTROPHORESIS

283

the separations obtained in the flowing curtain apparatus are given in terms of the evaluation of the collected fractions. Thus, Fig. 12.22 shows the collection pattern resulting from curtain flow electrophoresis of a crude peptide fraction precipitated with acetone from a nucleosol of Yoshida ascites carcinoma cells [27]. Fig. 12.23 shows four consecutive photometric tracings obtained after 2 0 , 2 8 , 5 0 and 70 s, respectively, by curtain flow electrophoresis of serum proteins [20]. The diameter of the injected streak was about 0.05 mm and the injected sample volume was 0.1-0.3 pl, containing 3-1Opg of protein. Fig. 12.24 shows an interesting group of tracings obtained by the same instrument [20] using pathological sera.

Viruses Relatively little work has been done on virus separations by continuous flow deviation electrophoresis. The separation of strains of fd3 bacteriophages by flowing curtain electrophoresis has been reported [28]. In this study, it was possible to isolate from the main component two mutants present in minute amounts, which differed from it by one and two electric charges, respectively, in their protein coats. Fig. 12.25 shows the rapid separation of a mixture of two strains of tobacco mosaic virus, U1 and U2, into two widely spaced streaks achieved by endless belt electrophoresis [ 181. The mixture contained about 2% of the U2 and 0.5% of the U1 fraction. The picture shown was obtained by ultraviolet light photography. The scarce component, U1, is the faster and appears on the left because, as in all endless belt photographs shown here, the electromigration is from right to left. The streaks corresponding to the two virus components of the mixture are clearly separated (top arrows) after travelling 4 cm downwards from the injector (not shown), that is, after about 8 s residence in the electric field. The two streaks on the left (lower arrows) show how far the separation has increased in the upward ascending streaks after an additional half-revolution about the iron core (about 20 s). Micro-organisms We saw in Fig. 12.1 3 an example of a separation of two species of yeast, Rhodotorula and Saccharomyces cerevisiae,by circular endless belt electrophoresis [7]. A photograph of a similar separation in the non-circular endless belt apparatus of bacteria (Escherichia coli) and a species of yeast (Rhodotomla)is given in Fig. 12.26, accompanied by a histogram which shows that there is no overlap between the fractions in the collector [18]. An interesting observation that can be made with a number of pure strains of bacteria is shown in Fig. 12.27. A streak of a pure strain of Escherichiafreundii is shown t o be split into two electrophoretically distinct and widely separated components [29]. Similar observations have also been made on some strains of Escherichia coli [30]. Electron microscopy revealed that the two fractions into which the pure bacterial strains were split represented a piliated and non-piliated fraction. Cultures of each of these fractions gave rise to offspring that again consisted of a mixture of piliated and non-piliated bateria exhibiting the same electrophoretic separation pattern. Preparative separations of bacteria differing in their Gram-staining characteristics and resistance to antibiotics by means of flowing curtain electrophoresis have also been reported [31].

284

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

R

Fig. 12.21. (A) Perspective view of serpentine path continuous flow deviation electrophoresis cell [ 31. (B) Simultaneous separation of two streaks of a mixture of three dyes (injected a t the bottom) through two separate injectors in the serpentine flow cell. The buffer flow is dkected upward. Streaks in the two separation patterns (from left to right): Evans Blue, Rose Bengal and “Brush” green recording ink. (C) Collection pattern for the separated components of the two sample streaks [3]. The sequence of colours is the same as in B. Evans Blue appears in test-tubes 2,3,16 and 17; Rose Bengal in 6,7,18,20 and 21 and the green dye in tubes 9,10,11,23,24 and 25. Tubes 8 and 22 contain a dilute mixture of Rose Bengal with the green dye. The collected fractions are very dilute in tubes 6, 8, 11, 18 and 25. Test-tubes 4 and 19 are empty.

Subcellular particles Most of the work on the separation of subcellular particles has been performed with the flowing curtain apparatus developed by Hannig and his numerous co-workers at the Max-Planck Institute of Biochemistry, Martinsried, near Munich, G.F.R. We shall refer to Hannig’s review papers [31,32] for complete surveys of the activities in this field and limit ourselves here, within the scope of our methodological objectives, to a few representative illustrations.

ENDLESS FLUID BELT ELECTROPHORESIS

285

286

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

2.5

t

0

m

1.5

N

w

b 0 N

w

0.5

20

40 Froct No

-

60

80

Fig. 12.22. Flat curtain flow electrophoresis o f a crude peptide fraction precipitated with acetone from the nucleosol of Yoshida ascites carcinoma cells [27].

rnobility~105~cm volt“ 2~

sec-’

Fig. 12.23. Separation of serum proteins by the analytical fluid curtain apparatus in pH 8.8 Tris-borate buffer [20]. Separation times: (a) 20 s; (b) 28 s; (c) 50s; (d) 7 0 s. The overlapping of the components diminishes with separation time.

Of particular interest for fundamental biological investigations and for the study of biological cell membranes in particular are the successes in generating and separating b y deviation electrophoresis artificially created vesicles surrounded by “insideaut” and “outside-out’’ membranes [33]. It proved possible to detach from erythrocytes small protuberances as isolated vesicles. These particles have the same outer surface as the erythrocytes carrying the sialic acid. They migrate electrophoretically with the same mobility as the erythrocytes. On the other hand, it also proved possible to detach from erythrocytes as isolated vesicles tiny invaginations formed in their surface. These vesicles carry the inside membrane surface of the erythrocytes on their outside, i.e., the siahc acid layer is now on the inside [34,35].Electrophoretic analysis in the flowing curtain apparatus showed that these “inside-out’’ vesicles have a smaller electric surface charge density than the “outside-out’’ vesicles, as indicated by their slower electromigration rate. Fig. 12.28 shows the distribution of vesicles among the fractions obtained by the flowing curtain apparatus from a mixture of both types of vesicles [34]. It turned out that such membrane inversions, with consequent creation of insideaut

ENDLESS FLUID BELT ELECTROPHORESIS 4orbus- Waldenstrorn Vb (121)-37.6

I Nephrotic Alb(

287

syndrome

al) - 43.0

-

16.6

- 12.4 - 29.2

IgD - Plasmoc ytoma

Liver cirrhosis

41b (UI) - 41.8 a2 - 15.8 PI 11.4 /32 - 6.0 71 - 14.3 7, -107

Alb(a1)- 21.2

-

? 7

-

7.7

-22.4 -48.7

Fig. 12.24. Separations of pathological sera within 42 s with the analytical h i d curtain apparatus [ 201.

Fig. 12.25. Separationof two strains, U1 and U2 (slower component), of tobacco mosaic virus in the endless fluid belt apparatus: after about 8 s (arrows at top right) and after about 20 s (arrows at bottom left) [ 181.

vesicles, were also possible with other biological entities such as E. coli bacteria and mitochondria whose inner membrane surface thus became accessible to investigation [36,37]. Of no lesser interest is the demonstration of the possibility of electrophoretic isolation of undamaged subcellular cell components, such as renin granules from rabbit kidney cortex [38], rat liver lysosomes [39] and rat liver ribosomes [40].

Cells Electrophoresis of somatic cells presents considerable experimental difficulties that are

288

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Fig. 12.26. Separation of bacteria and yeast cells: Escherichiu coli (faster component on the left) and Rhodotomla (slower component) [ 181. A: separation pattern; B: collection histogram.

Fig. 12.27. Splitting of Escherichiufreundii into fast (F) and slow ( S ) components observed with the endless fluid belt apparatus [ZS].F and S, F' and S' and F" and S" denote consecutive half-turns of the helix.

ENDLESS FLUID BELT ELECTROPHORESIS

289

I

6001

Outside-out

I

20

25

A

15

Fraction number

Inside-out

30

Fig. 12.28. Separation in the fluid curtain apparatus of “inside-out” and “outside-out” erythrocyte vesicles [ 34). Left peak: vesicles carrying sialic acid on the outside surface (as the intact erythrocytes). Right peak: a pure fraction of vesicles carrying the sialic acid on the inner surface.

not inherent in the electrophoretic process. They are based on the sensitivities of the cells to their chemical environment, the osmotic pressure of the ambient solution and the presence of factors that could affect the viability of the cell. Clumping of the cells, which accelerates sedimentation, should be carefully avoided. It is advisable to provide the instrumental parts t o which the cells could adhere (such as the collection tubing) with a special coating. Special buffers should be used to ensure the survival of the cells during the separation process. All of these factors have been reviewed in detail in connection with flowing curtain electrophoresis [26]. In the endless belt apparatus, cell losses due to cell adhesion in the collector system (including the collection tubes) vary greatly from one species of cell to another. Thus, with erythrocytes the cell recovery may be as high as 98%, while with lymphoid cells it may be below 50%. The following illustrations show cell separations in the non-circular endless belt apparatus. Fig. 12.29A and B shows two histograms for collection patterns of human blood cells from two hospital patients [18]. Fig. 12.30 shows an unusual separation pattern in which the erythrocyte streak splits into two distinct cell components. This blood sample

290

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

0 Erythrocytes 200 Granulocytes

Leukocytes

0 Erythrocytes

8

Fraction number

9

10

Fraction number

12

Fig. 12.29. Separation of human erythrocytes from leucocytes in the endless fluid belt apparatus [ 181. A : Histogram for collection pattern from a hospital patient. B: Histogram for collection pattern obtained from a different patient.

Fig. 12.30. Splitting of a donor’s erythrocytes into two electrophoretically distinct components as observed with the endless fluid belt apparatus [41].

was obtained from a donor who exhibited partial polyagglutinability [41]. Fig. 12.31 shows the distribution pattern in a collection of murine mesenteric node lymphocytes subjected to endless belt electrophoresis. This figure illustrates how the resolution is improved by reducing the injection rate, thus making the streak thinner, which confines the particles in it more to the central region of the annulus. Fig. 12.32A shows a photograph of a separation pattern of a contaminated suspension of human lymphocytes [5]. Fig. 12.32B shows the distribution of the lymphocytes and contaminants in the collection pattern, and Fig. 12.32C shows the distribution of E- and EA-rosette-forming cells in a lymphocyte collection pattern in which enrichment of B and T lymphocytes could be obtained in different fractions. The remaining illustrations were obtained with the fluid curtain apparatus. Fig. 12.33 shows a photograph of the separation path of a mixture of human and rabbit erythrocytes escaping at the top right from the meeting point of the separated streaks [42].

29 1

ENDLESS FLUID BELT ELECTROPHORESIS M u r t n e m e ~ e n t e r i cl y m p h node cells

0 P l o8/

30

M= High in) r a t e ( 1 0 0 ) e-e=tow inj r a t e ( 4 0 )

25 20 15 10 5 F r a c t i o n number - migration

0

Fig. 12.31. Separation of murine mesenteric lymph node cells with the endless fluid belt apparatus (51. The solid line represents a collection pattern obtained at high sample injection rate; the broken line, showing higher resolution, was obtained at a low injection rate.

This instrument was applied to investigations of significant biological problems and one of the important recent results is the isolation of a single-cell population by Heidrich and Dew [43] from a mixed-cell suspension derived from rabbit's kidney cortex. In this work it proved possible to isolate proximal and distal tubules as well as renin-active cells. Of particular importance is the work on the fractionation of lymphocytes into immunologically distinct sub-populations that has been carried out systematically with the fluid curtain apparatus. A complete bibliography can be found elsewhere [26] and we shall limit ourselves to the consideration of illustrative examples. Fig. 12.33 shows curves that represent the overall leucocyte distribution in a sample of human w h t e blood cells subjected to electrophoretic analysis and the distribution curves for granulocytes, lymphocytes, eosinophils and monocytes inferred from an analysis of the collected fractions [3 11 . Fig. 12.34 shows a trimodal electrophoretic distribution profile for murine spleen cells [44]. The electroinigration is towards the left. The peak of the fraction of highest mobility corresponds t o T cells, whereas the two peaks of lower mobility (to the right of it) are formed by surface immunoglobin-carrying lymphocytes (Ig') and other types of cells. The legend indicates the cell sub-populations as established by physical and imniunological tests on the collected fractions. These examples suffice to demonstrate that continuous flow deviation electrophoresis is a powerful technique for fundamental investigations in cell biology and has potential practical value in medical research and diagnosis. Measurement of electrophoretic mobility The principles according to which electrophoretic mobilities can be measured with a continuous flow deviation electrophoresis apparatus are essentially the same for fluid curtain electrophoresis as for endless fluid belt electrophoresis. In the former, the slope of a streak against the vertical is a measure of the electrophoretic mobility of the streak components, whereas in the latter the pitch of the helix (i.e., the distance between

292

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Fraction number - migration

Fig. 12.32. Separations of suspensions of human lymphocytes with contaminants by means of the endless fluid belt apparatus. The lymphocytes were prepared by the Ficoll-Isopaque interface centrifugation method [ S 1. (A) Photograph of separation pattern. (B) Collection patterns of lymphocytes and contaminants. (C) Distributions of the E-rosette- and EA-rosette-forming cells in a lymphocyte collection pattern obtained with the endless fluid belt apparatus. The vertical scale on the right applies to the curve drawn through the circles (0).The percentage scale on the left applies to the curves drawn through the crosses (x) and squares (B). WBC = White blood cells. (Graphs (B) and (C) have been obtained by Dr. R.C. Seeger and MI. J. Rosenblatt in collaborative experiments).

adjacent turns) measures the electrophoretic mobility. This statement is correct, however, only in the absence of electroosmotic streaming, which in fact is normally present. Thus, owing to electroosmotic convection even electrically neutral particles will deviate from a zero-deviation path. We must therefore establish a baseline, i.e., the path of an electrically neutral particle against which the deviation of streaks of charged particles will be measured [5, 181. The neutral dye ApolIon (Microchemical Specialities, Berkeley, Calif,, U.S.A.) can serve this purpose.

293

ENDLESS FLUID BELT ELECTROPHORESIS

Granulocytes

0

0

Lymphocytes

30

40

35

45

50

Fraction number

Fig. 12.33. Separation of human leucocytes by the fluid curtain apparatus in phosphate buffer (pH 7.3) with a field intensity o f 80V/cm [ 311. LJ--o--o: Overall distribution curve; -: granulocytes; . lymphocytes; -:eosinophile granulocytes; - - -: rnonocytes.

-._.-.

--

We shall now describe how absolute mobilities can be measured with the endless fluid belt apparatus. An analogous procedure could be used with the fluid curtain apparatus. To measure absolute mobilities, we must know the intensity of the electric field and the distance traversed in the field direction by the particles under observation against the zero-mobility reference streak in a known time. The electric field can be found as the ratio of the voltage between two points, such as between the metal tube, S , and a straight metal wire inserted through the capillary, Cap, in Fig. 12.8. The migration time can be chosen to equal one buffer revolution time in the annulus. This can be timed by injecting a pulse of dye through the injector, IN. The separation between the particles under test (mixed with Appollon dye) and the Apollon zero-mobility reference streak is measured after one turn. From these measurements, we can calculate the horizontal distance covered in 1 s due to electrornigration by the observed particles in a field of 1 V/cm, i.e., their mobility, p . Usually, it is more convenient to make relative mobility measurements, for which we require a standard of known mobility. The dye brilliant blue (K & K Labs., Hollywood,

294

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Fraction ( mrn) 380 3 5 8 335 313 29.1 2 6 8 2 4 6 Electrophoretic migration path

224

201

- -

Fig. 12.34. Electrophoretic distribution profie of spleen cells from conventionally raised C3D2F1 hybrid mice [44]. - - -: All nucleated cells; -A-A-: O* lymphocytes; - 0 - 0 -: all Ig+ lymphocytes; G-@-: ring-like fluorescing Ig+ lymphocytes; --o--o-: spot-like fluorescing Ig' lymphocytes.

Calif., U.S.A.) is suitable for this purpose. It preserves a constant ionic charge in the pH range 3.3-9.3. If we inject brilliant blue of mobility p B and Apollon together with a particle, U, of unknown mobility, we obtain a triple streak. If we consider a horizontal line intersecting with these streaks at some distance from the injector at the points A, B and U, the horizontal distance between the Apollon and brilliant blue streaks will then be AB and the distance between Apollon and the streak of unknown mobility will be AU. The unknown mobility, p u , is then given by

! % = -AU PB

AB

(12.14)

The electrophoretic mobility of a species of ions or biological particles depends on the nature of the ambient buffer solution, the chemical composition, ionic strength and pH of which must be specificed in order t o make the given mobility value meaningful. There is, however, another important parameter that must also be specified, viz., the temperature at which the mobility has been measured. This is a less simple matter as there is a temperature gradient within the fluid belt or curtain with a temperature maximum in the mid-plane between the walls. This problem has been solved by using brilliant blue (whose streak is centred in the annulus) as a thermometer, as it were [ 591. If we tabulate the absolute mobility of brilliant blue as a function of temperature, we can determine the temperature at the centre of the annulus from the deviation of brilliant blue streak in a known electric field [5,18,19]. As the material of unknown mobility lies in the same fluid layer as the brilliant blue streak, its mobility has thus been determined for the temperature indicated by the absolute mobility of brilliant blue. In this connection, we recommend the use of the newly proposed Tiselius unit of electrophoretic mobihty (1 TU = 1O-' cmz s-' V-' = 10-9m2 s-' V-'), w h c h has been introduced as an analogue to the Swedberg unit used in sedimentation [I].

CONCLUSION

295

CONCLUSION Continuous flow deviation electrophoresis can be conducted without recourse to porous media or similar means of stabilization against thermal convection in flow channels of different shapes. We have considered examples of a flat curtain, a serpentine path and circulation in an endless belt about a horizontal axis. In the last two cases, additional stabilization is achieved by periodic inversion of nascent thermal convection vortices. An additional advantage of the latter systems is the possibility of compressing a long migration path into a small space. The endless belt system possesses an additional, unique advantage, viz., the use of a magnetic field for generation of buffer flow. The smoothness and constancy of the flow produced by the interaction of the electrophoretic current with the constant field of a permanent magnet is not the rnain advantage of this system, which obviates the need for a complex pump for this purpose. The principal advantage of this magnetohydrodynamic drive is the possibility of onutting lateral confines in the flow channel, which thus becomes a sandwich with the fluid contained between two parallel dielectric walls. Thus, membranes, which are indispensable in the flat and serpentine fluid curtain apparatus, can be omitted. This omission not only leads to simpler operation and maintenance, but is also a very significant improvement because omission of membranes eliminates membrane-induced temporal and spatial shifts in pH and electrical conductivity in the separation space and permits convective streak colliniation 1451. The flat fluid curtain system has received the greatest amount of work in its development and has been put to many uses in research, not only in the laboratory of its origin in Munich but also, as a result of its commercial availability, in many other laboratories. The diverse uses and applications of this apparatus are apparent from the bibliography. The endless belt apparatus, however, is not yet available commercially and has been confiied, owing to the complexity of its construction, to instruments emanating from one laboratory. It has therefore been used by only a few workers in few investigations. The development of the fluid curtain and the endless belt apparatus has proceeded concurrently since about 1960, and not without mutual benefits, which can be seen from the chronology of new improvements although not always from the references. It is to be expected that this symbiotic relationship between these methods will continue in the future. The examples shown not only illustrate the great potential of these methods as powerful research tools, but also suggest the possibility of diagnostic uses in medicine and therapeutic uses through preparative separations on a large scale of components which could be administered to patients. ACKNOWLEDGEMENTS Our work o n cell separations has been greatly aided by the advice and cooperation of Dr. Charles W.Boone, Head of the Cell Biology Laboratory of the National Cancer Institute. In the separations of human B and T lymphocytes we enjoyed the cooperation of Dr. Robert Seeger of the UCLA Department of Pediatrics and of his assistant Mr. Joseph

29 6

CONTINUOUS FLOW DEVIATION ELECTROPHORESIS

Rosenblatt (cf. Fig. 32C). We received the greatly appreciated similar cooperation of Dr. Benjamin Bonavida of the UCLA Department of Medical Microbiology and Immunology in work aiming at separation of murine B and T lymphocytes. Last, but not least, I am indebted to Professor K. Hannig and Dr. H. G . Heidrich of the Max Planck Institute of Biochemistry in Martinsried near Munich for having so kindly provided numerous illustrations on the fluid curtain apparatus and its uses.

SYMBOLS AND UNITS E B J

Electric field intensity in V/cm Magnetic field intensity in gauss Electric current density in A/cm* Electric conductivity in (ohm cm)-' U Electrophoretic mobility in Tiselius units: TU (1 TU = (cm/s)/(v/cm)) [ 11 P W Electroosmotic mobility in TU 7) Fluid viscosity in poise U Fluid curtain velocity in cm/s Central maximum velocity in cm/s uo Ve Electrophoretic velocity in cm/s Electroosmotic velocity at the wall in cm/s v, Velocity o f a particle due to electro-osmosis and electrophoresis in cm/s u* X, Y, Z Coordinate axes Q! Angle of deviation in degrees Z Distance of a point from center of fluid ribbon in cm h Thickness of fluid ribbon in cm d Internal diameter of injector tube, or initial thickness of injected streak in cm R Resolving power Vertical force density (forcelunit volume) in dynes/cm3 fx Horizontal force density (forcelunit volume) in dynes/cm3 f Y

REFERENCES 1 2

N. Catsimpoolas, S. Hjertkn, A. Kolin and J. Porath, Nature (London), 259 (1976) 264. K. Hannig, tl. Wirth, B. H. Meyer and K. Zeiller, Hoppe-Seyler's 2.Physiol. Chem., 356 (1975)

3 4 5

A. Kolin and P. Cox, Proc. Nat. Acad. Sci. US.,52 (1964) 19. A. Kolin,Proc. Nat. Acud. Sci. US.,56 (1966) 1051. A. Kolin, in N. Catsirnpoolas (Editor),Methodsof Cell Separation, Vol. I, Plenum, Ncw York,

6 7 8

A. Kolin, Proc. Not. Acad, Sci. U S . , 46 (1960) 509. A. Kolin,J. Chromatogr., 26 (1967) 164.

1209.

1979, pp. 93-180.

A. Strickler and T. Sacks, in N. Catsimpoolas (Editor), Isoelecmk Focusingand Isorachophoresis, New York Academy of Sciences, New York, 1973, pp. 497-514. 9 H. L. Dryden, I;. P. Murnaghan and H. Bateman, in Hydrodynamics, Dover, New York, 1956, p. 184. 10 H. Lamb, in Hydrodynamics, Dover, New York, 1945, p. 582. 11 M. Smoluchowski, in L. Graetz (Editor), Handbuch der Eiektrizitat und Magnetismus, Vol. 2 , Barth, Leipzig, 1921, p. 366.

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12 C. C. Brinton and M. A. Lauffer, in M. Bier (Editor), Electrophoresis, Vol. 1, Academic Press, New York, 1959, pp. 428487. 13 H. Svensson and I. Brattsten, Ark. Kemi, 1 (1949) 401. 14 W. Grassman and K. Hannig, Naturwissenschaften, 37 (1950) 496. 15 J . Barrollier, E. Watzke and H. Gibian, 2. Naturforsch. B , 13 (1958) 754. 16 K. Hannig, Z. Anal. Chem., 181 (1961) 244. 17 K. Hannig, Hoppe-Seyler’s Z. Physiol. Chem., 338 (1964) 211. 18 A. Kolin and S. J. Luner, Anal. Biochem. ,30 (1969) 111. 19 S. J. Luner, Thesis, University of California, Los Angeles, 1969. 20 K. Hannig, H. Wirth, R. K. Schindler and K. Spiegel, Hoppe-Seyler’s 2. Physiol. a e m . , 358 (1977) 753. 21 A.Kolin,Proc. Nut. Acad. Sci. U.S.,51 (1964) 1110. 22 H. Lamb, in Hydrodynamics, Dover, New York, 1945, p. 582. 23 H. L. Dryden, F. P. Murnaghan and H. Bateman, in Hydrodynamics, Dover, New York, 1956, p. 184. 24 B. Bergrahm, Sci. Tools, 14 (1967) 34. 25 T. S. Fawcett, in N. Catsimpoolas (Editor), Isoelectric Focusing and Isotachophoresis, New York Academy of Sciences, New York 1973, pp. 209 and 112-126. 26 K. Zeiller, R. Loser, H. Pascher and K. Hannig, Hoppe-Seyler’s Z. Physiol. Chem., 356 (1975) 1225. 27 P. Spitzauer, A. Schweiger and K. Hannig, Hoppe-Seyler’s Z. Physiol. Chem., 354 (1973) 1327. 28 G. Braunizer, G. Hobom and K. Hannig, Hoppe-Seylerk Z. Physiol. Chem., 338 (1964) 278. 29 A. Kolin, Proc. 1st European Biophysics Congress, Baden, Austria, 1971, p. 481. 30 R. M. Owen, Thesis, Calif. State College, Long Beach, Calif., 1972. 31 K. Hannig, in D. Click and R. M. Rosenbaum (Editors), Techniques of Biochemical and Biophysical Morphology, Wiley-Interscience, New York, 1972, p. 21 1. 32 K. Hannig, Mitt. Max-Planck Ges., p. 185. 33 H. G. Heidrich, Mitt. Max-Planck Ges., 6 (1974) 464. 34 H. G. Heidrich and G. Leutner, Eur. J. Biochem., 41 (1974) 37. 35 G. Leutner and H. G. Heidrich, The mechanism of “inside-out” vesicle formation (in preparation). 36 H. G. Heidrich, R. Stahn and K. Hannig, J. Cell Biol., 46 (1970) 137. 37 H. G. Heidrich and W. L. Olsen, J. Cell Biol., 67 (1975) 444. 38 M. E. Dew and H. G. Heidrich, Hoppe-Seyler’s Z. Physiol. Chem., 356 (1975) 621. 39 K. Hannig, R. Stahn and K. P. Maier, Hoppe-Seyler’s Z. Physiol. Chem., 350 (1969) 784. 40 A. Schweiger and K. Hannjg, Hoppe-Seyler’s 2. Physiol. Chem., 348 (1967) 1005. 41 P. Sturgeon, A. Kolin, K. S. Kwak and S. J . Luner, Haematologia, 6 (1972) 93. 42 M. Ganser, K. Hannig, W. F. Kriismann, G. Pascher and G. Ruhenstroth-Bauer, Klin. Wochenschr., 46 (1968) 809. 43 H. G. Heidrich and M. E. Dew,J. Cell Biol., 74 (1977) 780. 44 K. Zeiller, G. Pascher and K. Hannig, Immunology, 31 (1974) 863. 45 A. Kolin, in P. G. Righetti, C. J. van Oss and J. W. Vanderhoff (Editors), Electrokinetic Separation Methods, Elsevier/North-Holland Biomedical Press, Amsterdam, New York, Oxford, 1979, p. 169.