Cross-section illuminator for a continuous particle electrophoresis cell

[57] ABSTRACT

In a continuous particle electrophoresis cell, the present invention comprises means for illuminating a section of the electrolyte curtain with a narrow blade of light rather than with a ^beam w^hich would flood the area of interest. Such blade of light illuminates the bands of sample particles only where they intersect the plane of light. By viewing the electrophoresis cell at a suitable angle, against a dark background, the cross-sectional pattern of the particle band distribution becomes fully visible. This provides a valuable, practical tool for gauging and optimizing instrument performance.

Claims, 15 Drawing Figures .204/299 .204/299

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Patented May 16, 1972

3,663,395 .2 Sheets-Sheet I

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ATTORNEY

Patented May 16, 1972

3,663,395

Sheets-Sheet 2

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3,663,395

CROSS-SECTION ILLUMINATOR FOR A CONTINUOUS PARTICLE ELECTROPHORESIS CELL

BACKGROUND OF THE INVENTION

1. Field of the Invention.

The present invention relates generally to continuous particle electrophoresis apparatus and, more particularly, to a method and means for illuminating a cross-section of the electrolyte curtain and sample particle bands.

2. Description of the Prior Art.

Electrophoresis, in general, is the phenomenon of migration of charged particles or ions in a liquid carrier medium under the influence of an electric field. This phenomenon can be used to fractionate small particles into separate bands dependent on the electrophoretic mobility or surface chemical properties of the particles.

In one form of continuous particle electrophoresis, a buffer solution or electrolyte is caused to flow freely as a thin film or curtain in an electrophoresis space between a pair of substantially flat plates of electrically insulating material mounted in substantially parallel, face-to-face relationship. The sample to be fractionated is injected continuously into the curtain in such a manner that it flows in a narrow band entrained within the electrolyte. An electric potential gradient is applied to the curtain at some angle to the flow, typically being perpendicular thereto. Such potential gradient causes the lateral separation of the sample particles into various particle groups or components, in the form of a steady-state band pattern,depending upon many factors including the electrophoretic mo tn ♦ r ,. - , ----^{l r}-------- '—**· '·*&lt;= bility of the respective particles, the strength of the field, etc. ³° °^{f followin}8 detailed description of the

Many factors, heretofore poorly understood, affect the performance of such continuous particle electrophoresis cells. These factors have limited instrument resolution as well as other aspects of performance. More specifically, it has now 35 been determined that due to the horizontal and vertical velocity profiles of the curtain, the cross-sections of the individual particle bands assume the form of crescents. The curvature^or lateral amplitude of these crescents varies as a funcbetween the particle band zeta potential and the cell wall zeta potential, the location of the sample injection tip, and the electrical conductivity of the sample, especially in relation to the conductivity of the curtain. The lateral amplitude of the crescents determines the resolution obtainable under given operating conditions. Also, variations in the forms of the crescents give valuable information on the zeta potential of the cell walls and on the nature of any instabilities which may be present. However, to date, these factors have been either . __________________ unrecognized or poorly understood. In addition, heretofore, ^{50 ofthe} properties of the crescents; ⁿ,° ^t°°,¹.^{has been ava}&gt;lable for examining the cross-sections of the individual particle bands.

SUMMARY OF THE INVENTION

In accordance with the present invention, there has been discovered a technique for curtain illumination which, for the first time, makes the cross-sections of the individual particle bands and the crescents located therein visible. With the present technique, one can now distinguish when an intrinsically broad band is present or whether a band which is apparently broad actually consists of one or more elongated crescents, which may be finely resolved and resting within each other. Further, the cause of crescent broadening may be identified, whether due to sample flow rate, the electrical conductivity of the sample, decentering of the sample injection tip, excessive difference between the zeta potentials of the particles and the walls, or inequality of the front and rear wall zeta potentials.

Briefly, the present technique of curtain illumination comprises means for illuminating a section of the curtain with a thin blade of light thereby illuminating only those particles in the particle bands which intersect the plane of the light. By viewing the cell at a suitable angle, against a dark background and seeing the light scattered from the illuminated particles^ the cross-sectional pattern of the particle distribution becomes fully visible. According to one embodiment of the present invention, a line source of light lying in a plane parallel to the curtain and preferably at right angles to the curtain flow direction is imaged by a lens in the plane of the curtain. According to a second embodiment of the present invention, the line source is replaced with a straight-edged slit which is illuminated in any desired manner and a lens which is used to focus an image of the slit in the plane of the curtain. In any event, the illuminated plane is viewed against a dark background whereupon the particle distribution becomes fully visible.

It is therefore an object of the present invention to provide a technique for improving performance in a continuous particle electrophoresis cell.

It is a further object of the present invention to provide a cross-section illuminator for a continuous particle electrophoresis cell.

It is a still further object of the present invention to provide means for illuminating the curtain in a continuous particle electrophoresis cell with a blade of light which is thin compared with the thickness of the curtain.

It is another object of the present invention to provide a 25 method and means for making the cross-sections of individual particle bands in a continuous particle electrophoresis cell visible.

Still other objects,features and attendant advantages of the present invention will become apparent to those skilled in the preferred embodiments constructed in accordance therewith, taken in conjunction with the accompanying drawings wherein like numerals designate like parts in the several figures and wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an element of the curtain in a - --------------------- _{a lu}„_v- continuous particle electrophoresis cell showing the liquid and tion ot such factors as the sample flow rate, the difference 40 P^artIcle velocity profiles in both the horizontal and vertical i,.u-------· . directions;

FIG. 2 is a schematic cross-sectional view of an electrophoresis curtain taken in a direction perpendicular to the direction of electrolyte flow, showing horizontal liquid circulation in the cell;

FIG. 3 is a view similar to that of FIG. 1, showing the crescent formation due to velocity profile effects;

FIGS. 4 a-4 c and 5-10 are schematic cross-sectional views of an electrophoresis curtain similar to FIG. 2, showing some nt the «Ρ.Ι._________ , ®

FIGS. 11 and 12 are schematic side elevation views of first and second embodiments of the present illuminator; and

FIG. 13 is a view taken in the direction of eye 69 in FIG. 11, showing the appearance of an electrophoresis cell using the il-&gt;·&gt; luminators of FIGS. 11 or 12.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present technique for curtain illumination for making 60 the cross-sections of the individual particle bands visible may be used with essentially all presently available continuous particle electrophoresis cells. For example, the present illuminator may be used with the apparatus described in my U S Pat No. 3,412,007 issued Nov. 19, 1968 for Continuous Flow Electrophoresis Apparatus. Alternatively, the present illuminator may be used with the system disclosed in co-pending application Ser. No. 630,980 of Victor H. Huebner, filed Apr 14, 1967, now U.S. Pat. No. 3,509,035 for Improved Continuous Particle Electrophoresis Cell and assigned to Beckman Instruments, Inc.,the assignee of the present application. Accordingly, only so much of such electrophoresis cells as is necessary for an understanding of the present invention will be described hereinafter.

In order to fully understand the manner in which the present cross-section illuminator may be used to optimize the per45

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3,663,395 formance of a continuous particle electrophoresis cell, it is first necessary to understand the nature of liquid and particle movement in such cells. Accordingly, and with reference now to the drawings and, more particularly, to FIG. 1 thereof, planes 1 and 2 represent, respectively, the front and rear faces, adjacent to the cell walls, of an element of the electrolyte curtain lying within the electric field. Planes 3 and 4 are arbitrary vertical sections of such a curtain element. Graph S shows the vertical velocity profile of the liquid assuming the electrolyte flows from top to bottom in FIG. 1. As shown in graph 5, the velocity is a maximum at the center of the cell and diminishes in parabolic fashion to zero at the cell walls. With a D.C. electric potential gradient applied across the curtain, the vertical velocity pattern is unchanged, but there is now established a pattern of horizontal liquid movement indicated as graph ABC. Such horizontal velocity pattern is also parabolic, but it has a finite velocity next to the walls, rather than zero. In the midplane 6 of the curtain, the movement is in a direction opposite to that at the walls. This is the pattern of electroosmotic flow in the cell, arising from the fact that the cell walls themselves are charged and have a definite zeta potential. Such velocity at the cell walls (lines DA or CG) is proportional to the wall zeta potential. If the wall is negative and the anode is at the right in FIG. 1, the liquid at the wall moves to the left.

The horizontal velocity pattern represented by graph ABC implies that there is horizontal circulation in the cell in the form of a closed double loop. This is shown in FIG. 2 by arrows 11. It has also been confirmed by experiment that there is negligible net horizontal flow through the cell. In other words, there is negligible mass transfer of liquid through the membranes at the edges of the cell. Therefore, area ADE in FIG. 1 plus area CFG (negative flow) equals area EBF (positive flow).

When a particle is introduced anywhere into the curtain, it will have two components of motion. Vertically, it will move at the velocity of the liquid, this depending on the point in the curtain in wich it is entrained (see curve 5 in FIG. 1). Horizontally, the particle will have a certain velocity relative to the liquid, which is generally proportional to its zeta potential and the field gradient, this velocity being the same at any depth in the curtain. However, the observed horizontal velocity will be the sum of this electrophoretic velocity and the velocity of the liquid itself. The observed horizontal particle velocity, therefore, varies with depth, and in a parabolic manner, as represented, for example, by the curve JHI in FIG. 1.

Referring now to FIG. 3, if a particle is introduced into the curtain at a point K in a plane 7, then with no electric field applied, it descends to a point Kj in a plane 8. With a D.C. electrical potential gradient applied to the curtain, a particle starting at point K is deflected to point K₂. The observed distance of the deflection, X, varies as the ratio of the observed horizontal to vertical velocity experienced by the particle. Since both these velocities vary with depth, particles injected at different depths, for example, at points L or M, are deflected by a different amount than particles injected at K. We therefore obtain a curved line such as M₂K₂L₂ for the deflected positions. If a stream of particles, N, is introduced in plane 7, such stream having a generally circular cross-section, the deflected band appears in plane 8 as a crescent, N„ in cross-section. This lateral spreading of the crescent is the main factor which inherently determines obtainable resolution in free-flow continuous particle electrophoresis cells.

With the present technique, to be described more fully hereinafter, these crescents have become visible, thereby revealing many important features of continuous flow electrophoresis behavior. Some of the properties of the crescents are illustrated in the materials which follow.

Each of FIGS. 4 a-4 c and S-10 represents a cross-sectional view taken through a typical electrophoresis cell having a pair of substantially flat plates 20 and 21 of electrically insulating material mounted in substantially parallel, face-to-face relationship. In each case, the sample to be fractionated is injected into an electrolyte which is flowing freely in laminar fashion as a thin curtain in a direction perpendicular to the plane of the drawings. An electric potential gradient is applied to the curtain in a direction parallel to walls 20 and 21 and perpendicu5 lar to the flow direction.

As shown in FIG. 4 a, if a sample is introduced at a low flow rate, the cross-section of the deflected band at a downstream point 23 is only slightly, if at all, distorted into a crescent shape. Dotted circle 22 represents the cross-section of the ⁰ band at the same downstream point in the absence of an applied electric field. At higher flow rates, the crescent is enlarged both in depth and in width as shown in FIGS. 4 b and 4 c . The increased lateral spreading is due to the introduction of ₁₅ some particles at increasingly greater distances from the midplane. At very high flow rates, as seen in FIG. 4 c, the crescent tips extend asymptotically toward walls 20 and 21 although as the crescent broadens, its crest or nose remains nearly fixed.

Referring now to FIG. 5, if several components are present, ₂₀ each having a different zeta potential, each band being electrophoretically homogenous, then each band forms its own crescent, shown as crescents 25,26 and 27, point 24 being the zero field position. Adjacent components which are close in zeta potential value may show nesting crescents as illustrated 25 ^by crescents 25 and 26. At times, a single band may cover a certain spread or distribution of zeta potentials. In this case, the band cross-section is, in effect,a series of compacted, unresolved crescents. Frequently, the result is a band with a cross-section of lenticular shape as shown at 28 in FIG. 6.

Referring now to FIG. 7, it has also been determined that a decentered sample injection tip may inject the sample too close to the back or the front wall 20 or 21, respectively. In this case, with a sample injected close to wall 20, for example, only one arm of the crescent may be seen at 31 and this may 35 be abnormally broad. Point 30 is the zero field position in FIG.

Referring now to FIG. 8, a sample of mixed individual components is introduced which covers a wide span of zeta potentials. Point 32 is the zero field position. One of the bands, at ⁴θ 33, is seen to be free of crescent effect. This typically results when such band has the same zeta potential as that of the walls. More exactly, this result occurs when the electrophoretic particle velocity is equal and opposite to the ₄₅ horizontal liquid velocity at the walls. Bands to the right, at 34, 35 and 36, which are of increasingly more negative zeta potential than the walls, form increasingly broader crescents. Bands to the left, at 37, 38 and 39, which are of increasingly less negative (or more positive) zeta potential than the walls, have ₅θ a reverse orientation. From this it may be concluded that if the zeta potential of the walls is subtracted from the zeta potential of the particles and the result is negative, the crescent is convex toward the cathode. If the result is positive, the crescent is convex toward the anode. As a result, it has been determined 55 that the particle zeta potential is a second factor, the sample flow rate being the first factor, which determines the crescent shape and width.

Referring now to FIG. 9, the effect of a difference between the two wall zeta potentials is shown for an injected sample 60 where point 40 is the zero field position. Assume that A is the position of a band with the same zeta potential as the front wall and that B is the band position when the zeta potential equals that of the back wall: Asymmetrical crescents are seen at 41-46 in regions outside of the area between positions A 65 and B. The arms of such crescents are unequal and their crests are displaced from the midplane. On the other hand, bands at equal potential positions A and B are curved segments showing no crests. A band between positions A and B shows an Sshaped figure. With increase of sample flow rate, any of these 70 cross-sections will be extended as shown by the dotted lines.

It has been seen that the amount of spread of a band depends, in part, on how far the particles extend from the midplane toward the walls. A primary factor, therefore, in crescent spreading is the sample flow rate. However, certain other effects may occur to move particles further toward the

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3,663,395 walls to further extend the crescents. The electrical conductivity of the sample, especially in relation to curtain conductivity, is one of these effects. The conductivity of a sample has two components. One of these is conductivity due to the ionic content of the medium; the other is due to surface conductance of the particles which arises mainly from the mobility of ions in the ionic counter-layer which surrounds the charged particles. The surface conductance increases with the particle zeta potential and the total particle surface area in a unit of sample volume. It is therefore greater for small particles and increases in proportion to particle concentration. If the total sample conductivity is appreciably greater than that of the curtain, in-depth spreading of the sample may occur. This effect starts to act on the sample as soon as it enters the electric field, causing a portion of the particles to move toward the walls as shown by dotted lines SO in FIG. 10. If the predisposing conditions are severe, spreading toward the walls may occur in a variable, unstable manner. The band may then blossom into artifacts as shown at SI. The crescent arms 52 and S3 become greatly extended and closely approach the walls. There is also uneven lumping or concentration of particles at different positions along the crescent curve as shown at 54 and 55. Under normal lighting, this banding within the crescent often looks like separate bands, but being actually a ₂₅part of a single crescent, they are, in fact, a single component only. In addition, these false bands may constantly change in position although at times rather slowly. Bands may fade and others appear, although the peak position of the crescent usually remains fixed. If the sample injection tip is badly 30 decentered or the zeta potential of the two walls is quite different, the artifact bands may extend along one wall only. On the other hand, if a given band has a zeta potential equal to or near that of the walls, then it has almost no tendency to spread laterally or form artifacts even at relatively high sample flow 35 rates.

Accordingly, it should now be apparent that there are many factors which affect the overall performance of a free-flow continuous particle electrophoresis cell. By way of summary, the performance of the instrument and, most importantly, its 40 resolution, is affected by the sample flow rate, the relationship between particle and wall zeta potentials, the location of the sample injection tip, and the electrical conductivity of the sample, especially in relation to curtain conductivity. Unfortunately, however, none of these effects could previously be ob- 45 served directly, and it was therefore not possible to apply effective techniques for compensating for the effects to increase instrument performance.

According to the present invention, there is provided means for illuminating a section of the electrolyte curtain with a narrow blade of light which illuminates the bands of sample particles only where they intersect the plane of the light. By viewing the electrophoresis cell at a suitable angle, and seeing the light scattered from the illuminated particles, the cross-sectional pattern of the particle band distribution becomes fully visible.

Referring now to FIG. 11, there is shown a side sectional view of a portion of an electrophoresis cell 60 including a front wall 61 and a rear wall 62 which are mounted in substantially parallel, face-to-face relationship to form an electrophoresis space 63 in which the electrolyte curtain flows downwardly as seen in FIG. 11. Positioned in wall 61 is an aperture 64 having a viewing window 65 for viewing the particle bands. According to a first embodiment of the present in- 65 vention, the illuminator consists of a line source of light 66, such as a straight incandescent filament, lying in a plane parallel to the curtain and preferably at right angles to the curtain flow direction. The source of light is imaged by a lens 67 in the plane of the curtain. Lens 67 may be radially symmetrical, for 70 example with spherical surfaces, or may be cylindrical, with the cylinder axis parallel to line source 66. The optic axis of the illuminator is preferably inclined to the plane of the curtain, as shown, and the curtain is viewed by the eye 69 at some angle may vary somewhat with the size of the particles and their optical properties. An aperture stop 68 is usually added and adjusted for the best compromise between illumination intensity and narrowness of the wedge or blade of light which intersects the curtain. In any event, the maximum thickness of the light wedge within the curtain is preferably made small compared with the curtain thickness.

Aperture stop 68 may be an iris diaphragm for the spherical j lens case or may be a pair of opaque blades 70 and 71, with their edges parallel to line source 66 in the cylindrical lens case. The parallel blade aperture could also be used with the spherical lens. By viewing cell 60 against a dark background 72 and seeing the light scattered from the illuminated particles, the cross-sectional pattern of the particle distribution becomes fully visible.

Referring now to FIG. 12, and in accordance with a second embodiment of the present invention, the line source may be replaced with a straight-edged slit 73, the latter being illuminated in any desired, efficient manner. For example, and as shown in FIG. 12, a source of light 74, which may again be a line source, may have its radiation focused or condensed by a lens 75, which is either spherical or cylindrical, onto slit 73. The only requirement is that the full width of the slit be illuminated. A second lens 67 focuses an image of the slit in the curtain plane. An aperture stop 68 in the system would again usually be used. In addition, a reflector 76 behind source 74 may be used to improve illumination.

As stated previously, the background 72 against which the cross-sectional pattern is viewed should be as dark as possible. In addition, the illuminating radiation may be other than visible light, e.g. ultraviolet, with detection, for example, by electronic or photographic means. Also re-emission of light from the particles may be by fluorescence rather than physical scattering. Finally, the cell surfaces adjacent to the illuminated part of the curtain may be lightly coated with a light-scattering film. As shown in FIG. 13, this produces two horizontal lines of illumination, the upper one 80 being formed where the beam intersects the front face of the back plate and the lower one 81 being formed where the beam intersects the rear surface of the front cell plate at the cell windows. This enhances the ability to see the location and size of the crescents with respect to the wall planes.

It can therefore be seen that in accordance with the present invention, there is disclosed a technique for curtain illumination in a continuous particle electrophoresis cell which, for the first time, makes the cross-sections of the individual particle bands and the crescents located therein visible. With the present technique, one can now distinguish intrinsically broad bands from bands which are only apparently broad and which may actually consist of a single homogeneous crescentic band or several closely spaced, nested crescents. One may also 5 5 ascertain to what extent band broadening is due to sample flow rate, die electrical conductivity of the sample, especially in relation to curtain conductivity, the decentering of a sample injection tip, or wall zeta potential effects.

While the invention has been described with respect to the 60 preferred physical embodiments constructed in accordance therewith, it will be apparent to those skilled in the art that various modifications and improvements may be made without departing from the scope and spirit of the invention.