U.S. patent number 3,663,395 [Application Number 04/885,941] was granted by the patent office on 1972-05-16 for cross-section illuminator for a continuous particle electrophoresis cell.
This patent grant is currently assigned to Beckman Instruments, Inc.. Invention is credited to Allen Strickler.
United States Patent |
3,663,395 |
Strickler |
May 16, 1972 |
CROSS-SECTION ILLUMINATOR FOR A CONTINUOUS PARTICLE ELECTROPHORESIS
CELL
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
broad beam which 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.
Inventors: |
Strickler; Allen (Fullerton,
CA) |
Assignee: |
Beckman Instruments, Inc.
(N/A)
|
Family
ID: |
25388039 |
Appl.
No.: |
04/885,941 |
Filed: |
December 17, 1969 |
Current U.S.
Class: |
204/645;
356/344 |
Current CPC
Class: |
G01N
27/44721 (20130101); G01N 21/45 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); G01N 21/41 (20060101); G01N
21/45 (20060101); B01k 005/00 () |
Field of
Search: |
;204/180,299 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Mack; John H.
Assistant Examiner: Prescott; A. C.
Claims
I claim:
1. In a continuous particle electrophoresis apparatus having a thin
curtain of liquid carrier medium which flows in an electrophoresis
space between a pair of spaced plates, the improvement
comprising:
means for illuminating a cross-section of said curtain transverse
to the curtain flow direction with a narrow blade of radiation;
and
means for observing said curtain cross-section.
2. In a continuous particle electrophoresis apparatus according to
claim 1, the improvement wherein said radiation is visible
light.
3. In a continuous particle electrophoresis apparatus according to
claim 2, the improvement wherein said means for observing said
curtain cross-section comprises:
a transparent window in one of said plates of said apparatus, the
other of said plates being transparent; and
a dark background positioned on the outside of said other plate
whereby said curtain cross-section may be viewed through said
transparent window against said dark background.
4. In a continuous particle electrophoresis apparatus according to
claim 1, the improvement wherein said radiation is in the
ultraviolet region of the spectrum.
5. In a continuous particle electrophoresis apparatus according to
claim 1, the improvement wherein said blade of light is thin
compared with the thickness of said curtain.
6. In a continuous particle electrophoresis apparatus according to
claim 1, the improvement wherein said means for illuminating a
cross-section of said curtain comprises:
a line source of light lying in a plane parallel to said curtain;
and
means for imaging said light source substantially in the plane of
said curtain.
7. In a continuous particle electrophoresis apparatus according to
claim 6, the improvement wherein the axial plane defined by said
line source and its image at said curtain is inclined to the plane
of said curtain, and wherein said means for observing said curtain
cross-section permits observation of said curtain cross-section at
an angle with respect to said optic axis.
8. In a continuous particle electrophoresis apparatus according to
claim 1, the improvement wherein said blade of radiation intersects
said curtain along a line perpendicular to the direction of said
flow, and has an angle of incidence on said curtain of less than
90.degree..
9. In a continuous particle electrophoresis apparatus according to
claim 1, the improvement wherein said means for illuminating a
cross-section of said curtain comprises:
first and second spaced,co-planar blades forming a straight-edged
slit therebetween;
means for illuminating said slit; and
means for focusing an image of said slit in the plane of said
curtain.
10. In a continuous particle electrophoresis apparatus having a
thin curtain of liquid carrier medium which flows in an
electrophoresis space between a pair of spaced plates, means for
illuminating a cross-section of the liquid curtain comprising:
a line source of visible light; and
means for imaging said line source in the plane of said curtain
perpendicular to the curtain flow direction.
11. In a continuous particle electrophoresis apparatus according to
claim 10, wherein said line source and said focusing means
illuminate said cross-section of said curtain with a blade of light
which is thin compared with the thickness of said curtain.
12. In a continuous particle electrophoresis apparatus having a
thin curtain of liquid carrier medium flowing in an electrophoresis
space between a pair of spaced plates, a method for observing the
performance of said apparatus comprising:
illuminating a cross-section of said curtain transverse to the
curtain flow direction with a narrow blade of radiation.
13. In a continuous particle electrophoresis apparatus according to
claim 12, the method wherein said radiation is visible light.
14. In a continuous particle electrophoresis apparatus according to
claim 13, the method further comprising:
viewing said illuminated cross-section against a dark
background.
15. In a continuous particle electrophoresis apparatus according to
claim 12, the method wherein said step of illuminating a
cross-section of said curtain comprises the step of:
illuminating a cross-section of said curtain with a blade of light
which is thin compared with the thickness of said curtain.
16. In a continuous particle electrophoresis apparatus according to
claim 12, the method wherein said step of illuminating a
cross-section of said curtain comprises the step of:
illuminating said cross-section at an angle which is inclined to
the plane of said curtain.
Description
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 mobility of the respective particles,
the strength of the field, etc.
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 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 function of such factors as the sample flow rate, the
difference between 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, no tool
has been available 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 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 art
from a reading of the following detailed description of 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
continuous particle electrophoresis cell showing the liquid and
particle velocity profiles in both the horizontal and vertical
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 of the
properties of the crescents;
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
illuminators of FIGS. 11 or 12.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present technique for curtain illumination for making 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 performance
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 5 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 K.sub.1 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 K.sub.2. 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 M.sub.2 K.sub.2 L.sub.2 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.sub.1, 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 5-10 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
perpendicular 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 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 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, 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 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 be abnormally broad.
Point 30 is the zero field position in FIG. 7.
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 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 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 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 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 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 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 S-shaped figure. With increase of sample flow rate, any of these
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 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 50 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 51. The crescent arms 52 and 53 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 part 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 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
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 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 observed 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 invention, 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 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 with
respect to the illumination direction. The optimum 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 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 ascertain to what extent band
broadening is due to sample flow rate, the 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
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.
* * * * *