U.S. patent number 3,773,645 [Application Number 05/198,130] was granted by the patent office on 1973-11-20 for electrophoresis device.
This patent grant is currently assigned to Colora Messtechnik GmbH. Invention is credited to Stephan Hubertus Anton Nees, Wilhelm Schmidt.
United States Patent |
3,773,645 |
Nees , et al. |
November 20, 1973 |
ELECTROPHORESIS DEVICE
Abstract
An electrophoresis device having a separating chamber, and two
buffer containers one of which is attached to one end of said
separating chamber and the other one is connected to the other end,
and an electrode in each in said buffer containers, said device
essentially having a box-shaped housing and a box area which is
divided up into two partial areas serving as buffer containers by
means of an essentially horizontal intermediate bottom of lesser
depth than the box together with an intermediate wall which extends
essentially vertically from the intermediate bottom.
Inventors: |
Nees; Stephan Hubertus Anton
(Erlangen, DT), Schmidt; Wilhelm (Erlangen,
DT) |
Assignee: |
Colora Messtechnik GmbH
(Lorch/Wurtt., DT)
|
Family
ID: |
27182987 |
Appl.
No.: |
05/198,130 |
Filed: |
November 12, 1971 |
Foreign Application Priority Data
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|
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|
|
Nov 14, 1970 [DT] |
|
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P 20 56 129.9 |
Nov 14, 1970 [DT] |
|
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P 20 56 128.8 |
Nov 14, 1970 [DT] |
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P 20 56 127.7 |
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Current U.S.
Class: |
204/606;
204/610 |
Current CPC
Class: |
G01N
27/44704 (20130101); G01N 27/44756 (20130101); B01D
57/02 (20130101) |
Current International
Class: |
B01D
57/02 (20060101); G01N 27/447 (20060101); B01k
005/00 () |
Field of
Search: |
;204/299,18G,18R,301 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Mack; John H.
Assistant Examiner: Prescott; A. C.
Claims
What is claimed is:
1. A gel-electrophoresis device comprising a vertical separating
chamber, the cross section of which is elongated and has a
substantially rectangular shape, two buffer containers, one being
in communication with the upper end of said separating chamber,
elution means interconnected between the lower end of said
separating chamber and the other buffer container, said elution
means comprising an elongated elution chamber, means at each of the
opposed ends of said elongated chamber for supplying and removing
an elution fluid to and from said elution chamber, an electrode in
each of said buffer containers, and means for connecting the
electrodes to a source of direct current.
2. A device as claimed in claim 1 wherein said elution means
further comprises a second elongated chamber beneath said elongated
elution chamber for receiving a concentrated buffer fluid.
3. A device as claimed in claim 1 wherein the lower wall of said
elongated elution chamber is composed of semipermeable membrane
material.
4. A device as claimed in claim 2 wherein said second elongated
chamber has an upper and a lower wall each composed of
semipermeable membrane material, said lower wall separating said
second chamber from said other buffer container and said upper wall
constituting the lower wall of said elution chamber.
5. A device as claimed in claim 2 and further comprising means at
each of the opposed ends of said second elongated chamber for
supplying and removing the concentrated buffer fluid.
6. A device as claimed in claim 1 wherein said elution means
comprises a plate having a recess therein which constitutes said
elongated elution chamber, the shape of said recess conforming with
the shape of the lower end of said separating chamber and means for
tightly securing the upper side of said plate to the lower end of
said separating chamber.
7. A device as claimed in claim 6 wherein said recess extends
through said plate and further comprising means for closing the
lower side of said recess.
8. A device as claimed in claim 6 wherein said elution means
further includes a second plate beneath said first mentioned plate
and having a recess therein for receiving a concentrated buffer
fluid and a semipermeable membrane firmly gripped between said
first and second plates and constituting the lower wall of said
elongated elution chamber.
9. A device as claimed in claim 8 wherein the recess of said second
mentioned plate corresponds in shape to the recess of said first
plate.
10. A device as claimed in claim 8 wherein said elution means
further includes a third plate and a second semipermeable membrane
gripped between said third plate and said second plate to form the
lower wall of the recess in said second plate.
11. A device as claimed in claim 10 wherein the recess of said
third plate corresponds in shape to the recess in said first
plate.
12. A device as claimed in claim 6 wherein the means for supplying
and removing an elution fluid comprises channels leading to and
from the opposed ends of the recess in said plate.
13. A device as claimed in claim 8 and further comprising channel
means in said second plate for supplying and removing the
concentrated buffer fluid to and from the opposed ends of the
recess in said second plate.
14. A device as claimed in claim 10 wherein said first, second and
third plates are superposed with semipermeable membranes disposed
therebetween, said recesses being in register and conforming with
the shape of the lower end of said separating chamber and further
comprising means for pressing the superposed plates together and
for pressing the assembled plates against the bottom of said
separating chamber.
15. A device as claimed in claim 1 and further comprising baffle
rods disposed within said elongated elution chamber, arranged
alternately on opposite sides thereof and extending diagonally to
the direction of flow of the elution fluid passing
therethrough.
16. A device as claimed in claim 1 and further comprising vertical
walls defining said separating chamber, said walls being provided
with channels for supplying and removing the elution fluid to and
from said elongated elution chamber.
17. A device as claimed in claim 1 and further comprising
adjustable means within the other buffer container for supporting
said elution means and pressing it against the lower end of said
separating chamber.
18. A gel electrophoresis device comprising two laterally spaced
parallel, vertical walls forming a vertical separating chamber
therebetween, two buffer containers on opposed sides of said
separating chamber, one being only in communication with the upper
end of said separating chamber and the other only in communication
with the lower end of said separating chamber, whereby each
container may be easily filled with buffer fluids to the same level
in each container, an electrode in each of said containers and
means for connecting said electrodes to a source of direct
current.
19. A device as claimed in cliam 18 wherein each of the laterally
spaced walls form one wall of each of the buffer containers.
20. A device as claimed in claim 18, wherein one of said laterally
spaced walls extends upwardly to a higher point than the other of
said walls.
21. A device as claimed in claim 18 and further comprising a lid
member disposed above said separating chamber, and wherein said
laterally spaced walls extend upwardly to the same level, said lid
member forming a fluid tight seal with one of said laterally spaced
walls.
22. A device as claimed in claim 18 and further comprising a
substantially rectangular box-like housing having a bottom and side
elements and including an intermediate bottom element above the
bottom of said housing and extending partially but not less than
one half of the distance across said housing, the bottom of one of
said laterally spaced walls being connected to the inner end of
said intermediate bottom, whereby the intermediate bottom element
and said one laterally spaced wall together with side portions of
said housing form one of said buffer containers, the other of said
buffer containers being defined by the other of said laterally
spaced walls, the bottom and side elements of said housing, and the
intermediate bottom element.
23. A device as claimed in claim 22 wherein the space between said
laterally spaced walls is adjustable.
24. A device as claimed in claim 22 wherein a portion of said other
buffer container is disposed beneath the lower end of said
separating chamber.
25. A device as claimed in claim 22 and further comprising a
detachable lid for said housing and wherein the side element of
said housing spaced from said intermediate bottom element is
detachable.
26. A device as claimed in claim 22 wherein the one laterally
spaced wall forming a part of said one buffer container is provided
with a window therethrough, and further comprising a cover plate
for covering said window on that side facing said separating
chamber and means for securing said cover plate over said window in
said one spaced wall, said means also securing said other spaced
wall in spaced relation to said cover plate to form said separating
chamber.
27. A device as claimed in claim 26 and further comprising packing
means between said cover plate and said other laterally spaced
wall.
28. A device as claimed in claim 27 wherein said packing means
comprises at least two groups of vertically extending parallel
members, said groups being spaced apart to divide said separating
chamber into at least two parallel, vertical chambers, said packing
means being composed of the same material as the material facing
said separating chamber.
29. A device as claimed in claim 26 and further comprising a lid
member disposed above said separating chamber, said lid member
forming a fluid tight seal with the top of said other-spaced wall
to prevent communication between said other buffer container and
said one buffer container.
30. A device as claimed in claim 26 wherein said securing means
comprises bolts extending outwardly from said one laterally spaced
wall and through said cover plate and said other laterally spaced
wall and nuts for securing the cover plate and spaced walls
together.
31. A device as claimed in claim 22 and further comprising a recess
on the inside of one wall of each of said two buffer containers for
receiving each of said electrodes.
32. A device as claimed in claim 18 wherein a wall of the other
buffer container in communication with the lower end of said
separation chamber is provided with a recess for receiving one of
said electrodes.
33. A device as claimed in claim 32 wherein the wall of the one
buffer container in communication with the upper end of said
separation chamber is provided with a recess for receiving the
other of said electrodes.
34. A device as claimed in claim 31 wherein each of said recesses
in the walls is provided with an upwardly inclined upper surface
and said walls are provided with channels extending from the
uppermost part of said recesses to the top of said walls for
removing gas bubbles formed at said electrodes.
35. A device as claimed in claim and further comprising detachable
cooling means disposed in each of said buffer containers adjacent
said laterally spaced walls for cooling said separation
chamber.
36. A device as claimed in claim 35 wherein said cooling means
comprises cooling plates arranged parallel to said laterally spaced
walls.
Description
BACKGROUND OF THE INVENTION
This invention relates to an electrophoresis device with two buffer
working tanks one of which is connected to one end of a separating
chamber and the other one of which is connected to the other end of
a separating chamber, and with one electrode each in the buffer
tanks.
Prior art electrophoresis devices are built in such a way that a
vertical separating chamber follows a first box-shaped container
for the buffer solution. A second buffer container, which can also
be box-shaped, is located on the vertical separating chamber. The
production of these components, which are preferably circular in
shape, is expensive. Furthermore, because of the upper buffer tank,
the overall design of the device os very unstable so that the
danger of damage is quite great. In these prior art devices good
accessibility to the upper and lower chamber limitation, which is
necessary in various electrophoresis methods, is not present.
Also prior art preparative electrophoresis devices are essentially
constructed of axisymmetric parts they consequently have a
cylindrically shaped separating chamber, bowl-shaped buffer
containers and an elution chamber which is also circularly shaped
in the cross section. The parts of the prior art devices are
balanced to one another in such a way that preparative
electrophoreses can be carried out. For the carrying out of
analytical tasks, however, these devices are not suitable.
The yields of known preparative electrophoresis devices are also
relatively low even in more modern devices. Also the selectivity
which is achieved with known devices is still not optimum so that
substances with practically the same electrophoretic rate of travel
cannot be securely separated.
OBJECTS OF THE INVENTION
One object of this invention to create an electrophoresis device in
which these prior art disadvantages are not present, in which
therefore a compact form of construction is available, simple parts
can be used, the danger of damage from a low degree of stability
and difficult construction is excluded and a good accessibility of
the upper and lower separating chambers is provided for. It is a
further object of this invention to provide a simple and efficient
cooling of the separating chambers, as well as a hydrostatic
pressure equalization in the two buffer containers without special
means, such as lifting pipes or similar, which are required for
this purpose.
It is a still further object of this invention to eliminate the
disadvantages of known preparative electrophoresis devices so that
a device is created with which higher yields and greater
selectivities can be achieved.
A still further object of the invention is to provide a device
which makes it possible, without problems, to carry out not only
preparative electrophoreses but also analytical
electrophoreses.
A yet still further object of this invention is to permit an
operation according to the most varied known electrophoresis
methods so that the range of application of the proposed device is
expanded.
SUMMARY OF THE INVENTION
The objects of this invention are achieved by means of an
essentially box-shaped design of the electrophoresis device. The
device of this invention is provided with a box-shaped area by
means of an essentially horizontal intermediate bottom which is of
smaller depth than the box, can be split up together with an
intermediate wall which goes out from the intermediate bottom and
extends essentially vertically to it into two partial areas serving
as a buffer container. It is particularly advantageous to have the
intermediate wall extending vertically upwards into the box because
when both partial areas are open upwardly ithe buffer fluids can be
brought practically to the same level whereby the hydrostatic
pressure equalization is brought about in the simplest manner. The
design according to this invention permits an arrangement of the
separating chamber essentially in the plane of the intermediate
wall. The thickness of the separating chamber may be varied in a
particularly simple manner when parallel to the intermediate wall
and in the region of the greater angle formed by the intermediate
bottom and the intermediate wall. A plate can be applied which can
be adjustable in its distance and which, together with the
intermediate wall, forms the separating chamber.
The accessibility to the buffer containers and the separating
chamber is particularly simplified in that the front side wall of
the box-shaped housing and the lid of the housing are removable.
According to a further embodiment of this invention the device can
be used for the application of the most varied electrophoresis
methods, for example the continuous electrophoresis method, the
discontinuous electrophoresis method, the isoelectric focusing and
the iso-tachoelectrophoresis when means for pressing in inserts
(such as elution insert, isoelectric focusing insert, and the like)
in the partial space located below the separating chamber to the
lower limitation of the separating chamber. These pressing means
are achieved preferably by means of a pressing device which
operates in the manner of a lifting stand.
This invention has a further advantage by permitting the cooling
means which are provided for cooling the separating chamber to
engage the buffer solutions in both buffer containers. It then
suffices for the buffer solutions to be cooled in the region of the
separating chamber since the buffer solutions themselves can act as
a cooling medium. Within the framework of the invention it is
advantageous to arrange a laminar cooling plate or hose parallel to
the separating chamber walls in order to be able to achieve in this
way the greatest possible cooling area.
For this purpose the invention is based on the fact that with the
known construction principle of preparative electrophoresis devices
neither the selectivity nor the yields can be increased to any
considerable degree. This is due to the fact that in the
amplification of the separating chamber diameter for increasing the
yields the cooling of the carrier substance becomes problematical
and also the elution of greater circular-shaped or circular-ring
shaped cross sections can no longer occur in a satisfactory manner.
Inadequate cooling of the carrier substance in the separating
chamber results namely in the fact that a temperature gradient
occurs between the inside, the center of the cylindrical separating
chamber and its cooled outside wall.
Since the rate of travel in the separating chamber depends upon the
temperature and the flushing occurs with different flow velocities
it is not possible to increase the quality of the electrophoresis
devices when maintaining the known principal design.
According to this invention the separating chamber has an essential
rectangular cross section and the elution chamber is attachable to
one end of the separating chamber. By means of the steps according
to this invention it becomes possible to cool large separating
chamber volumes effectively owing to their essentially greater
surfaces and also to carry out the flushing on the elution side of
the separating chamber with uniform flow velocities. With this,
however, the preconditions for a considerable improvement in
quality of the known preparative electrophoresis devices are given.
The rectangular separating chamber construction can be considerably
simplified and made more economical in comparison to most of the
blown cylindrical separating chambers by means of the utilization
of simple glass plates. The rectangular separating chamber design
also permits a compact design of the entire device including the
buffer container. In addition, by removing the elution chamber the
device can easily be used immediately for analytical
electrophoresis. An interchanging of the elution chamber against
other attachments to be placed on the underside of the separating
chamber makes it possible to operate according to various
electrophoresis methods and simplifies the work in that, for
example, by means of an attachment formed as a fastener, the
separating chamber can be filled with carrier substance without
having to be removed from the device and the electrophoresis can
immediately be started in the device after the polymerization and
the removal of the fastener attachment without the disturbing
transport of the carrier substance and without any difficulties of
installation into the device.
Within the framework of this invention it is appropriate to
construct the elution chamber in such a way that its form
essentially corresponds to the lower separating chamber opening,
i.e., so that the elution chamber is rectangular. It is
advantageous to construct the elution chamber out of a plate which
has a recess which corresponds to the lower separating chamber
opening so that the lower separating chamber limitation should be
tightly attachable to the upper side of the plate over the recess.
When the recess corresponds to the diameter of the plate, the plate
is slit, and means are provided for closing the lower side of the
slit opposite the separating chamber. Connections for the feeding
and removal of the elution fluid are provided in the region of the
small sides of the recess or of the slit. This procedure permits
uniform flow velocity over the entire cross section of the chamber,
therefore uniform washing out of the electrophoretically separated
substances and consequently a high degree of selectivity on the
part of the device. The feeding and removal of the elution fluid on
the narrow sides of the rectangular elution chamber also causes a
high concentration of the eluted substance.
The slit may be provided with a semipermeable membrane as a means
for closing the separating chamber of the plate. This membrane is
preferably squeezed, by means of a second plate, between the second
plate and the first plate. In this manner the second plate can have
a corresponding recess as in the first plate. Preferably, in the
region of the narrow sides of the recess of the second plate,
attachments are provided for the feeding and removal of a buffer
fluid hwich is particularly highly concentrated within the
framework of the invention. The recess in the second plate can also
be formed as a slit, whereby the slit on the side opposite that of
the elution chamber is closed by means of a second membrane. In a
similar manner the second membrane is also squeezed, by means of a
third plate which is also slit, between that plate and the second
plate. There is thus obtained a plate stack composed of three
plates with a slit which passes through and which corresponds in
size to the separating chamber outlet, whereby a continuous
semipermeable membrane is located betwen the plates. Such a plate
stack is produced in the simplest manner and combined with the
elution chamber according to the invention and also inserted into
the device. For this purpose, within the framework of the
invention, means are provided for the compact pressing together of
the stack and for the tight pressing of the stack against the
bottom side of the separating chamber.
In the framework of the device, the elution chamber and the means
for bearing pressure upon the elution chamber are preferably
provided for in the lower located region serving as the buffer
container in the case of a compact box-shaped construction of the
electrophoresis devices in which, by means of an intermediate
bottom and an intermediate wall, the housing is divided up into two
areas forming the buffer container between which regions the
separating chamber is located. The means for pressing against the
elution chamber has a lifting stand which is preferably formed of
threaded bolts. These bolts are located on the bottom of the
lower-situated region, lie vertically and the stand plate can move
to and fro on these bolts by means of threaded nuts. In this
manner, the elution chamber can be pressed against the lower side
of the separating chamber.
It is particularly advantageous, for the cooling of the separating
chamber which is rectangular in cross section, to arrange the
cooling agents parallel to the large sides of the separating
chamber which can be composed, for example, of a cooling house
proceeding in a plane with immersion frames.
In the case of the prior art electrophoresis devices, in the upper
and lower buffer container, locking elements are provided for a
frame in which an electrode is housed. These frames are placed
immediately above and below the separating chamber. The placement
below the separating chamber has the disadvantage that gas bubbles
rising up on the electrode reach the lower side of the separating
chamber and in this way cause a disruption in the electrophoresis.
In order to eliminate this disadvantage it is necessary to provide,
between the lower side of the separating chamber and the electrode
in the buffer container, means which prevent the gas bubbles from
reaching the lower side of the separating chamber. This is achieved
by holding in another frame a deflecting means such as an
absorptive paper which is shaped into an acute-angled groove in
such a manner that the point points downwards and the upper edges
of the groove lie laterally outside of the lower limit of the
separating chamber. Gas bubbles rising up are pushed laterally
upwards from the lower side of the groove and cannot reach the
separating chamber.
It can be seen that in this kind of construction the structure and
assembly of the device are complicated and that there is the danger
that the absorptive paper is mistakenly not inserted and that
impurities can reach the buffer solution through the paper.
In accordance with a further embodiment of this invention at least
the buffer container, which is in contact with the lower end of the
separating chamber, has a recess in the inner container wall,
preferably the container backwall, for housing the electrode.
This feature prevents the rising gas bubbles from reaching the
bottom side of the separating chamber. There is furthermore
achieved that no special frame and no special holding means in the
buffer container are needed for the electrode so that the
construction and erection of this part of the electrophoresis
device are considerably simplified.
Prior art devices also have a considerable disadvantage in that the
separating chamber is not easily accessible from above when the
upper electrode attachment is placed in the buffer container. The
subsequent insertion of the electrode attachment into the buffer
container is disadvantageous, however, since the buffer fluid is
placed into motion through the insertion and thus the fluid
substances which are piled up on the uppper side of the separating
chamber can easily be mixed. Bearing this situation in mind, this
invention makes it possible for the accessibility to the upper
limit of the separating chamber to be unhampered and also makes it
possible to do away with a subsequent insertion of the electrodes
into the buffer 0containers.
This is achieved by also providing a recess for the electrode in
the buffer container which comes into contact with the upper end of
the separating chamber. This thereby provides the advantage of good
accessibility and the elimination of the necessity of having to
insert the upper electrode subsequently.
Examples of construction of the invention are described in what
follows based on the drawing.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a side view of the electrophoresis device of this
invention in which the side wall has been removed:
FIG. 2 shows an alternate form of a separating chamber wall for use
with this invention;
FIG. 3 shows the assembly of a separating chamber for use with this
invention;
FIG. 4 shows a special design of a separating chamber part for use
in this invention;
FIG. 5 shows the elution attachment of the device of this
invention;
FIG. 6 shows a special form of an elution chamber part for use with
this invention;
FIG. 7 is a view of the inside of the backwall of the
electrophoresis device of this invention;
FIG. 8 is a cross section view through the backwall of the device
according to FIG. 7, and
FIG. 9 is a cross section view through an addition for working
according to the method of iso-electric focusing.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In FIG. 1 there is shown an electrophoreses device having a bottom
1, lid 2, backwall 3 and front side wall 4 of a box-shaped housing
5 with a side wall removed. Inside of the housing 5 an intermediate
bottom 6 has been inserted into about one-third of the overall
height of the housing. It extends beyond half of the depth of the
fronting to the front side wall 4. An intermediate wall 8 extends
vertically upwards from its free end 7. The intermediate bottom 6
and the intermediate wall 8 provide the boundary, together with the
backwall 3 and the side wall, not shown, of the housing 5, for a
buffer container 9, and bound it tightly against the remaining area
which forms a second buffer container 10. Parallel to the
intermediate wall 8 in the direction of the front side wall 4 of
the housing 5 with a distance, there is provided a plate 11 which
forms the separating chamber 12 of the electrophoresis device
together with the intermediate wall 8. The plate 11 is somewhat
higher than the intermediate wall 8 so that the
hatched-line-indicated buffer solution in the buffer container 9
can reach the upper end of the separating chamber. The lower end of
the separating chamber 12 is surrounded by the fluid which is
located in the buffer container 10. An electrode 13 or 14 is
provided in the backwall 3 in the region of the two buffer
containers 9 and 10.
By means of the described construction of the device, in which
according to the invention the two buffer containers 9 and 10 are
located essentially laterally, preferably on both sides of the
separating chamber 12, a pressed construction form is given, only
simple parts are used, and there is no danger that the device will
be exposed to the risk of damage because of inadequate stability or
complicated construction. When the lid 2 is removed, the device is
easily accessible from above so that the extra shifting or layering
of substances, which is often to be undertaken with great
sensitivity, is easily possible. Also the introduction of the
buffer solution from above into both buffer containers is easily
possible. Complicated and expensive pressure equalization devices
are therefore not necewsary.
The front side wall 4 is detachable from the two side walls. the
detachability of the front side wall provides good access to the
lower end of the separating chamber 12 so that it can be filled
with carrier substance without having to be removed from the
device, when the easily accessibly lower separating chamber opening
is closed. The preparation of the carrier substance can therefore
take place inside of the device, whereas in the case of preivously
known devices this had to be done outside of the device. The loaded
separating chamber can also remain in the device during the time of
the polymerization of the carrier substance.
The described construction also makes it possible to use the buffer
fluid in the containers 9 and 10 as a cooling agent, in which a
cooling device, for example cooling plates or cooling hoses 15, 16
are placed parallel to the surfaces 8 and 11 limiting the
separating chamber 12 at a distance from the side walls of the
housing. This arrangement is particularly simple in the utilization
of an immersion frame which can be introduced into the device from
above.
Advantageously, the intermediate wall 8 forms at least partially a
part of the separating chamber 12. Thus the erection of the device
becomes particularly easy.
The plate 11, which is placed parallel to the intermediate wall 8
and in the region of the larger angle which is formed by the
intermediate bottom 6 and the intermediate wall 8, and which,
together with the intermediate wall, forms the separating chamber
12, is adjustable with respect to the intermediate wall in which
corresponding means, such as studs, are provided on the
intermediate wall and return-leading elements, such as boreholes,
which are suitable for this are provided on the plate. These have
not been represented for purposes of simplicity. In this manner
there is achieved in the simplest manner the variation of thickness
of the carrier substance in the separating chamber 12 according to
the particular requirements.
The electrodes 13 and 14 which are provided on the inside housing
backwall 3, preferably in a recess for each one, are provided with
connecting elements (not shown) which project through the backwall
of the housing. As will be described in greater detail later on,
the recesses in which the electrodes are placed are connected to
the outside air preferably by means of a canal which runs upwards
in the housing backwall so that air bubbles rising on the electrode
can escape. The removability of the lid 2 and the front sidewall 4
of the housing 5 is solved by any known manner. The tightness of
the removable parts with respect to the rest of the housing is also
achieved by any known means.
The described arrangement makes it possible to provide means for
pressing attachments onto the lower limit of the separating chamber
in the partial space located below the separating chamber 12. These
means can be used to close the bottom side of the separating
chamber by means of a special attachment in order to make possible
the already-described introduction of the carrier substance inside
of the device. Preferably, the pressing means are shaped in the
manner of a lifting stand 17 which can be easily activated from the
front with the front side wall 4 removed. A motorized height
adjustability of the lifting stand is possible, but it is hardly
necessary in practice since the manual activation, for example, by
means of one or more threaded spindles 18 and spindle nuts 19 can
be carried out with few movements of the hand.
The cooling of the separating chamber 12 is particularly easy and
efficient, in the case of this invention, in comparison to the
already known preparative electrophoresis devices which operate
basically with cylinder-shaped separating chambers. Because of the
large side surfaces of the separating chamber which is rectangular
in shape in the cross section, provision is made for a good heat
exchange and it is achieved in that no significant temperature
gradient occurs in the separating chamber. It is thus provided that
inside of the separating chamber no temperature-caused differences
in rate of travel occur so that the individual bands do not undergo
any distortions. It thus also becomes possible to cleanly separate
from one another even the bands which are located close to one
another.
Within the framework of the invention another form of the
intermediate wall 8 and of the separating chamber 12 is also
possible. This possibility is described in further detail based on
FIG. 2.
The intermediate wall 8 is provided with a window 20, which is
closed or closeable from the front side 4 of the box-shaped housing
5 through the attachment of a plate 21 and which, together with a
plate 22 which corresponds to the plate 11, forms the separating
chamber. Between the plate 21 which closes the window 20 and the
window frame (intermediate wall 8), packing means are provided
(shown by dashes in FIG. 2) so that no buffer fluid can enter from
the container 9 into the remaining area of the housing 5.
Preferably the side walls of the housing 5 are not used as side
limit of the separating chamber 12, but between the two plates 21
and 22 which form the separating chamber there are provided
laterally limiting packing means which may be composed of glass
rods 24. The glass rods 24 are quadrangular or rectangular shaped
in the cross section (see FIG. 4) and carry compressible packing
means 25 running in corresponding nuts on their two sides opposite
the two plates 21 and 22. When the two plates, which are also
preferably made of glass, are pressed together, then the separating
chamber 12 is practically of the same material on all of its
limiting walls, so that, for example, expansion phenomena or other
disturbances do not cause the carrier substance to detach from the
chamber walls and creeping currents to occur which could disrupt
the electrophoresis. As already shown in FIG. 1, the plate 8 or 12
which closes the buffer container 9 towards the front is arranged
in such a way that a distance is present between its upper edge and
the housing lid 2, whereas the second plate 11 or 22 is closer to
the housing lid 2 than the upper edge of the previously mentioned
plate 8 or 21. This thus causes the buffer fluid in the buffer
container 9 to only be able to reach the upper end of the
separating chamber 12 but not the other buffer container 10.
It is also possible within the scope of this invention to make the
two plates which form the separating chamber 12 of the same height
and to see to it that the buffer container 9, limited by the
housing backwall 3 and the plate 21 which closes the window 20,
together with the upper opening of the separating chamber 12 is
made tight in comparison with the other buffer container 10 through
the housing lid 2. Naturally, instead of the housing lid an
intermediate lid can also be used.
It can be seen from FIG. 3 that on the window frames (intermediate
wall 8) there are provided threaded bolts 26 projecting towards the
front side 4 of the housing 5 and onto which there are placed nuts
27. When these bolts are fastened securely the outside plate 22 is
pressed against the packing means 24, 25 so that the plate 21 which
closes the window 20 is also pressed against the window frame.
There thus occurs a tight separating chamber in the housing 5
having a thickness which corresponds to the thickness of the glass
rods 24. The thickness of the separating chamber can be adjusted in
a simple manner by varying the thickness of the glass rods. For use
in connection with this invention it may be advantageous in
analytical devices to provide for a number of packing means (24,
25) running parallel to the lateral packings in order to have
several separating chamber stretches available under otherwise
equal iontophoresis conditions.
With the already-mentioned lifting stand 17 it is also possible to
attach to the bottom side of the separating chamber 12 a so-called
elution attachment which is described in detail below. The yields
in the device according to this invention are considerably greater
than those of known devices which leads to the result that a
considerably greater surface can be elutriated, whereby
consequently the amounts of elution fluid which are used can be
kept small. The elution takes place from the narrow side wall of
the separating chamber 12 or the elution attachment and on the
other narrow side there occurs the removal of the elution fluid.
The yields can be increased to a still greater extent by means of
meander-shaped path. Not only is the yield in the device according
to this invention considerably increased in comparison to that of
known electrophoresis devices but the selectivity of bands closely
following one another is also increased. This is also a result of
the design according to this invention since, in comparison with
devices with cylindrical separating chambers, a uniform elution
current can be provided. In the case of cylindrical separating
chambers the elution does not take place with uniform rate of flow
since the feeding and removal of the elution current through a
circular shaped or circular ring shaped cross section is not
possible with prior art means and consequently irregular flushings
occur which have an adverse effect upon the selectivity of the
separation. In the design example which is represented, the plate
of the lifting stand 17 is composed of a frame into which the
elution attachment 30 shown in FIG. 5 can be inserted. The frame is
rectangular and extends over the entire separating chamber cross
section. It is open from below so that the buffer fluid which is
present in the container 10 can reach the elution attachment from
below. With the aid of the lifting stand arrangement, the
attachment 30 can be pressed tightly against the lower separating
chamber opening.
The elution attachment 30 is composed of a first plate 31, a second
plate 32, a third plate 33, a first semipermeable membrane 34 and a
second semipermeable membrane 35. The plates 31, 32, 33 are
rectangular and are provided in the center of the plates with a
slit 36 which goes through the thickness of the plate and the
dimensions of which correspond to those of the bottom side of the
separating chamber. The slit 36 need not extend over the entire
length of the plate.
On the narrow sides 39, 40 of the plates 31 and 32 there are leads
41, 42, 43, 44 to the slit ends 36. At 41 elution fluid is fed to
the elution chamber 37 formed in the plate 31 and at 43
concentrated buffer solution is fed and removed at 44. The
concentrated buffer solution serves the purpose of greatly
diminishing the electrical field intensity and in this way achieves
a braking effect for the substances which are to be elutriated and
results in an increase in the effective flushing. An absorption of
the proteins which are to be elutriated on the membrane 34 and
their electrophoresis through the membrane is thus effectively
prevented. By means of the flowing of the highly concentrated
buffer fluid through the buffer chamber 38 formed in the plate 32
provision is made to keep the concentration of the buffer fluid
uniform at the membrane 34.
The plate 31 in the vicinity of its upper side above the slit 36
has a net 46 which serves for supporting the gel which is located
in the separating chamber 12. Baffle rods 45 may be provided
diagonally to the flow direction in the chambers 37 and 38
interchangeably from both sides of the slit 36 in order to force a
meander-shaped flow. Thus, at a relatively high rate of flow only a
slight drive is necessary and the obtention of highly-concentrated
eluates is possible. The baffle rods 45 also serve to keep the
membranes 34 and 35 tight (see FIG. 6).
It can be seen from FIG. 5 that the plates 31 and 32 must possess a
thickness which corresponds at least to the strength of the
connections 41 to 44. Thereby there is realized a relatively large
elution chamber. It is, however, worthwhile to keep the elution
chamber as small as possible so that high elution speeds can be
achieved with a low elution fluid weight rate of flow in order to
provide for a good dissolution and quality of the device. According
to a further development of the invention a reduction in the
elution chamber volume is achieved by providing the connections for
the feeding and removal of the elution fluid on the ends of the
separating chamber 12 which are adjacent to the elution chamber.
The connections are achieved by means of boreholes (from outside of
the separating chamber 12 to the elution chamber inside). In this
way the plate 31 can be kept very thin and consequently the elution
chamber volume can be kept very small so that the desired quality
improvement (better dissolving power) is achieved.
FIG. 7 shows a view of the inside of the backwall 3. It can be seen
that the electrodes 13 and 14, which are shaped in the form of a
wire here, are housed in recesses 51, 52 which are milled in the
backwall 3. The recesses are somewhat wedge-shaped so that their
upper edges 53 and 54 rise upwards obliquely. Gas bubbles rising to
the electrodes 13, 14 travel along these upper edges 53, 54 which
run obliquely upwards and gather at the highest point. For
diverting the existing gas, therefore, at the highest points of the
wedge-shaped recesses 51, 52 a channel 55 or 56 is bored upwards
through the backwall 3 through which the gas can escape into the
open air. From FIG. 8, which shows a cross section through the
backwall 3 it can be seen that the upper edges 53 and 54 are also
designed obliquely in the direction of the outside of the backwall
3. In this way the recesses 51, 52 are given a defined highest
position at which the gas collects and at which point the canals
55, 56 engage into the recesses. For purposes of simplicity only
the canal 55 of the upper recess 51 is shown in FIG. 8.
The aforementioned type of arrangement and design of the electrodes
13, 14, as well as their housing into recesses 51, 52 in the
backwall 3 is recommended, for example, in disc
electrophoresis.
A further advantage of this invention is that the device can also
be used for the method of iso-electric focusing in which
particularly small buffer container chambers are used. It is
possible with the aid of the lifting stand 17 to press a
corresponding attachment 57 onto the lower end of the separating
chamber 12 which only has a small volume and contains an electrode
63. In this way it is possible to use one and the same device for
all known electrophoresis methods and for analytical and
preparative purposes only by using individual attachments.
When using the method of iso-electric focusing the electrodes
should not be far from the separating chamber so that the potential
gradient lies essentially within the separating chamber which is
filled with the carrier means. The attachment of the electrode to
the lower separating chamber opening entails, however, the danger
that the gas bubbles rising from the electrode can reach the
underside of the separating chamber and disturb the
electrophoresis.
This danger is eliminated by means of the iso-electric focusing
attachment according to this invention. FIG. 9 shows sectional view
through the attachment 57 which is essentially composed of a
square-shaped container the length of which is at least the length
of the lower opening of the separating chamber 12. At its upper
side the container has the opening 59 which creates a connection of
the lower opening of the separating chamber 12 and the inside 60 of
the container. By means of a closeable opening (not shown) the
container or the inside 60 of the container can be filled with
buffer fluid. The attachment 57 has an adjacent chamber 61 towering
above the lower separating chamber limit laterally and which is
partially delimited from the remaining part of the buffer container
by means of a diaphragm 62 which is essentially vertical and ending
over the bottom of the buffer container. The electrode 63 is housed
in the adjacent chamber 61 and the gas bubbles which go out from it
rise to the upper limit of the adjacent chamber 61 where they can
escape through a canal 64. The opening 59 at the upper side 58 of
the attachment 57 is surrounded by packing material which is
arranged in such a way that it can be pressed against the lower
limit of the separating chamber 12. FIG. 1 schematically shows how
the pressing can occur with the aid of the lifting stand 17. For
this purpose, there can be used vertically standing threaded bolts
located at the housing bottom 1, therefore inside of the large
buffer container 10 which is not filled during an iso-electric
focusing procedure. The bolts can be moved back and forth on the
threaded bolts with the aid of which the lifting stand 17 can move
back and forth on the bolts.
In the case of iso-electric focusing a correspondingly smaller
buffer container with electrode can be attached on the upper end of
the separating chamber 12. The advantage of the small volume is
thus obtained and also the danger of the gas bubble contact with
the upper end of the separating chamber 12 does not occur. The
elimination of the developed gas by means of a ventilation canal
provided over the electrode in its adjacent chamber according to
the invention is nevertheless useful so that no disturbing excess
pressure occurs in the buffer container. The adjacent chamber can
be kept very small, by the way, so that it is only insignificantly
different from the volume of the iso-electric focusing
attachment.
Obviously, the embodiments shown are exemplary only and a wide
variety of embodiments may be devised without departing from the
spirit and scope thereof.
* * * * *