U.S. patent application number 11/088797 was filed with the patent office on 2005-08-04 for electrophoresis apparatus and method.
This patent application is currently assigned to Gradipore Ltd.. Invention is credited to Ogle, David, Rylatt, Dennis Brian, Vigh, Gyula.
Application Number | 20050167270 11/088797 |
Document ID | / |
Family ID | 25646454 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050167270 |
Kind Code |
A1 |
Vigh, Gyula ; et
al. |
August 4, 2005 |
Electrophoresis apparatus and method
Abstract
An electrophoretic apparatus comprising: a first electrolyte
chamber containing a first electrode; a second electrolyte chamber
containing a second electrode; a first sample chamber disposed
between the first and second electrolyte chambers and proximate to
the first electrolyte chamber; a second sample chamber disposed
between the first sample chamber and the second electrolyte; three
ion-permeable barriers separating the first electrolyte chamber,
the first sample chamber, the second sample chamber, and the second
electrolyte chamber, respectively, wherein the ion-permeable
barriers impede convective mixing of the contents in each of the
respective chambers; a first electrolyte reservoir and a second
electrolyte reservoir in fluid communication with the first and
second electrolyte chambers, respectively; a first sample reservoir
and a second sample reservoir in fluid communication with the first
and second sample chambers, respectively; means adapted for
communicating a first electrolyte and a second electrolyte between
the respective electrolyte chambers and reservoirs; means adapted
for communicating a first fluid and a second fluid between the
respective sample chambers and reservoirs, wherein at least one of
the first and second fluid contains at least a sample, wherein
application of an electric potential causes migration of at least
one component through at least one of the ion-permeable
barriers.
Inventors: |
Vigh, Gyula; (Magnolia,
TX) ; Ogle, David; (Cowan, AU) ; Rylatt,
Dennis Brian; (New South Wales, AU) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Gradipore Ltd.
North Ryde
TX
Texas University System, The
College Station
|
Family ID: |
25646454 |
Appl. No.: |
11/088797 |
Filed: |
March 25, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11088797 |
Mar 25, 2005 |
|
|
|
09961591 |
Sep 24, 2001 |
|
|
|
Current U.S.
Class: |
204/518 ;
204/520; 204/627 |
Current CPC
Class: |
B01D 57/02 20130101;
G01N 27/44769 20130101; G01N 27/44795 20130101 |
Class at
Publication: |
204/518 ;
204/520; 204/627 |
International
Class: |
B01D 057/02; B01D
059/42 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 22, 2000 |
AU |
PR0313 |
Dec 22, 2000 |
AU |
PR2299 |
Claims
What we claim is:
1. An electrophoretic apparatus comprising: a first electrolyte
chamber containing a first electrode; a second electrolyte chamber
containing a second electrode, wherein the second electrolyte
chamber is disposed relative to the first electrolyte chamber so
that the electrodes are adapted to generate an electric field in an
electric field area upon application of a selected electric
potential between the electrodes; a first sample chamber disposed
between the first and second electrolyte chambers and proximate to
the first electrolyte chamber so as to be at least partially
disposed in the electric field area; a second sample chamber
disposed between the first sample chamber and the second
electrolyte chamber so as to be at least partially disposed in the
electric field area; a first ion-permeable barrier separating the
first and second sample chambers so as to impede convective mixing
of the contents in each of the first and second sample chambers; a
second ion-permeable barrier separating the first electrolyte
chamber and the first sample chamber so as to impede convective
mixing of the contents in each of the first sample chamber and the
first electrolyte chamber; a third ion-permeable barrier separating
the second electrolyte chamber and the second sample chamber so as
to impede convective mixing of the contents in each of the second
sample chamber and the second electrolyte chamber; a first
electrolyte reservoir and a second electrolyte reservoir in fluid
communication with the first and second electrolyte chambers,
respectively; a first sample reservoir and a second sample
reservoir in fluid communication with the first and second sample
chambers, respectively; means adapted for communicating an
associated first electrolyte between the first electrolyte chamber
and the first electrolyte reservoir; means adapted for
communicating an associated second electrolyte between the second
electrolyte chamber and the second electrolyte reservoir; means
adapted for communicating a first fluid between the first sample
chamber and the first sample reservoir; and means adapted for
communicating a second fluid between the second sample chamber and
the second sample reservoir, wherein at least one of the first and
second fluid contains at least a sample; wherein application of the
selected electric potential causes migration of at least one
component through at least one of the ion-permeable barriers.
2. The apparatus according to claim 1 wherein the first
ion-permeable barrier is a membrane having a characteristic average
pore size and pore size distribution.
3. The apparatus according to claim 2 wherein the first
ion-permeable barrier is an isoelectric membrane having a
characteristic pI value.
4. The apparatus according to claim 1 wherein the second
ion-permeable barrier and the third ion-permeable barrier are
membranes having characteristic average pore sizes and pore size
distributions.
5. The apparatus according to claim 1 wherein at least one of the
second ion-permeable barrier and the third ion-permeable barrier is
an isoelectric membrane.
6. The apparatus according to claim 1 wherein both the second
ion-permeable barrier and the third ion-permeable barrier are
isoelectric membranes.
7. The apparatus according to claim 1 wherein each of the
isoelectric membranes have a different characteristic pI value.
8. The apparatus according to claim 1 further comprising means
adapted for circulating electrolyte from each of the first and
second electrolyte reservoirs through the first and second
electrolyte chambers forming first and second electrolyte streams;
and means adapted for circulating the contents from each of first
and second sample reservoirs through the respective first and
second sample chambers forming first and second sample streams.
9. The apparatus according to claim 8 wherein the means adapted for
communicating the first electrolyte, the second electrolyte, the
first fluid, and the second fluid comprise pumping means which are
separately controlled for independent movement of the respective
fluids.
10. The apparatus according to claim 1 further comprising means
adapted for at least one of removing contents from and replacing
contents in at least one of the first and second sample
reservoirs.
11. The apparatus according to claim 1 further comprising means
adapted to maintain the temperature of the first electrolyte,
second electrolyte, contents of the first sample chamber, and
contents of the second sample chamber.
12. The apparatus according to claim 1 wherein the first
electrolyte chamber, second electrolyte chamber, first sample
chamber, and second sample chamber are contained in a separation
unit wherein the separation unit is selected from the group
consisting of a cassette and a cartridge and such separation unit
is fluidly connected to the electrolyte reservoirs and the sample
reservoirs.
13. A method for selectively removing at least one component from a
selected sample comprising: communicating a first electrolyte to a
first electrolyte chamber containing a first electrode wherein the
first electrolyte chamber is in fluid communication with a first
electrolyte reservoir; communicating a second electrolyte to a
second electrolyte chamber containing a second electrode, wherein
the second electrolyte chamber is disposed opposite the first
electrolyte chamber and wherein the second electrolyte chamber is
in fluid communication with a second electrolyte reservoir;
communicating a first fluid to a first sample chamber disposed
between the first and second electrolyte chambers and proximate to
the first electrolyte chamber, wherein the first sample chamber is
in fluid communication with a first sample reservoir; communicating
a second fluid to a second sample chamber disposed between the
first sample chamber and the second electrolyte chamber, wherein
the second sample chamber is in fluid communication with a second
sample reservoir, wherein a first ion-permeable barrier separates
the first and second sample chambers, a second ion-permeable
barrier separates the first electrolyte chamber and the first
sample chamber, and a third ion-permeable barrier separates the
second sample chamber and the second electrolyte chamber, wherein
the ion-permeable barriers impede convective mixing between the
respective chambers, wherein at least one of the first and second
fluids contains at least a sample; and applying a selected electric
potential to cause migration of at least one selected component
through at least one of the ion-permeable barriers.
14. The method according to claim 13 wherein at least one of the
components in the first and second sample chambers has a pI
value.
15. The method according to claim 13 wherein the first
ion-permeable barrier is a membrane having a characteristic average
pore size and pore size distribution.
16. The method according to claim 13 wherein the first
ion-permeable barrier is an isoelectric membrane having a
characteristic pI value.
17. The method according to claim 13 wherein the second
ion-permeable barrier and the third ion-permeable barrier are
membranes having characteristic average pore sizes and pore size
distributions.
18. The method according to claim 13 wherein at least one of the
second ion-permeable barrier and the third ion-permeable barrier is
an isoelectric membrane.
19. The method according to claim 13 wherein both the second
ion-permeable barrier and the third ion-permeable barrier are
isoelectric membranes.
20. The method according to claim 13 further comprising removing at
least one component from at least one of the first and second
sample reservoirs.
21. The method according to claim 13 further comprising replacing
the components in at least one of the first and second sample
reservoirs.
22. The method according to claim 13 wherein substantially all
trans-barrier migration of the components is initiated upon the
application of the selected electric potential.
23. The method according to claim 13 wherein the application of the
selected electric potential is maintained until at least one
desired component reaches the desired purity in at least one of the
first and second sample chambers.
24. An electrophoretic separation unit comprising: a first
electrolyte chamber containing a first electrode; a second
electrolyte chamber containing a second electrode, wherein the
second electrolyte chamber is disposed relative to the first
electrolyte chamber so that the electrodes are adapted to generate
an electric field in an electric field area upon application of a
selected electric potential between the electrodes; a first sample
chamber disposed between the first and second electrolyte chambers
and proximate to the first electrolyte chamber so as to be at least
partially disposed in the electric field area; a second sample
chamber disposed between the first sample chamber and the second
electrolyte chamber so as to be at least partially disposed in the
electric field area; an isoelectric barrier separating the first
and second sample chambers so as to impede convective mixing of the
contents in each of the first and second sample chambers; a first
ion-permeable barrier separating the first electrolyte chamber and
the first sample chamber so as to impede convective mixing of the
contents in each of the first sample chamber and the first
electrolyte chamber; a second ion-permeable barrier separating the
second electrolyte chamber and the second sample chamber so as to
impede convective mixing of the contents in each of the second
sample chamber and second electrolyte chamber; means adapted for
communicating an associated first electrolyte to the first
electrolyte chamber; means adapted for communicating an associated
second electrolyte to the second electrolyte chamber; means adapted
for communicating a first fluid to the first sample chamber; and
means adapted for communicating a second fluid to the second sample
chamber, wherein at least one of the first and second fluids
contains at least a sample; wherein application of the selected
electric potential causes migration of at least one component
through at least one of the ion-permeable barriers.
25. The unit according to claim 24 wherein the isoelectric barrier
is an isoelectric membrane.
26. The unit according to claim 24 wherein the first ion-permeable
barrier and the second ion-permeable barrier are membranes having
characteristic average pore sizes and pore size distributions.
27. The unit according to claim 24 wherein at least one of the
first ion-permeable barrier and the second ion-permeable barrier is
an isoelectric membrane.
28. The unit according to claim 24 wherein both the first
ion-permeable barrier and the second ion-permeable barrier are
isoelectric membranes.
29. The unit according to claim 28 wherein each of the isoelectric
membranes have a different characteristic pI value.
30. A method for selectively removing at least one component from a
selected sample comprising: communicating a first electrolyte to a
first electrolyte chamber containing a first electrode;
communicating a second electrolyte to a second electrolyte chamber
containing a second electrode; communicating a first fluid to a
first sample chamber disposed between the first and second
electrolyte chambers and proximate to the first electrolyte
chamber; communicating a second fluid to a second sample chamber
disposed between the first sample chamber and the second
electrolyte chamber, wherein an isoelectric barrier separates the
first and second sample chambers, a first ion-permeable barrier
separates the first electrolyte chamber and the first sample
chamber, and a second ion-permeable barrier separates the second
sample chamber and the second electrolyte chamber, wherein the
barriers impede convective mixing between the respective chambers,
wherein at least one of the first and second fluids contains a
sample; and applying a selected electric potential to cause
migration of at least one selected component though at least one of
the barriers.
31. The method according to claim 30 wherein at least one of the
components in the first and second sample chambers has a pI
value.
32. The method according to claim 30 wherein the isoelectric
barrier is an isoelectric membrane.
33. The method according to claim 30 wherein the first
ion-permeable barrier and the second ion-permeable barrier are
membranes having characteristic average pore sizes and pore size
distributions.
34. The method according to claim 30 wherein at least one of the
first ion-permeable barrier and the second ion-permeable barrier is
an isoelectric membrane.
35. The method according to claim 30 wherein both the first
ion-permeable barrier and the second ion-permeable barrier are
isoelectric membranes.
36. The method according to claim 30 wherein each of the
isoelectric membranes have a different characteristic pI value.
37. The method according to claim 30 wherein substantially all
trans-barrier migration of the components is initiated upon the
application of the selected electric potential.
38. An electrophoretic apparatus comprising: a first electrolyte
chamber containing a first electrode; a second electrolyte chamber
containing a second electrode, wherein the second electrolyte
chamber is disposed relative to the first electrolyte chamber so
that the electrodes are adapted to generate an electric field in an
electric field area upon application of a selected electric
potential between the electrodes; a first sample chamber disposed
between the first and second electrolyte chambers and proximate to
the first electrolyte chamber so as to be at least partially
disposed in the electric field area; a second sample chamber
disposed between the first sample chamber and the second
electrolyte chamber so as to be at least partially disposed in the
electric field area; a first isoelectric barrier having a
characteristic pI separating the first and second sample chambers
so as to impede convective mixing of the contents in each of the
first and second sample chambers; a second isoelectric barrier
having a characteristic pI separating the first electrolyte chamber
and the first sample chamber so as to impede convective mixing of
the contents in each of the first sample chamber and the first
electrolyte chamber; a membrane having a characteristic average
pore size and pore size distribution separating the second sample
chamber and the second electrolyte chamber so as to impede
convective mixing of the contents in each of the second sample
chamber and the second electrolyte chamber; a first electrolyte
reservoir and a second electrolyte reservoir in fluid communication
with the first and second electrolyte chambers, respectively; a
first sample reservoir and a second sample reservoir in fluid
communication with the first and second sample chambers,
respectively; means adapted for communicating an associated first
electrolyte between the first electrolyte chamber and the first
electrolyte reservoir; means adapted for communicating an
associated second electrolyte between the second electrolyte
chamber and the second electrolyte reservoir; means adapted for
communicating a first fluid between the first sample chamber and
the first sample reservoir; and means adapted for communicating a
second fluid between the second sample chamber and the second
sample reservoir, wherein at least one of the first and second
fluids contains at least a sample; wherein application of the
selected electric potential causes migration of at least one
component through at least one of the barriers.
39. An electrophoretic apparatus comprising: a first electrolyte
chamber containing a first electrode; a second electrolyte chamber
containing a second electrode, wherein the second electrolyte
chamber is disposed relative to the first electrolyte chamber so
that the electrodes are adapted to generate an electric field in an
electric field area upon application of a selected electric
potential between the electrodes; a first sample chamber disposed
between the first and second electrolyte chambers and proximate to
the first electrolyte chamber so as to be at least partially
disposed in the electric field area; a second sample chamber
disposed between the first sample chamber and the second
electrolyte chamber so as to be at least partially disposed in the
electric field area; a first isoelectric barrier having a
characteristic pI separating the first and second sample chambers
so as to impede convective mixing of the contents in each of the
first and second sample chambers; a second isoelectric barrier
having a characteristic pI separating the first electrolyte chamber
and the first sample chamber so as to impede convective mixing of
the contents in each of the first sample chamber and the first
electrolyte chamber; a third isoelectric barrier having a
characteristic pI separating the second electrolyte chamber and the
second sample chamber so as to impede convective mixing of the
contents in each of the second sample chamber and second
electrolyte chamber; a first electrolyte reservoir and a second
electrolyte reservoir in fluid communication with the first and
second electrolyte chambers, respectively; a first sample reservoir
and a second sample reservoir in fluid communication with the first
and second sample chambers, respectively; means adapted for
communicating an associated first electrolyte between the first
electrolyte chamber and the first electrolyte reservoir; means
adapted for communicating an associated second electrolyte between
the second electrolyte chamber and the second electrolyte
reservoir; means adapted for communicating a first fluid between
the first sample chamber and the first sample reservoir; and means
adapted for communicating a second fluid between the second sample
chamber and the second sample reservoir, wherein at least one of
the first and second fluids contains at least a sample; wherein
upon application of the selected electric potential causes
migration of at least one component through at least one of the
barriers.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to an electrophoresis
apparatus and method suitable for electrophoretically altering the
original composition of a mixture that contains at least one
ampholytic component.
[0002] When ampholytic compounds, such as amino acids, peptides,
oligopeptides, proteins, and the like are present in a solution at
a low concentration, their charge-state depends on the pH of their
environment. At a certain characteristic pH value, the net
charge--and consequently, the electrophoretic mobility--of an
ampholytic compound becomes zero. That pH value is called the pI
value of the ampholytic compound. When two ampholytic compounds
have different pH values, their net charge becomes zero at
different pH values. Thus, if a pH gradient is established in an
electric field, the two ampholytic species achieve zero net charge
at different points of the pH gradient that can result in their
separation. Such separations are called isoelectric focusing (IEF)
separations. IEF separations have been achieved in (i) artificial
pH gradients created from non-amphoteric buffers either at constant
or spatially varying temperatures, (ii) natural pH gradients
created from carrier ampholytes or from the very components of the
mixture to be separated (autofocusing), and (iii) immobilized pH
gradients.
[0003] IEF separations typically rely on anti-convective means to
preserve the stability of the pH gradient. The IEF principle has
been utilized for both analytical and preparative-scale separation
of both simple and complex mixtures of ampholytic components. IEF
separations have been obtained in thin-layer format, column-format
and in multi-compartment format, in both static and flowing media.
In flowing media, separations have been achieved in both
straight-through and recycling format. IEF separations often take
considerable time because the electrophoretic mobility of each
ampholytic species becomes low as they approach the point in the pH
gradient where they become isoelectric.
[0004] Therefore, there is a need for IEF separation schemes and
equipment which (i) minimize the distance the components have to
migrate electrophoretically to achieve separation, (ii) maximize
the electric field strength that brings about the electrophoretic
separation without causing detrimental heating effects, (iii)
maximize the production rate that can be achieved in unit
separation space and time, and (iv) minimize the use of auxiliary
agents needed for the electrophoretic separation.
[0005] The present invention provides an apparatus and method which
can electrophoretically alter the original composition of a mixture
that contains at least one ampholytic component.
SUMMARY OF THE INVENTION
[0006] In accordance with the present invention, there is provided
an electrophoresis apparatus and method which can
electrophoretically alter the original composition of a mixture
that contains at least one ampholytic component.
[0007] Further, in accordance with the present invention, there is
provided an electrophoresis apparatus and system which (i)
minimizes the distance the components have to migrate
electrophoretically to achieve separation, (ii) maximizes the
electric field strength that brings about the electrophoretic
separation without causing detrimental heating effects, (iii)
maximizes the production rate that can be achieved in unit
separation space and time, and (iv) minimizes the use of auxiliary
agents needed for the electrophoretic separation.
[0008] Still further, in accordance with the present invention,
there is provided an electrophoretic apparatus comprising:
[0009] a first electrolyte chamber containing a first
electrode;
[0010] a second electrolyte chamber containing a second electrode,
wherein the second electrolyte chamber is disposed relative to the
first electrolyte chamber so that the electrodes are adapted to
generate an electric field in an electric field area upon
application of a selected electric potential between the
electrodes;
[0011] a first sample chamber disposed between the first and second
electrolyte chambers and proximate to the first electrolyte chamber
so as to be at least partially disposed in the electric field
area;
[0012] a second sample chamber disposed between the first sample
chamber and the second electrolyte chamber so as to be at least
partially disposed in the electric field area;
[0013] a first ion-permeable barrier separating the first and
second sample chambers so as to impede convective mixing of the
contents in each of the first and second sample chambers;
[0014] a second ion-permeable barrier separating the first
electrolyte chamber and the first sample chamber so as to impede
convective mixing of the contents in each of the first sample
chamber and the first electrolyte chamber;
[0015] a third ion-permeable barrier separating the second
electrolyte chamber and the second sample chamber so as to impede
convective mixing of the contents in each of the second sample
chamber and the second electrolyte chamber;
[0016] a first electrolyte reservoir and a second electrolyte
reservoir in fluid communication with the first and second
electrolyte chambers, respectively;
[0017] a first sample reservoir and a second sample reservoir in
fluid communication with the first and second sample chambers,
respectively;
[0018] means adapted for communicating an associated first
electrolyte between the first electrolyte chamber and the first
electrolyte reservoir;
[0019] means adapted for communicating an associated second
electrolyte between the second electrolyte chamber and the second
electrolyte reservoir;
[0020] means adapted for communicating a first fluid between the
first sample chamber and the first sample reservoir; and
[0021] means adapted for communicating a second fluid between the
second sample chamber and the second sample reservoir, wherein at
least one of the first and second fluid contains at least a
sample,
[0022] wherein application of the selected electric potential
causes migration of at least one component through at least one of
the ion-permeable barriers.
[0023] In one preferred form, the first ion-permeable barrier is a
membrane having a characteristic average pore size and pore size
distribution. In one form, all the ion-permeable barriers are
membranes having a characteristic average pore size and pore size
distribution. This configuration of the apparatus is suitable for
separating compounds on the basis of charge and or size.
[0024] In another preferred form, the first ion-permeable barrier
is an isoelectric membrane having a characteristic pI value.
Preferably, the isoelectric membrane has a pI value in a range of
about 2 to about 12.
[0025] In another preferred form, the second and third
ion-permeable barriers are membranes having a characteristic
average pore size and pore-size distribution.
[0026] In another preferred form, at least one of the second or
third ion-permeable barriers is an isoelectric membrane having a
characteristic pI value. Preferably, the at least one isoelectric
membrane has a pI value in a range of about 2 to about 12. In
another preferred form, both the second and third ion-permeable
barriers are isoelectric membranes each having a characteristic pI
value. Preferably, the isoelectric membranes have a pI value in a
range of about 2 to about 12. When both the second and third
ion-permeable barriers are isoelectric membranes, the membranes can
have the same or different characteristic pI values.
[0027] The isoelectric membranes are preferably
polyacrylamide-based membranes. It will be appreciated, however,
that other isoelectric membranes would also be suitable for the
present invention.
[0028] In another preferred form, the apparatus further comprises
means for circulating electrolyte from each of the first and second
electrolyte reservoirs through the respective first and second
electrolyte chambers forming first and second electrolyte streams
in the respective electrolyte chambers; and means for circulating
contents from each of the first and second sample reservoirs
through the respective first and second sample chambers forming
first and second sample streams in the respective sample
chambers.
[0029] Preferably, means for circulating the electrolyte and sample
streams are pump arrangements separately controllable for
independent movement of the electrolyte streams and the sample
streams.
[0030] The apparatus may further include means for removing and
replacing sample in the first or second sample reservoirs. The
apparatus may also further include means to maintain temperature of
electrolyte and sample solutions.
[0031] In another preferred form, the separation unit is provided
as a cartridge or cassette fluidly connected to the electrolyte
reservoirs and the sample reservoirs. In one preferred form, the
separation unit is provided as a cartridge or cassette connected to
the electrolyte reservoirs and the sample reservoirs.
[0032] Still further, in accordance with the present invention,
there is provided a method for selectively removing at least one
component from a selected sample comprising:
[0033] communicating a first electrolyte to a first electrolyte
chamber containing a first electrode wherein the first electrolyte
chamber is in fluid communication with a first electrolyte
reservoir;
[0034] communicating a second electrolyte to a second electrolyte
chamber containing a second electrode, wherein the second
electrolyte chamber is disposed opposite the first electrolyte
chamber and wherein the second electrolyte chamber is in fluid
communication with a second electrolyte reservoir;
[0035] communicating a first fluid to a first sample chamber
disposed between the first and second electrolyte chambers and
proximate to the first electrolyte chamber, wherein the first
sample chamber is in fluid communication with a first sample
reservoir;
[0036] communicating a second fluid to a second sample chamber
disposed between the first sample chamber and the second
electrolyte chamber, wherein the second sample chamber is in fluid
communication with a second sample reservoir, wherein a first
ion-permeable barrier separates the first and second sample
chambers, a second ion-permeable barrier separates the first
electrolyte chamber and the first sample chamber, and a third
ion-permeable barrier separates the second sample chamber and the
second electrolyte chamber, wherein the ion-permeable barriers
impede convective mixing between the respective chambers, wherein
at least one of the first and second fluids contains at least a
sample; and
[0037] applying a selected electric potential to cause migration of
at least one selected component through at least one of the
ion-permeable barriers.
[0038] Preferably, at least one sample component has a pI
value.
[0039] In a preferred form, electrolyte from at least one of the
first and second electrolyte reservoirs is circulated through the
first or second electrolyte chamber forming a first or second
electrolyte stream.
[0040] The choice of electrolyte in the first and second
electrolyte chambers will depend on the compound or compounds to be
treated, separated or transferred from a sample chamber to the
other sample chamber, or one or both of the electrolyte chambers.
Similarly, the choice of the pI of the isoelectric membranes will
also depend on the compound or compounds to be treated, separated
or transferred from a given sample.
[0041] Electrolytes such as acetic acid as the anolyte, and
triethanol amine as the catholyte, have been found to be suitable
for the separation of a number of components from biological
samples. Salt such as NaCl may also be added to the electrolyte to
assist. It will be appreciated, however, that other electrolytes
would also be applicable, depending on the desired separation or
treatment.
[0042] In another preferred form, electrolyte from both the first
and second electrolyte reservoirs is circulated through the first
and second electrolyte chambers forming first and second
electrolyte streams.
[0043] In another preferred form, the contents of the first or
second sample reservoir is circulated through the first or second
sample chamber forming a first or second sample stream through the
first or second sample chamber. In another preferred form, sample
or liquid in the first or second sample reservoir is removed and
replaced with fresh sample or liquid.
[0044] Preferably, substantially all transbarrier migration occurs
upon the application of the electric potential. In another
preferred form, the application of the electric potential is
maintained until at least one desired component reaches a desired
purity in at least one of the first and second sample chamber or in
the first or second sample reservoirs.
[0045] Still further, in accordance with the present invention,
there is provided an electrophoretic separation unit
comprising:
[0046] a first electrolyte chamber containing a first
electrode;
[0047] a second electrolyte chamber containing a second electrode,
wherein the second electrolyte chamber is disposed relative to the
first electrolyte chamber so that the electrodes are adapted to
generate an electric field in an electric field area upon
application of a selected electric potential between the
electrodes;
[0048] a first sample chamber disposed between the first and second
electrolyte chambers and proximate to the first electrolyte chamber
so as to be at least partially disposed in the electric field
area;
[0049] a second sample chamber disposed between the first sample
chamber and the second electrolyte chamber so as to be at least
partially disposed in the electric field area;
[0050] an isoelectric barrier separating the first and second
sample chambers so as to impede convective mixing of the contents
in each of the first and second sample chambers;
[0051] a first ion-permeable barrier separating the first
electrolyte chamber and the first sample chamber so as to impede
convective mixing of the contents in each of the first sample
chamber and the first electrolyte chamber;
[0052] a second ion-permeable barrier separating the second
electrolyte chamber and the second sample chamber so as to impede
convective mixing of the contents in each of the second sample
chamber and second electrolyte chamber;
[0053] means adapted for communicating an associated first
electrolyte to the first electrolyte chamber;
[0054] means adapted for communicating an associated second
electrolyte to the second electrolyte chamber;
[0055] means adapted for communicating a first fluid to the first
sample chamber; and
[0056] means adapted for communicating a second fluid to the second
sample chamber, wherein at least one of the first and second fluids
contains at least a sample;
[0057] wherein application of the selected electric potential
causes migration of at least one component through at least one of
the ion-permeable barriers.
[0058] Still further, in accordance with the present invention,
there is provided a method for selectively altering the
concentration of a selected sample:
[0059] communicating a first electrolyte to a first electrolyte
chamber containing a first electrode;
[0060] communicating a second electrolyte to a second electrolyte
chamber containing a second electrode;
[0061] communicating a first fluid to a first sample chamber
disposed between the first and second electrolyte chambers and
proximate to the first electrolyte chamber;
[0062] communicating a second fluid to a second sample chamber
disposed between the first sample chamber and the second
electrolyte chamber, wherein an isoelectric barrier separates the
first and second sample chambers, a first ion-permeable barrier
separates the first electrolyte chamber and the first sample
chamber, and a second ion-permeable barrier separates the second
sample chamber and the second electrolyte chamber, wherein the
barriers impede convective mixing between the respective chambers,
wherein at least one of the first and second fluids contains a
sample; and
[0063] applying a selected electric potential to cause migration of
at least one selected component through at least one of the
barriers.
[0064] An advantage of the present invention is that the apparatus
and method have scale-up capabilities, increased separation speed,
lower cost of operation, lower power requirements, and increased
ease of use.
[0065] Yet another advantage of the present invention is that the
apparatus and method have improved yields of the separated
component, and improved purity of the separated component.
[0066] These and other advantages will be apparent to one skilled
in the art upon reading and understanding the specification.
[0067] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element, integer or step, or group of elements, integers or
steps, but not the exclusion of any other element, integer or step,
or group of elements, integers or steps.
[0068] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed in Australia before the priority date of
each claim of this application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0069] FIG. 1 is a schematic diagram of a separation unit for use
in the present invention.
[0070] FIG. 2 is a schematic diagram of an apparatus according to
the present invention utilizing the separation unit of FIG. 1.
[0071] FIG. 3 is an exploded view of a cartridge which may be used
with the separation unit of FIG. 1.
[0072] FIG. 4A is a plan view of a grid element which may be
incorporated as a component of a cartridge of a separation
unit.
[0073] FIG. 4B is a reverse plan view of the grid element of FIG.
4A.
[0074] FIG. 5 is a cross-sectional view on the lines X-X of FIG.
4A.
[0075] FIG. 6 is a cross-sectional view on the lines XI-XI of FIG.
4A.
[0076] FIG. 7 is a cross-sectional view on the lines XII-XII of
FIG. 4A.
[0077] FIG. 8 is a plan view of an alternative embodiment of a grid
element which may be incorporated as a component of a cartridge of
a separation unit.
[0078] FIG. 9 shows an apparatus utilizing the separation unit of
FIG. 1.
[0079] FIG. 10 shows the image, at 280 nm, of the protein bands
separated by the iCE280 full-column-imaging capillary isoelectric
focusing instrument, from a chicken egg-white sample, used as the
starting material for the electrophoretic separation experiments
described in Examples 1 to 3. The peaks labeled pI 3.52 and pI 9.61
correspond to dansyl phenylalanine and terbutaline, respectively,
used as isoelectric point markers. The egg-white sample was diluted
1:25 with deionized water and filtered prior to analysis. Analysis
conditions: instrument: iCE280 full-column imaging capillary IEF
system, separation capillary: 5 cm long, 100 micrometer I.D. fused
silica, focusing medium: 8% carrier ampholytes for pH 3-10 in
aqueous 0.1% methylcellulose solution, focusing time: 5 minutes,
applied potential: 3,000 V.
[0080] FIG. 11 shows the image, at 280 nm, of the protein bands
separated by the iCE280 full-column-imaging capillary isoelectric
focusing instrument, from aliquots collected at the end of the
experiment from the first sample reservoir (bottom panel) and
second sample reservoir (top panel) of the electrophoretic
apparatus disclosed here and described in Example 1. Separation
conditions: anolyte: 60 mL of 80 mM acetic acid, pH=2.9, catholyte:
60 mL 8 mM triethanol amine, pH=9.9, sample: 60 mL aqueous
egg-white sample diluted 1:25 in deionized water, separation time:
15 minutes, applied potential: 250 V, first ion-permeable barrier
between the first electrolyte chamber and the first sample chamber:
pI=4.0 isoelectric membrane, second ion-permeable barrier between
the first sample chamber and the second sample chamber: pI=5.0
isoelectric membrane, third ion-permeable barrier between the
second sample chamber and the second electrolyte chamber: pI=7.0
isoelectric membrane. The peaks labeled pI 3.52 and pI 9.61
correspond to dansyl phenylalanine and terbutaline, respectively,
used as isoelectric point markers. Analysis conditions: instrument:
iCE280 full-column imaging IEF system, capillary: 5 cm long, 100
micrometer I.D. fused silica, focusing medium: 8% carrier
ampholytes for pH 3-10 in aqueous 0.1% methylcellulose solution,
focusing time: 5 minutes, applied potential: 3,000 V.
[0081] FIG. 12 shows the image, at 280 nm, of the protein bands
separated by the iCE280 full-column-imaging capillary isoelectric
focusing instrument, from aliquots collected at the end of the
experiment described in Example 2 from the first sample reservoir
(bottom panel) and the second sample reservoir (top panel) of the
electrophoretic apparatus disclosed here. Separation conditions:
anolyte: 60 mL 2 mM acetic acid, catholyte: 60 mL 8 mM triethanol
amine, sample: 60 mL aqueous egg-white sample diluted 1:25 in
distilled water, separation time: 15 minutes, applied potential:
250 V, first ion-permeable barrier between the first electrolyte
chamber and the first sample chamber: polyacrylamide membrane with
a nominal molecular mass cut-off of 5,000 dalton, second
ion-permeable barrier between the first sample chamber and the
second sample chamber: pI=5.0 isoelectric membrane, third
ion-permeable barrier between the second sample chamber and the
second electrolyte chamber: polyacrylamide membrane with a nominal
molecular mass cut-off of 5,000 dalton. The peaks labeled pI 3.52
and pI 9.61 correspond to dansyl phenylalanine and terbutaline,
respectively, used as isoelectric point markers. Analysis
conditions: instrument: iCE280 full-column imaging IEF system,
capillary: 5 cm long, 100 micrometer I.D. fused silica, focusing
medium: 8% carrier ampholytes for pH 3-10 in aqueous 0.1%
methylcellulose solution, focusing time: 5 minutes, applied
potential: 3,000 V.
[0082] FIG. 13 shows the image, at 280 nm, of the protein bands
separated by the iCE280 full-column-imaging capillary isoelectric
focusing instrument, from aliquots collected at the end of the
experiment described in Example 3 from the first sample reservoir
(bottom panel) and the second sample reservoir (top panel) of the
electrophoretic apparatus disclosed here. Separation conditions:
anolyte: 60 mL 2 mM acetic acid, catholyte: 60 mL 8 mM triethanol
amine, sample: 60 mL aqueous egg-white sample diluted 1:25 in
deionized water, separation time: 15 minutes, applied potential:
250 V, first ion-permeable barrier between the first electrolyte
chamber and the first sample chamber: polyacrylamide membrane with
a nominal molecular mass cut-off of 1,000,000 dalton, second
ion-permeable barrier between the first sample chamber and the
second sample chamber: pI=5.0 isoelectric membrane, third
ion-permeable barrier between the second sample chamber and the
second electrolyte chamber: polyacrylamide membrane with a nominal
molecular mass cut-off of 1,000,000 dalton. The peak labeled pI
3.52 corresponds to dansyl phenylalanine, used as isoelectric point
marker. Analysis conditions: instrument: iCE280 full-column imaging
IEF system, capillary: 5 cm long, 100 micrometer I.D. fused silica,
focusing medium: 8% carrier ampholytes for pH 3-10 in aqueous 0.1%
methylcellulose solution, focusing time: 5 minutes, applied
potential: 3,000 V.
[0083] FIG. 14 shows the image of an SDS-PAGE gel used to analyze
the protein bands present in the aliquots collected from the first
and second sample reservoirs of the electrophoretic apparatus
disclosed here and described in Example 4 during the isolation of
IgG from human plasma. Molecular weight markers (Sigma, St. Louis,
Mo., USA) were applied onto Lane 1, a pharmaceutical-grade IgG
preparation used as reference material onto Lane 2. Samples taken
at 0, 10, 20, and 40 minutes respectively from the first sample
reservoir were applied onto Lanes 3, 4, 5, and 6. Samples taken at
0, 10, 20, and 40 minutes respectively from the second sample
reservoir were applied onto Lanes 7, 8, 9, and 10. Separation
conditions: anolyte: 2 L 2 mM 5-aminocaproic acid adjusted to pH
4.8 with HCl, also containing 5 mM NaCl, catholyte: 2 L 2 mM MOPSO
adjusted to pH 6.8 with NaOH, also containing 5 mM NaCl, sample: 10
mL human plasma sample diluted 1 to 3 with deionized water,
separation time: 40 minutes, applied potential: 250 V, first
ion-permeable barrier between the first electrolyte chamber and the
first sample chamber: polyacrylamide membrane with a nominal
molecular mass cut-off of 150,000 dalton, second ion-permeable
barrier between the first sample chamber and the second sample
chamber: pI=5.8 isoelectric membrane, third ion-permeable barrier
between the second sample chamber and the second electrolyte
chamber: polyacrylamide membrane with a nominal molecular mass
cut-off of 150,000 dalton.
DETAILED DESCRIPTION OF THE INVENTION
[0084] Before describing the preferred embodiments in detail, the
principal of operation of the apparatus will first be described. An
electric field or potential applied to ions in solution will cause
the ions to move toward one of the electrodes. If the ion has a
positive charge, it will move toward the negative electrode
(cathode). Conversely, a negatively-charged ion will move toward
the positive electrode (anode).
[0085] In the apparatus of the present invention, ion-permeable
barriers that substantially prevent convective mixing between the
adjacent chambers of the apparatus or unit are placed in an
electric field and components of the sample are selectively
transported through the barriers. The particular ion-permeable
barriers used will vary for different applications and generally
have characteristic average pore sizes and pore size distributions
and/or isoelectric points allowing or substantially preventing
passage of different components.
[0086] Having outlined some of the principles of operation of an
apparatus in accordance with the present invention, an apparatus
itself will be described.
[0087] Referring to FIG. 1, a schematic representation of
separation unit 2 is shown for the purpose of illustrating the
general functionality of a separation device utilizing the
technology of the present invention. Separation unit 2 comprises
first electrolyte inlet 4, and second electrolyte inlet 6, first
sample inlet 8, and second sample inlet 10, first electrolyte
outlet 12, and second electrolyte outlet 14, and first sample
outlet 16 and second sample outlet 18. Between first electrolyte
inlet 4 and first outlet 12 is first electrolyte chamber 22.
Likewise, between second electrolyte inlet 6 and second electrolyte
outlet 14 is second electrolyte chamber 24. First sample and second
sample inlets and outlets also have connecting chambers. First
sample chamber 26 running adjacent to first electrolyte chamber 22
connects first sample inlet 8 to first sample outlet 16. Similarly,
second sample chamber 28 running adjacent to second electrolyte
chamber 24 connects second sample inlet 10 to second sample outlet
18. Ion-permeable barriers 30 and 32 separate electrolyte chambers
22 and 24 from first sample and second sample chambers 26 and 28,
respectively. Between first sample and second sample chambers 26
and 28 is ion-permeable barrier 34. In one embodiment, when in use,
first and second electrolyte 36 and 38 occupy first and second
electrolyte chambers 22 and 24. It should be understood that during
operation, first and second electrolyte 36 and 38, as well as first
and second sample 56 and 66 may be stagnant in, or flow through,
the respective chambers.
[0088] A schematic diagram of an apparatus utilizing separation
unit 2 of FIG. 1 is shown in FIG. 2 for the purpose of illustrating
the general functionality of an apparatus utilizing the technology
of the present invention. In this purely illustrative example, four
chambers (first electrolyte chamber 22, second electrolyte chamber
24, first sample chamber 26, and second sample chamber 28) are
connected to four flow circuits. First electrolyte flow circuit 40
comprises first electrolyte reservoir 42, electrolyte tubing 44,
and electrolyte pump 46. Second electrolyte flow circuit 41
comprises second electrolyte reservoir 43, electrolyte tubing 45,
and electrolyte pump 47. In the configuration shown in FIG. 2,
electrolyte flow circuits 40 and 41 are running independently from
each other so that the composition, temperature, flow rate and
volume of first electrolyte 36 and second electrolyte 38 can be
suitably adjusted independently of one another.
[0089] In the embodiment shown, first electrolyte 36 flows from
first electrolyte reservoir 42 through tubing 44 to pump 46 to
first electrolyte chamber 22. Second electrolyte 24 flows from
second electrolyte reservoir 43 through tubing 45 to pump 47 to
second electrolyte chamber 24. First electrolyte 36 flows through
inlet 4 and second electrolyte 38 flows through inlet 6. First
electrolyte 36 exits separation unit 2 through outlet 12 and second
electrolyte 38 exits separation unit 2 through outlet 14. After
exiting separation unit 2, electrolytes 36 and 38 flow through
tubing 44 and 45 back into respective electrolyte reservoirs 42 and
43. In one embodiment, electrolytes 36 and 38 are held stagnant in
electrolyte chambers 22 and 24 during separation. Electrolytes 36
and 38 can also act as a cooling medium and help prevent a build up
of gases generated during electrophoresis.
[0090] First sample flow circuit 48 contains first sample reservoir
50, tubing 52 and pump 54. First sample 56 flows from first sample
reservoir 50 through tubing 52 to pump 54, then through inlet 8
into first sample chamber 26. In one embodiment, the flow
directions of first sample 56 and electrolytes 36 and 38 in first
sample chamber 26 are opposite. First sample 56 exits separation
unit 2 at outlet 16 and flows through tubing 52, then heat
exchanger 68 that passes through second electrolyte reservoir 43
before returning to first sample reservoir 50 through tubing 52. In
an alternative embodiment, heat exchanger 68 passes through first
electrolyte reservoir 42. In another embodiment, the flow
directions of first sample 56 and electrolytes 36 and 38 in first
sample chamber 26 are the same.
[0091] In addition to components of interest, first sample 56 may
contain any suitable electrolyte or additive known in the art as
demanded by the procedure, application, or separation being
performed to substantially prevent or cause migration of selected
components through the ion-permeable barriers. In a preferred
embodiment, sample from which constituents are to be removed is
placed into first sample reservoir 50. However, it is understood
that in an alternative embodiment, sample from which constituents
are to be removed is placed into second sample reservoir 60.
[0092] Similarly, second sample flow circuit 58 contains second
sample reservoir 60, tubing 62 and pump 64. Second sample 66 flows
from second sample reservoir 60 through tubing 62 to pump 64, then
through inlet 10 into second sample chamber 28. In one embodiment,
the flow directions of second sample 66 and electrolytes 36 and 38
in second sample chamber 28 are opposite. Second sample 66 exits
separation unit 2 at outlet 18 and flows through tubing 62, then
heat exchanger 70 that passes through second electrolyte reservoir
43 before returning to second sample reservoir 60 through tubing
62. In an alternative embodiment, heat exchanger 70 passes through
first electrolyte reservoir 43.
[0093] Second sample 66 may contain any suitable electrolyte or
additive known in the art as demanded by the procedure,
application, or separation being performed to substantially prevent
or cause migration of selected components through the ion-permeable
barriers. In a preferred embodiment, sample from which constituents
are to be removed is placed into second sample reservoir 60.
However, it is understood that in an alternative embodiment, sample
from which constituents are to be removed is placed into first
sample reservoir 50.
[0094] Individually adjustable flow rates of first sample, second
sample, first electrolyte and second electrolyte, when employed,
can have a significant influence on the separation. Flow rates
ranging from zero through several milliliters per minute to several
liters per minute are suitable depending on the configuration of
the apparatus and the composition, amount and volume of sample
processed. In a laboratory scale instrument, individually
adjustable flow rates ranging from about 0 mL/minute to about
50,000 mL/minute are used, with the preferred flow rates in the 0
mL/min to about 1,000 mL/minute range. However, higher flow rates
are also possible, depending on the pumping means and size of the
apparatus. Selection of the individually adjustable flow rates is
dependent on the process, the component or components to be
transferred, efficiency of transfer, and coupling of the process
with other, preceding or following processes.
[0095] Preferably, all tubing 44, 52, and 62 is peristaltic tubing
that is autoclavable, chemically resistant, and biologically inert.
One such tubing is Masterflex.RTM. C-FLEX.RTM. 50 A tubing. Also,
pumps 46, 47, 54 and 64 are preferably peristaltic pumps. In the
presently preferred embodiment, heat exchangers 68 and 70 are
constructed from stainless steel, although other materials known in
the art are suitably used. Preferably, heat exchangers 68 and 70
are autoclavable, chemically resistant, biologically inert and
capable of facilitating heat exchange.
[0096] Furthermore, it is preferable that first sample flow circuit
48, second sample flow circuit 58, first electrolyte flow circuit
40 and second electrolyte flow circuit 41 are completely enclosed
to prevent contamination or cross-contamination. In a preferred
embodiment, reservoirs 42, 43, 50, and 60, are completely and
individually enclosed from the rest of the apparatus.
[0097] The separation unit further comprises electrodes 88a and
88b. Preferably, the respective electrodes are located in the first
and second electrolyte chambers and are separated from the first
and second sample chambers by ion-permeable barriers.
[0098] Electrodes 88a and 88b are suitably standard electrodes or
preferably are formed from platinum coated titanium expanded mesh,
providing favorable mechanical properties, even distribution of the
electric field, long service life and cost efficiency. Electrodes
88a and 88b are preferably located relatively close to
ion-permeable barriers 30 and 32 providing better utilization of
the applied potential and diminished heat generation. A distance of
about 0.1 to 6 mm has been found to be suitable for a laboratory
scale apparatus. For scaled-up versions, the distance will depend
on the number and type of ion-permeable barriers, and the size and
volume of the electrolyte and sample chambers. Preferred distances
would be in the order of about 0.1 mm to about 10 mm.
[0099] Separation unit 2 also preferably comprises electrode
connectors 78 that are used for connecting separation unit 2 to
power supply 72. Preferably, power supply 72 is external to
separation unit 2, however, separation unit 2 is configurable to
accept internal power supply 72. Electrode connectors 78 are
preferably autoclavable.
[0100] Separation is achieved when an electric potential is applied
to separation unit 2. Selection of the electric field strength
varies depending on the separation. Typically, the electric field
strength varies between 1 V/cm to about 5,000 V/cm, preferably
between 10 V/cm to 2,000 V/cm and leads to currents of up to about
1 A. It is preferable to maintain the total power consumption of
the unit at the minimum, commensurable with the desired separation
and production rate.
[0101] In one embodiment, the applied electric potential is
periodically stopped and reversed to cause movement of components
that have entered the ion-permeable barriers back into at least one
of the fluid streams, while substantially not causing re-entry of
any components that have entered other fluid streams. In another
embodiment, a resting period is utilized. Resting (a period during
which fluid flows are maintained but no electric potential is
applied) is an optional step that suitably replaces or is included
after an optional reversal of the electric potential. Resting is
often used for protein-containing samples as an alternative to
reversing the potential.
[0102] Separation unit 2 is suitably cooled by various methods
known in the art such as ice bricks or cooling coils (external
apparatus) placed in one or both electrolyte reservoirs 42 and 43,
or any other suitable means capable of controlling the temperature
of electrolytes 36 and 38. Because both first sample flow circuit
48 and second sample flow circuit 58 pass through either
electrolyte reservoir 42 or 43, heat is exchanged between first and
second samples and one or both of first and second electrolytes.
Heat exchange tends to maintain the temperature in first sample 56
and second sample 66 at the preferred, usually low levels.
[0103] In another form, there is provided an electrophoresis unit
that comprises four chambers (first electrolyte chamber 22, second
electrolyte chamber 24, first sample chamber 26, and second sample
chamber 28). Ion-permeable barriers 30 and 32 separate electrolyte
chambers 22 and 24 from first sample and second sample chambers 26
and 28, respectively. Between first sample and second sample
chambers 26 and 28 is ion-permeable barrier 34. Electrodes are
housed in the first and second electrolyte chambers and sample
and/or fluid is placed into first sample chamber 26 and second
sample chamber 28. In use, an electric potential is applied between
the electrodes and one or more components in the first sample
chamber 26 or second sample chamber 28 are caused to move to the
other sample chamber or to one of the electrolyte chambers.
[0104] FIG. 3 is an exploded view of cartridge 100 which is
preferably a modular component of separation unit 2. When
configured as a modular unit, cartridge 100 preferably comprises
housing 102 for holding in place or encasing the component parts of
cartridge 100. In a presently preferred embodiment, cartridge 100
is generally elongated and has side walls 104 which are generally
parallel to one another and the longitudinal axis A of cartridge
100. The cartridge is suitably generally octagonal, hexagonal, or
ovular. In an octagonal configuration, cartridge 100 has three end
walls 106 on each side of side walls 104 forming an octagon.
However, two end walls on each side 106 are suitably used to form a
hexagon, or one curved end wall 106 on each side is suitably used
to form a generally ovular shape. Furthermore, end walls 106 are
suitably either straight or generally curved.
[0105] Extending around the base of side walls 104 and end walls
106 is a small flange 108 that is generally perpendicular to side
walls 104 and end walls 106 and projects inward toward the center
of cartridge 100. Along the exterior of either side walls 104 or
end walls 106 is preferably a handle 110 to facilitate placement of
cartridge 100 into separation unit 2. Flange 108 is preferably
configured to interact with lower gasket 112. In a preferred
embodiment, lower gasket 112 is generally planar and configured to
fit inside walls 104 and 106 of cartridge 100. In a presently
preferred embodiment, lower gasket 112 is made from silicon rubber.
Lower gasket 112 may be configured so that it has an aperture 114
extending in an elongated manner through the center of lower gasket
112. Also extending through and adjacent each end of lower gasket
112 are alignment holes 116. In a preferred embodiment, alignment
holes 116 are circular, forming generally cylindrical channels
through lower gasket 112. However, it is also contemplated that
alignment holes 116 are suitably triangular, square, rectangular,
hexagonal, octagonal, or similarly shaped.
[0106] Above lower gasket 112 is a generally planar lower
ion-permeable barrier 32. The external shape of ion-permeable
barrier 32 is generally the same as that of lower gasket 112 and
the interior of cartridge 100 so that ion-permeable barrier 32 is
configured to fit inside cartridge 100. Like lower gasket 112,
ion-permeable barrier 32 preferably has two alignment holes of the
same location and configuration as alignment holes 116 in lower
gasket 112. Ion-permeable barrier 32 substantially prevents
convective mixing of the contents of first electrolyte chamber 22
and first sample chamber 26, while permits selective trans-barrier
transport of selected constituents upon application of the electric
potential.
[0107] In one embodiment, ion-permeable barrier 32 is formed from a
membrane with a characteristic average pore size and pore-size
distribution. The average pore size and pore size distribution of
the membrane is selected to facilitate trans-membrane transport of
certain constituents, while substantially preventing trans-membrane
transport of other constituents.
[0108] In another embodiment, ion-permeable barrier 32 is an
isoelectric ion-permeable barrier, such as an isoelectric membrane
that substantially prevents convective mixing of the contents of
first electrolyte chamber 22 and first sample chamber 26, while
permits selective trans-barrier transport of selected constituents
upon application of the electric potential. Suitable isoelectric
membranes can be produced by copolymerizing acrylamide,
N,N'-methylene bisacrylamide and appropriate acrylamido derivatives
of weak electrolytes yielding isoelectric membranes with pI values
in the 2 to 12 range, and average pore sizes that either facilitate
or substantially prevent trans-membrane transport of components of
selected sizes.
[0109] Above lower ion-permeable barrier 32 is lower grid element
118 that is generally planar and also shaped like lower gasket 112
and the interior of cartridge 100 so that lower grid element 118 is
configured to fit inside cartridge 100. One of the functions of
lower grid element 118 is to separate lower ion-permeable barrier
32 from ion-permeable barrier 34. Another function of lower grid
element 118 is to provide a flow path for first sample 56. Like
lower ion-permeable barrier 32 and lower gasket 112, lower grid
element 118 suitably also has alignment holes 116.
[0110] Above lower grid element 118 is generally planar
ion-permeable barrier 34. The external shape of ion-permeable
barrier 34 is generally the same as that of lower gasket 112 and
the interior of cartridge 100 so that ion-permeable barrier 34 is
configured to fit inside cartridge 100. Ion-permeable barrier 34
substantially prevents convective mixing of the contents of first
sample chamber 26 and second sample chamber 28, while permits
selective trans-barrier transport of selected constituents upon
application of the electric potential.
[0111] In one embodiment, ion-permeable barrier 34 is formed from a
membrane with a characteristic average pore size and pore-size
distribution. The average pore size and pore size distribution of
the membrane is selected to facilitate trans-membrane transport of
certain constituents, while substantially preventing trans-membrane
transport of other constituents.
[0112] In another embodiment, ion-permeable barrier 34 is an
isoelectric ion-permeable barrier, such as an isoelectric membrane
that substantially prevents convective mixing of the contents of
first sample chamber 26 and second sample chamber 28, while permits
selective trans-barrier transport of selected constituents upon
application of the electric potential. Suitable isoelectric
membranes can be produced by copolymerizing acrylamide,
N,N'-methylene bisacrylamide and appropriate acrylamido derivatives
of weak electrolytes yielding isoelectric membranes with pI values
in the 2 to 12 range, and average pore sizes that either facilitate
or substantially prevent trans-membrane transport of components of
selected sizes.
[0113] Above ion-permeable barrier 34 are three upper components:
upper grid element 120, upper ion-permeable barrier 38, and upper
gasket 124. These three components are placed so that upper grid
element 120 is immediately above ion-permeable barrier 34,
ion-permeable barrier 38 is immediately above upper grid element
120, and upper gasket 124 is immediately above ion-permeable
barrier 38. The configuration of the three upper components
suitably mirrors that of the lower three components.
[0114] Components below ion-permeable barrier 34 having alignment
holes 116 may be connected together with a fastener, which is any
type of connector configured to interact with alignment holes 116
and facilitate through flow of first sample 56. Similarly,
components above ion-permeable barrier 34 having alignment holes
116 may be connected together with a fastener, which is any type of
connector configured to interact with alignment holes 116 and
facilitate through flow of second sample 66.
[0115] Components of cartridge 100 are suitably held in cartridge
100 by clip 126. Clip 126 is suitably snap fitted or glued around
the top of walls 104 and 106 of cartridge 100.
[0116] Ion-permeable barrier 38 substantially prevents convective
mixing of the contents 6f second electrolyte chamber 24 and second
sample chamber 28, while permits selective trans-barrier transport
of selected constituents upon application of the electric
potential.
[0117] In one embodiment, ion-permeable barrier 38 is formed from a
membrane with a characteristic average pore size and pore-size
distribution. The average pore size and pore size distribution of
the membrane is selected to facilitate trans-membrane transport of
certain constituents, while substantially preventing trans-membrane
transport of other constituents.
[0118] In another embodiment, ion-permeable barrier 38 is an
isoelectric ion-permeable barrier, such as an isoelectric membrane
that substantially prevents convective mixing of the contents of
second electrolyte chamber 24 and second sample chamber 28, while
permits selective trans-barrier transport of selected constituents
upon application of the electric potential. Suitable isoelectric
membranes can be produced by copolymerizing acrylamide,
N,N'-methylene bisacrylamide and appropriate acrylamido derivatives
of weak electrolytes yielding isoelectric membranes with pI values
in the 2 to 12 range, and average pore sizes that facilitate or
substantially prevent trans-membrane transport of components of
selected sizes.
[0119] Preferred grid elements 118 and 120 are shown in more detail
in FIGS. 4 to 7. FIG. 4A shows a plan view of a preferred grid
element which is incorporated as a component of cartridge 100 for
separation unit 2. An elongate rectangular cut-out portion 128
which incorporates lattice 131 is defined in the center of the grid
element. At each end of the grid element, an alignment hole 116 is
suitably provided for alignment with the other components of
cartridge 100. Preferably, a triangular channel area 130 having
sides and a base, extends and diverges from each alignment hole 116
to cut-out portion 128. Upstanding ribs 132, 134, and 136 (best
shown in FIGS. 6 and 7) are defined in channel area 130. Liquid
flowing through hole 116 thus passes along triangular channel area
130 between ribs 132, 134, and 136 and into lattice 131. Ribs 132,
134, and 136 direct the flow of liquid from hole 116 so that they
help ensure that liquid is evenly distributed along the
cross-section of lattice 131. Ribs 132, 134, and 136 also provide
support to ion-permeable barrier 34 disposed above or below the
grid element.
[0120] Lattice 131 comprises a first array of spaced parallel
members 138 extending at an angle to the longitudinal axis of the
grid disposed above and integrally formed with a second lower set
of spaced parallel members 140 extending at approximately twice the
angle of the first array of parallel members 138 to the
longitudinal axis of the grid. In the presently preferred
embodiment, the first array of parallel members 138 extend at
approximately a 45 degree angle from the longitudinal axis and the
second array of parallel members 140 extend at approximately 90
degrees to the first array of parallel members 138, however, other
angles are also suitably used.
[0121] Referring to FIG. 4B, the reverse side of the grid element
is illustrated. The reverse side is suitably relatively smooth and
flat aside from cut-out area 128 and alignment holes 116. The
smooth, flat surface tends to ensure sealing between ion-permeable
barriers 32 and grid element 118, and ion-permeable barrier 38 and
grid element 120, respectively.
[0122] Referring to FIG. 5, the upper and lower surfaces of first
and second parallel members 138 and 140 are preferably rounded.
When parallel members 138 and 140 are rounded, the absence of any
sharp edges help prevent damage to ion-permeable barrier 34 and
provide extra support. Lattice 131 evenly distributes the flow of
liquid over the surface of ion-permeable barrier 34. The use of a
first set of members 138 disposed above a second set of members 140
tends to ensure that the liquid in a stream is forced to move up
and down, changing direction frequently, which helps to encourage
mixing of the liquid and tends to inhibit static flow zones.
[0123] The thickness of the grid element is preferably relatively
small. In one presently preferred embodiment, exterior areas 144 of
the element are 0.8 mm thick. Sealing ridge 142 (also shown in
FIGS. 4A and 4B) extends around the periphery of lattice 131 to
improve sealing. Ridge 142 is preferably approximately 1.2 mm thick
measured from one side of the grid element to the other. The
distance between the opposite peaks of lattice elements 138 and 140
measured from one side of the grid to the other is preferably
approximately 1 mm. The relatively small thickness of the grid
provides several advantages. First, it results in a more even
distribution of liquid over ion-permeable barrier 34 and assists in
inhibiting its fouling by macromolecules.
[0124] Also, the volume of liquid required is decreased by the use
of a relatively thin grid which enables relatively small sample
volumes to be used for laboratory-scale separations, a significant
advantage over prior art separation devices.
[0125] Finally, if the electric field strength is maintained
constant, the use of a relatively thinner grid element enables less
electrical power to be deposited into the liquid. If less heat is
transferred into the liquid, the temperature of the liquid remains
lower. This is advantageous, since high temperatures may destroy
both the sample and the desired product.
[0126] FIG. 8 illustrates grid element 144 for an alternative
embodiment of the present invention. Grid element 144 utilizes an
ion-permeable barrier having a much larger surface area than that
of grid elements 118 and 120. The principal operation of grid
element 144 is suitably generally the same as that of the smaller
grid elements although holes 146 through which first sample 56 or
second sample 66 are fed are located in two opposite corners of
grid 144 and there are many more channels 148 feeding streams from
holes 146 to central portion 150 of grid 144. The cartridge,
cartridge casing, and other components are increased in size and
shape so as to match that of grid 144.
[0127] FIG. 9 is a diagram of a presently preferred embodiment of a
separation apparatus 200 for use in accordance with the present
invention. The separation apparatus comprises separation unit 2
configured to accept cartridge 100 and clamp 86. Clamp 86 is used
to fix separation unit 2 in place once a component cartridge is
placed into separation unit 2. In the presently preferred
embodiment, clamp 86 is constructed from aluminum and is preferably
anodized. Clamp 86 is preferably a simple screw clamp unit so that
a screw-operated knob may be used to open and close clamp 86. The
separation apparatus shows first sample reservoir 50, second sample
reservoir 60, and first and second electrolyte reservoirs 42 and 43
in electrolyte compartment 202.
[0128] In order that the present invention may be more clearly
understood, examples of separation methodology are described with
reference to the preferred forms of the separation technology as
described.
EXAMPLE 1
[0129] An apparatus according to the present invention, shown in
FIG. 9, was used to separate the proteins present in chicken
egg-white into two fractions. An electrophoresis separation
cartridge, shown in FIGS. 3 to 7, was adapted to be used in the
apparatus. The first ion-permeable barrier placed between the first
electrolyte chamber and the first sample chamber was a pI=4.0
isoelectric membrane prepared from Immobiline chemicals (Pharmacia,
Sweden), acrylamide and N-N'-methylene bis-acrylamide. The second
ion-permeable barrier placed between the first sample chamber and
the second sample chamber was a pI=5.0 isoelectric membrane
prepared from Immobiline chemicals (Pharmacia, Sweden), acrylamide
and N-N'-methylene bis-acrylamide. The third ion-permeable barrier
placed between the second sample chamber and the second electrolyte
chamber was a pI=7.0 isoelectric membrane prepared from Immobiline
chemicals (Pharmacia, Sweden), acrylamide and N-N'-methylene
bis-acrylamide.
[0130] The first electrolyte reservoir was filled with 60 mL of an
80 mM acetic acid solution, pH 2.9. The second electrolyte
reservoir was filled with 60 mL of an 8 mM triethanol amine
solution, pH 9.9. The first and second sample reservoirs were
filled with 30 mL each of a filtered chicken egg-white solution,
diluted with deionized water at a rate of 1 to 25. The anode was
placed into the first electrolyte chamber, the cathode into the
second electrolyte chamber. The applied potential was 250 V, the
separation time was 15 minutes. Aliquots were taken for analysis
from the sample reservoirs before separation and at the end of the
separation.
[0131] Full-column-imaging capillary isoelectric focusing on an
iCE280 instrument (Convergent Bioscience, Toronto, Canada) was used
to analyze the egg-white samples. The fused silica separation
capillary was 5 cm long, its internal diameter was 100 micrometer.
The focusing medium contained 8% carrier ampholytes to cover the pH
3-10 range, in an aqueous, 0.1% methylcellulose solution.
Seventy-five microliter of the sample to be analyzed was mixed with
150 microliter of the focusing medium, filled into the capillary
and focused for 5 minutes at 3,000 V. Dansyl phenylalanine
(pI=3.52) and terbutaline (PI=9.61) were used as pI markers.
[0132] FIG. 10 shows the results for the egg-white feed sample. The
peaks between pixels 650 and 850 correspond to ovalbumin isoforms,
those between pixels 1250 and 1350 correspond to ovotransferrin
isoforms.
[0133] As a result of the electrophoretic separation, proteins with
pI values lower than 5.0, such as ovalbumin (pI=4.7), accumulated
in the first sample reservoir, on the anodic side of the pI=5.0
isoelectric membrane (bottom panel in FIG. 11). Proteins with pI
values greater than 5.0, such as ovotransferrin (pI=6.1)
accumulated in the second sample reservoir, on the cathodic side of
the isoelectric membrane (top panel in FIG. 11).
EXAMPLE 2
[0134] The same apparatus as in Example 1 was used to separate the
proteins present in chicken egg-white into two fractions. An
electrophoresis separation cartridge, shown in FIGS. 3 to 7, was
adapted to be used in the apparatus. The first ion-permeable
barrier placed between the first electrolyte chamber and the first
sample chamber was a polyacrylamide membrane with a nominal
molecular mass cut-off of 5,000 dalton. The ion-permeable barrier
between the first sample chamber and the second sample chamber was
a pI 5.0 isoelectric membrane prepared from Immobiline chemicals
(Pharmacia, Sweden), acrylamide and N-N'-methylene bis-acrylamide
as in Example 1. The third ion-permeable barrier placed between the
second sample chamber and the second electrolyte chamber was a
polyacrylamide membrane with a nominal molecular mass cut-off of
5,000 dalton.
[0135] The first electrolyte reservoir was filled with 60 mL of a 2
mM acetic acid solution, pH 3.8. The second electrolyte reservoir
was filled with 60 mL of an 8 mM triethanol amine solution, pH 9.9.
The first and second sample reservoirs were filled with 30 mL each
of a filtered chicken egg-white solution, diluted with deionized
water at a rate of 1 to 25. The anode was placed into the first
electrolyte chamber, the cathode into the second electrolyte
chamber. The applied potential was 250 V, the separation time was
15 minutes. Aliquots were taken for analysis from the sample
reservoirs at the end of the separation.
[0136] Full-column-imaging capillary isoelectric focusing on an
iCE280 instrument (Convergent Bioscience, Toronto, Canada) was used
to analyze the egg-white samples. The fused silica separation
capillary was 5 cm long, its internal diameter was 100 micrometer.
The focusing medium contained 8% carrier ampholytes to cover the pH
3-10 range, in an aqueous, 0.1% methylcellulose solution.
Seventy-five microliter of the sample to be analyzed was mixed with
150 microliter of the focusing medium, filled into the capillary
and focused for 5 minutes at 3,000 V. Dansyl phenylalanine
(pI=3.52) and terbutaline (pI=9.61) were used as pI markers.
[0137] As a result of the electrophoretic separation, proteins with
pI values lower than 5.0, such as ovalbumin (pI=4.7), accumulated
in the first sample reservoir, on the anodic side of the pI=5.0
isoelectric membrane (bottom panel in FIG. 12). Proteins with pI
values greater than 5.0, such as ovotransferrin (pI=6.1)
accumulated in the second sample reservoir, on the cathodic side of
the pI=5.0 isoelectric membrane (top panel in FIG. 12). Neither
ovalbumin nor ovotransferrin were lost into the first or second
electrolyte chambers despite the fact that the first and third
ion-permeable barriers were not isoelectric membranes as in Example
1. At the end of the separation, the solution pH in the first and
second sample reservoirs was 4.7 and 6.7, respectively.
EXAMPLE 3
[0138] The same apparatus as in Example 1 was used to separate the
proteins present in chicken egg-white into two fractions. The first
ion-permeable barrier placed between the first electrolyte chamber
and the first sample chamber was a polyacrylamide membrane with a
nominal molecular mass cut-off of 1,000,000 dalton. The
ion-permeable barrier between the first sample chamber and the
second sample chamber was a pI 5.0 isoelectric membrane prepared
from Immobiline chemicals (Pharmacia, Sweden), acrylamide and
N-N'-methylene bis-acrylamide as in Example 1. The third
ion-permeable barrier placed between the second sample chamber and
the second electrolyte chamber was a polyacrylamide membrane with a
nominal molecular mass cut-off of 1,000,000 dalton.
[0139] The first electrolyte reservoir was filled with 60 mL of a 2
mM acetic acid solution, pH 3.8. The second electrolyte reservoir
was filled with 60 mL of an 8 mM triethanol amine solution, pH 9.9.
The first and second sample reservoirs were filled with 30 mL each
of a filtered chicken egg-white solution, diluted with deionized
water at a rate of 1 to 25. The anode was placed into the first
electrolyte chamber, the cathode into the second electrolyte
chamber. The applied potential was 250 V, the separation time was
15 minutes. Aliquots were taken for analysis from the sample
reservoirs at the end of the separation, and analyzed by
full-column-imaging capillary isoelectric focusing on an iCE280
instrument.
[0140] As a result of the electrophoretic separation, proteins with
pI values lower than 5.0, such as ovalbumin (pI=4.7), accumulated
in the first sample reservoir, on the anodic side of the pI=5.0
isoelectric membrane (bottom panel in FIG. 13). Proteins with pI
values greater than 5.0, such as ovotransferrin (pI=6.1)
accumulated in the second sample reservoir, on the cathodic side of
the pI=5.0 isoelectric membrane (top panel of FIG. 13). Neither
ovalbumin nor ovotransferrin were lost into the first or second
electrolyte chambers despite the fact that the average pore size of
the first and third ion-permeable barriers was large enough to
permit their passage through these barriers. At the end of the
separation, the solution pH in the first and second sample
reservoirs was 4.7 and 6.2, respectively.
EXAMPLE 4
[0141] The same apparatus as in Example 1 was used to purify
immunoglobulin G (IgG) from human plasma. The first ion-permeable
barrier placed between the first electrolyte chamber and the first
sample chamber was a polyacrylamide membrane with a nominal
molecular mass cut-off of 150,000 dalton. The ion-permeable barrier
between the first sample chamber and the second sample chamber was
a pI 5.8 isoelectric membrane prepared from Immobiline chemicals
(Pharmacia, Sweden), acrylamide and N-N'-methylene bis-acrylamide.
The third ion-permeable barrier placed between the second sample
chamber and the second electrolyte chamber was a polyacrylamide
membrane with a nominal molecular mass cut-off of 150,000
dalton.
[0142] The first electrolyte reservoir was filled with 2 L of a 2
mM 5-amino caproic acid solution that also contained 5 mM NaCl, its
pH was adjusted to 4.8 with HCl. The second electrolyte reservoir
was filled with 2 L of a 2 mM MOPSO solution that also contained 5
mM NaCl, its pH was adjusted to 6.8 with NaOH. The anode was placed
into the first electrolyte chamber, the cathode into the second
electrolyte chamber. The applied potential was 250 V. Initially,
both sample reservoirs were filled with deionized water. Potential
was applied for 2 minutes to remove any unpolymerized material from
the membranes. After 2 minutes, all reservoirs were emptied, the
electrolyte reservoirs were refilled with fresh electrolytes, the
sample reservoirs were filled with 15 mL each of human plasma
diluted 1 to 3 with deionized water. Potential was applied for 40
minutes. Aliquots were taken for analysis from the sample reservoir
chambers at 0, 10, 20, and 40 minutes, respectively.
[0143] FIG. 14 shows the image of an SDS-PAGE gel used to analyze
the protein bands present in the aliquots collected from the first
and second sample reservoirs of the electrophoretic apparatus
during the isolation of IgG from human plasma. Molecular weight
markers (Sigma, St. Louis, Mo., USA) were applied onto Lane 1, a
pharmaceutical-grade IgG preparation used as reference material
onto Lane 2. Samples taken at 0, 10, 20, and 40 minutes
respectively from the first sample reservoir were applied onto
Lanes 3, 4, 5, and 6. Samples taken at 0, 10, 20, and 40 minutes
respectively from the second sample reservoir were applied onto
Lanes 7, 8, 9, and 10. IgG was purified within 40 minutes.
[0144] These examples indicate that remarkably rapid separation of
ampholytic components can be achieved using the apparatus and
method disclosed here. The high production rates are attributed to
the short electrophoretic migration distances, high electric field
strength and good heat dissipation characteristics of the
system.
[0145] The invention has been described herein by way of example
only. It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive. Other features and
aspects of this invention will be appreciated by those skilled in
the art upon reading and comprehending this disclosure. Such
features, aspects, and expected variations and modifications of the
reported results and examples are clearly within the scope of the
invention where the invention is limited solely by the scope of the
following claims.
* * * * *