Ampholyte separation method and apparatus

Claims

What is claimed is:

1. An ampholyte separation apparatus comprising:

a lower base plate;

an upper base plate;

at least two transversely extending barriers positioned between said upper and lower base plates and defining at least one transversely extending channel therebetween;

a first semipermeable membrane abutting one end of each of said at least two transverse barriers;

a second semipermeable membrane adjacent to and spaced-apart from the other end of each of said at least two transverse barriers so as to define a sample flow channel therebetween in fluid communication with said at least one transversely extending channel;

a first electrode adjacent to said first semipermeable membrane and separated from said transversely extending barriers by said first semipermeable membrane;

a second electrode adjacent to said second semipermeable membrane and separated from said sample flow channel by said second permeable membrane;

a first temperature controllable thermal mass positioned in proximity to said first electrode; and

a second temperature controllable thermal mass positioned in proximity to said second electrode;

whereby an electric field and a pH gradient may be established along the length of said at least two transverse barriers so as to cause ampholytes flowing through said sample flow channel to be forced into said at least one transversely extending channel to differential positions corresponding to the isoelectric points of the ampholytes.

2. An apparatus according to claim 1 wherein said upper and lower base plates, said at least two transverse barriers and said thermal masses comprise copper.

3. An apparatus according to claim 2 wherein said upper and lower base plates, said at least two transverse barriers and said thermal masses are coated with polyethylene.

4. An apparatus according to claim 3 wherein acrylamide is grafted onto said polyethylene coating.

5. An apparatus according to claim 1 wherein said semipermeable membranes comprise cellulose acetate.

6. An apparatus according to claim 1 wherein said first electrode is a cathode and said second electrode is an anode.

7. An apparatus according to claim 6 wherein said electrodes comprise platinum.

8. An ampholyte separation apparatus comprising:

a lower copper base plate;

an upper copper base plate;

a plurality of transversely extending copper barriers positioned between said upper and lower base plates and defining a plurality of transversely extending channels therebetween;

a first semipermeable membrane of cellulose acetate extending along the length of said base plates and abutting one end of each of said transverse barriers;

a second semipermeable membrane of cellulose acetate extending along the length of said base plates and being spaced-apart from the other end of each of said transverse barriers so as to define a sample flow channel therebetween in fluid communication with said transversely extending channels;

a platinum cathode extending along and parallel to said first semi-permeable membrane and being separated from said transverse barriers by said first semi-permeable membrane;

a platinum anode extending along and parallel to said second semi-permeable membrane and being separated from said flow channel by said second semi-permeable membrane;

a first copper thermal mass positioned adjacent said cathode, said first copper thermal mass having an independent temperature control means; and

a second copper thermal mass positioned adjacent said anode, said second copper thermal mass having an independent temperature control means;

whereby an electric field and a pH gradient may be established along the length of said transverse barriers so as to cause ampholytes traveling through said flow channel to be forced into said transversely extending channels to differential positions corresponding to the isoelectric points of the ampholytes.

9. An ampholyte separation apparatus comprising: at least two elongate barriers defining at least one elongate channel therebetween;

a first semi-permeable membrane positioned transverse to said at least two elongate barriers and adjacent to one end of each of said at least two elongate barriers;

a second semi-permeable membrane positioned transverse to said at least two elongate barriers and adjacent to the other end of each of said at least two elongate barriers;

means for creating an electric field along the length of said at least two elongate barriers, said means for creating an electric field being positioned adjacent to said semi-permeable membranes so as to be separated from said elongate barriers by said semi-permeable membranes; and

at least one flow channel extending substantially parallel to said semi-permeable membranes and substantially transverse to said at least two elongate barriers.

10. An ampholyte separation apparatus comprising:

at least two elongate barriers defining at least one elongate channel therebetween;

a first semi-permeable membrane positioned transverse to said at least two elongate barriers and adjacent to one end of each of said at least two elongate barriers;

a second semi-permeable membrane positioned transverse to said at least two elongate barriers and adjacent to the other end of each of said at least two elongate barriers;

means for creating an electric field along the length of said at least two elongate barriers, said means for creating an electric field being positioned adjacent to said semi-permeable membranes so as to be separated from said elongate barriers by said said semi-permeable membranes;

at least one flow channel extending substantially parallel to said semi-permeable membranes and substantially transverse to said at least two elongate barriers; and

means for thermally establishing and controlling a pH gradient along the length of said at least two elongate barriers.

11. A process for separating ampholytes comprising:

directing a solution containing one or more ampholytes through a flow channel which is transverse to at least two elongate barriers defining at least one elongate channel therebetween;

establishing an electric field along the length of said elongate barriers;

thermally engendering a pH gradient along the length of said elongate barriers so as to cause at least one of the ampholytes to migrate from the flow channel into said at least one elongate channel and stabilize at a position corresponding to the isoelectric point of the at least one ampholyte; and

altering the pH gradient so as to cause the at least one ampholyte stabilized in said at least one elongate channel to reenter said flow channel and to be eluted and separately collected.

12. A process according to claim 11 wherein the pH gradient is thermally engendered so as to cause all of the ampholytes in solution to migrate into said at least one elongate channel and to stabilize at positions corresponding to their isoelectric points and wherein the pH gradient is altered so as to cause the ampholytes to reenter said flow channel at different times and to be separately eluted and collected.

13. A process according to claim 11 wherein the pH gradient is thermally engendered so as to cause one or more selected ampholytes to migrate into said at least one elongate channel and stabilize at positions corresponding to their isoelectric points while the other ampholytes remain in said flow channel so as to be eluted, and wherein the pH gradient is altered so as to cause the one or more selected ampholytes to reenter said flow channel at different times and to be separately eluted and collected.

14. A process for separating ampholytes comprising:

directing a solution containing two or more ampholytes through a flow channel which is transverse to a plurality of elongate barriers, said elongate barriers defining a plurality of elongate channels;

providing a first electrode adjacent one end of the transverse barriers and extending in the direction of the flow channel, said first electrode being separated from the transverse barriers by a first semi-permeable membrane which abuts the one end of said transverse barriers;

providing a second electrode spaced-apart from the other end of the transverse barriers and extending in the direction of the flow channel, said second electrode being separated from the transverse barriers by a second semi-permeable membrane which is also spaced-apart from the other end of the transverse barriers and is adjacent to the flow channel;

generating an electric field along the length of the transverse barriers with the first and second electrodes;

providing a first temperature controllable thermal mass adjacent to said first electrode so that said first electrode is positioned between said first semi-permeable membrane and said first thermal mass;

providing a second temperature controllable thermal mass adjacent to said second electrode so that said second electrode is positioned between said second semi-permeable membrane and said second thermal mass;

thermally engendering a pH gradient along the transverse barriers with the first and second thermal masses so as to cause at least one of the ampholytes to migrate from the flow channel into the elongate channels and stabilize at a position corresponding to the isoelectric point of the at least one ampholyte; and

altering the pH gradient so as to cause the at least one ampholyte stabilized in the elongate channels to reenter the flow channel and to be eluted and separately collected.

15. A process according to claim 14 wherein said flow channel, said transverse barriers and said thermal masses comprise copper.

16. A process according to claim 15 wherein said flow channel, said transverse barriers and said thermal masses are coated with polyethylene.

17. A process according to claim 16 wherein acrylamide is grafted onto said polyethylene coating.

18. A process according to claim 14 wherein the semi-permeable membranes comprise cellulose acetate.

19. A process according to claim 14 wherein the first electrode is a cathode and the second electrode is an anode.

20. A process according to claim 19 wherein the electrodes comprise platinum.

21. A process according to claim 14 wherein the pH gradient is thermally engendered so as to cause all of the ampholytes contained in the solution to migrate into the elongate channels and stabilize at positions corresponding to their isoelectric points and wherein the pH gradient is altered so as to cause the ampholytes to reenter the flow channel at different times and to be separately eluted and collected.

22. A process according to claim 14 wherein the pH is thermally engendered so as to cause one or more selected ampholytes to migrate into the elongate channels and stabilize at positions corresponding to their isoelectric points while the other ampholytes remain in the flow channel so as to be eluted and wherein the pH gradient is altered so as to cause the one or more selected ampholytes to reenter the flow channel at different times and to be separately eluted and collected.

23. A process for separating ampholytes comprising:

directing a solution containing two or more ampholytes with at least two of the ampholytes having identical isoelectric points but different mobilities through a flow channel which is transverse to a plurality of elongate barriers defining a plurality of elongate channels;

establishing an electric field along the length of said elongate barriers;

thermally engendering a pH gradient along the length of said elongate barriers so as to cause at least one of the ampholytes to migrate from the flow channel into the elongate channels and stabilize at a position corresponding to the isoelectric point of the at least one ampholyte and so as to cause the at least two ampholytes having identical isoelectric points but different mobilities to migrate into different elongate channels due to the different mobilities of the at least two ampholytes; and

altering the pH gradient so as to cause the at least one ampholyte to reenter the flow channel and to be separately eluted and collected and so as to cause the at least two ampholytes having identical isoelectric points to reenter the flow channel at different positions along the flow channel due to their location in different elongate channels and to also be separately eluted and collected.

24. A process for separating ampholytes comprising:

directing a solution containing one or more ampholytes through one of one or more flow channels which are transverse to at least two elongate barriers which define at least one elongate channel between said barriers;

establishing an electric field along the length of said elongate barriers so as to cause at least one of the ampholytes to migrate at a speed according to its electrophoretic mobility from said one of one or more flow channels through said at least one elongate channel and into one of the one or more flow channels to be eluted and separately collected.

25. A process for separating ampholytes comprising:

directing a solution containing one or more ampholytes through one of one or more flow channels which are transverse to at least two elongate barriers defining at least one elongate channel therebetween;

establishing an electric field along the length of said elongate barriers;

thermally engendering a pH gradient along the length of said elongate barriers so as to cause at least one of the ampholytes to migrate from the flow channel into said at least one elongate channel; and

altering the pH gradient so as to cause the at least one ampholyte in said elongate channel to enter one of said one or more flow channels and to be eluted and separately collected.

26. A process for separating ampholytes comprising:

directing a first solution containing one or more ampholytes through one of one or more flow channels which are transverse to at least two elongate barriers defining at least one elongate channel therebetween;

establishing an electric field along the length of said elongate barriers;

thermally engineering a pH gradient along the length of said elongate barriers so as to cause at least one of the ampholytes to migrate from the flow channel into said at least one elongate channel; and

flushing a second solution through the transverse channels so as to cause the at least one ampholyte in said elongate channel to enter one of said one or more flow channels and to be eluted and separately collected.

27. A process for separating ampholytes comprising:

directing a solution containing one or more ampholytes through a flow channel which is transverse to at least two elongate barriers defining at least one elongate channel therebetween, said two elongate barriers having a semi-permeable membrane abutting the ends of said elongate barriers opposite said flow channel;

establishing an electric field along the length of said elongate barriers so as to cause at least one of the ampholytes to migrate from the flow channel into said at least one elongate channel and stabilize at the boundary defined by said semipermeable membrane; and

reversing the electric filed so as to cause the at least one ampholyte stabilized at said boundary to migrate through said at least one elongate channel towards said flow channel at a speed according to the electrophoretic mobility of the ampholyte and to reenter said flow channel to be eluted and separately collected.

DESCRIPTION

1. Technical Field

This invention relates to the separation of biological materials and more specifically, to an ampholyte separation method and apparatus wherein a novel arrangement of a flow channel transverse barriers and electrodes are utilized with a readily controllable pH gradient to provide for the efficient separation of ampholytes having varying isoelectric points.

2. Background Art

The separation of biological materials from their natural environment is becoming increasingly important in light of the current advancements being made in biotechnology and related fields. Separation and purification techniques are especially important since the effectiveness of biological materials, such as proteins, is frequently dependent on the purity with which the material may be isolated.

Amphoteric biological materials, commonly known as ampholytes, maintain a positive charge in acidic media nd a negative charge in basic media. Each ampholyte has a characteristic isoelectric point which corresponds to the pH at which the ampholyte has a zero net charge. In the presence of a pH gradient and an electric field, an ampholyte will migrate electrophoretically to a position within the pH gradient where the net charge of the molecule is zero. Traditional isoelectric focusing (IEF) techniques utilize an established pH gradient and an electric field so as to focus the ampholyte into zones defined by the pH of the medium and the electric field applied.

For example, U.S. Pat. No. 4,204,929 describes an isoelectric focusing method whereby the fluid to be separated is pumped through generally parallel channels which are composed of permeable microporous membranes. The fluid is pumped in a direction parallel to the permeable channels and an electrical potential is applied across the streamlined channels. A pH gradient is established across the channels such that the electrical field causes various ampholytes to flow through the membranes of the separating channels and eventually migrate to the channel corresponding to the pH of the isoelectric point of each ampholyte. The fluid in each of the streamlined channels is recirculated such that the fluid flowing out of each channel is recirculated back to the entrance of the channel. The fluids are also cooled in the process of recirculation so as to minimize problems with dissipation of Joulian heat during isoelectric focusing.

A horizontal isoelectric focusing apparatus is described in U.S. Pat. No. 4,234,404. The apparatus includes a hollow elongated electrophoresis vessel which has an electrode at each opposite end. An unobstructed zone is provided in the interior of the vessel and is located at the bottom of the vessel when the vessel is in the normal position for electrophoresis. Located above the unobstructed zone are a plurality of upright, parallel, horizontally spaced partitions defining isolated compartments. The separation is carried out by depositing the fluid to be separated into the vessel such that it forms a liquid layer in the unobstructed zone. An electric potential is applied across the electrodes so that the components of the fluid migrate horizontally due to their different electrophoretic mobilities and eventually stabilize corresponding to their isoelectric points. The electric potential is then discontinued and the vessel is rotated about its horizontal longitudinal axis so as to bring the partitions into contact with the fluid layer. The respective fractions of separated components are thereby captured within isolated compartments and can then be recovered from the vessel.

An isoelectric focusing method and apparatus are also described in U.S. Pat. No. 4,670,119. The IEF device includes a series of parallel electrodes located around the border of a focusing chamber. The voltages of the electrodes may be controlled by the user so as to maintain various voltages along the length of the chamber. The ionic species present in the buffer solution will be concentrated at either the anode or cathode end of the chamber. The resulting charge imbalance will cause the dissociation of water thereby establishing a pH gradient. This technique allows the formation of a wide range of pH gradients, and the gradient may be altered during the isoelectric focusing process without disturbing the components being separated.

Also of interest, an apparatus for continuous electrochromatographic separation is described in U.S. Pat. No. 4,315,812. A vertical sandwich of rectangular glass containing a filter material is provided and a sample to be separated is pumped into the top of the vertical chamber such that the fluid flows under the force of gravity through the chromatographic column. Simultaneously, an electric field is applied across the fluid path such that the charged molecules migrate horizontally according to their respective electrophoretic mobilities. Therefore, instead of the fluid sample traveling down the column in a single path, the electric field causes the fluid to separate into different bands of components which travel vertically in separate paths. The separate samples can then be collected along the lower edge of the glass sandwich.

In general, isoelectric focusing techniques are disadvantageous in that prefocusing of the pH gradients typically requires several hours of tedious preparation. The long preparation times required further prolong the already lengthy IEF separation process. Additionally, the gels and other materials utilized to stabilize the pH gradient create difficulties in detecting and recovering the separated ampholytes.

Field-flow fractionation (FFF) is a method for separating particles according to size and is based on the differential flow profile through a channel. The particles are concentrated by an applied force (e.g., electric, magnetic, thermal, centrifugal, etc.) in various regions of the flow and will travel through the channel at different speeds thus being eluted at varying times. However, FFF techniques have been limited to analyses of small samples by the fact that the separation channels used are necessarily of small dimension in the direction of fractionation in order to maintain the differential flow profiles needed.

A need therefore exists for a method and apparatus for separating amphoteric species that utilizes a readily established and controllable pH gradient and that will facilitate the detection and recovery of the separated ampholyte molecules. The novel procedure and corresponding apparatus described herein should solve many of the problems associated with presently existing isoelectric focusing and field-flow fractionation techniques.

DISCLOSURE OF THE INVENTION

The separation apparatus of the present invention comprises a flow channel and a series of evenly and closely-spaced channels positioned transversely to the flow channel. Two electrodes are positioned along the length of the separation apparatus so that one is adjacent each of the outside ends of the closely-spaced channels and the other electrode is spaced-apart from the inside ends of the channels and positioned adjacent the flow channel. A thermal mass is located adjacent to each of the electrode compartments and extends along the length of the separation apparatus. Semipermeable membranes separate the electrode compartments from the sample flow channel and the closely-spaced transverse channels. A bottom plate and a top plate enclose the apparatus so as to create a fluid-tight separation chamber.

The method of the present invention comprises pumping a solution of mixed ampholytes through the flow channel while utilizing the thermal masses to create a temperature and corresponding pH gradient along the length of the closely-spaced transverse channels. The electrodes are utilized in order to generate an electric field along the length of the transverse channels so that each ampholyte will migrate from the flow channel into the transverse channels to a position corresponding to its isoelectric point. The thermal masses are then manipulated so as to change the pH gradient and cause the separated ampholytes to reenter the flow stream at different times so as to be separately eluted and collected.

It is therefore an object of the present invention to provide an ampholyte separation method and apparatus that will facilitate the detection and recovery of the separated ampholytes.

It is another object of the present invention to provide an ampholyte separation method and apparatus that will minimize the separation process time by utilizing a readily established and easily controllable pH gradient in free buffer solution.

It is still another object of the present invention to provide an ampholyte separation method and apparatus that will allow for the analysis of relatively large samples of mixed ampholytes.

DESCRIPTION OF THE DRAWINGS

Some of the objects of the invention having been stated, other objects will become evident as the description proceeds, when taken in connection with the accompanying drawings, in which:

FIG. 1 is a perspective view taken of a section along the length of the separation apparatus of the invention with the top plate removed for clarity of illustration;

FIG. 2 is a top plan view of the separation apparatus of the invention with the top plate removed for clarity of illustration; and

FIG. 3 is an end elevation view of the separation apparatus of the invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Referring now more specifically to the drawings, a preferred embodiment of an ampholyte separation apparatus according to the present invention is shown in FIGS. 1-3. More specifically, FIG. 1 is a perspective view of a section 10 taken from the medial portion of the separation apparatus. Section 10 comprises an upper plate 12 and a lower plate 14 with lower plate 14 having transverse barriers 16 which form closely-spaced channels 17 therebetween (see FIG. 2). A semipermeable membrane 18 extends along and abuts the ends of transverse barriers 16 while a semipermeable membrane 20 extends along and is spaced-apart from the other ends of transverse barriers 16. The space between semipermeable membrane 20 and the ends of transverse barriers 16 defines a sample flow channel 21 (see FIGS. 2 and 3) through which the sample to be separated is directed as shown by F. A fully permeable membrane (not shown) may be placed along the ends of transverse barriers 16 defining flow channel 21 in order to streamline sample flow F and to minimize turbulence within transverse channels 17.

It shuld be noted that the semipermeable membranes and transverse barriers can be arranged in various configurations so as to allow for different positioning of the flow channel. For example, both semipermeable membranes may be spaced-apart from both ends of the transverse barriers so as to create a flow channel at both ends of the transverse barriers. Alternatively, the semipermeable membranes may abut both ends of the transverse barriers with a flow channel extending parallel to the membranes and through the middle portion of the transverse barriers.

As shown clearly in FIGS. 2 and 3, semipermeable membrane 18 and semipermeable membrane 20 define compartments or channels wherein electrodes are positioned. Specifically, a cathode 22 is contained within an electrode compartment 23 such that cathode 22 is separated from transverse barriers 16 and transverse channels 17 by semipermeable membrane 18. Likewise, an anode 24 is contained within an electrode compartment 25 such that anode 24 is separated from sample flow channel 21 by semipermeable membrane 20. During the separation process, a solution of buffer is passed through electrode compartments 23 and 25 as shown by B.

A thermal mass 26 (see FIGS. 2 and 3) is positioned adjacent to anode compartment 23 and may be integral with lower plate 14. Similarly, a thermal mass 28 is positioned ad