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1. WO1979000002 - PERFECTIONNEMENTS A L"ELECTROPHORESE AVEC MEMBRANE

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CLAIMS
1. An electrophoresis membrane of known isoelectric point
and having buffering capacity at its isoelectric point whereby the tendency for electroendosmosis to occur during electrophoresis is reduced.
2. A membrane according to claim 1 having high concentrations of acidic and basic groups the level of which has been adjusted in preparation to give the membrane an isoelectric point in a defined pH range.
3. A membrane according to claim 1 or 2, being a chemically treated agarose or polyacrylamide gel.
4. A membrane according to claim 1, 2 or 3» 'being a supported membrane.
5- A membrane according to any of claims 1 to 4, comprising ampholyte molecules attached to a substrate.
6. A membrane according to claim 5. in which the ampholyte molecules are chemically attached to the substrate.
7. A membrane according to claim 6, in which the attachment is by means of an intermediate linking group.
8. A membrane according to any of the preceding claims, having an isoelectric point in the range pH 4 to 6.
9. A method of separating a substance by membrane electrophoresis in which the substance is isoelectrically focussed in an electrolyte pH gradient by means of a membrane according to any of claims 1 to

8 which is located between electrolyte of pH lower than its isoelectric point on the anodic (+) side of the -membrane and electrolyte of pH higher than its isoelectric point on its
cathodic (-) side.

10. A method according to claim 9. using a series of such
membranes having isoelectric points increasing towards the cathode and an electrolyte pH gradient of increasing pH in the same direction.

11. A method according to claim 9 or 10, in which the electrolyte pH or pH gradient is provided by monovalent buffering ions.
12. A method according to claim 11, in which the buffering ions comprise ions of a weak organic acid, or a weak organic base, or both.
13- A method according to any of claims 9 to 12, in which the pH difference between opposite sides of the or each membrane
is of the order of 0.5 units.
14. A method according to any of claims 9 to 13, in which proteins or peptides are separated.
15. A membrane electrophoresis apparatus for isoelfectric focussing comprising a series of membranes according to any of claims 1 to

8 arranged in sequence of increasing isoelectric points towards the cathode (negative electrode) and means for establishing a series of circulating electrolyte zones having a pH gradient of increasing pH in the same direction.
16. Apparatus according to claim 15, comprising means for mounting membranes in a generally vertical series.
17. Apparatus according to claim 16, in which the membranes are in sheet form and arranged in a nesting configuration with edge sealing.
18. Apparatus according to claim 17, in which electrolyte flow through the compartments between the membranes is by gravity.

19. Apparatus according to any of claims 15 to 18, in which each electrolyte zone in the pH gradient is provided with cooling means.

20. Apparatus according to any of claims 15 to 19, having an anode in an acidic compartment separated from the membrane series by an anion exclusion membrane so that electricity is carried through the latter exclusively by hydrogen ions.
21. Apparatus according to claim 20 in which the anode is lead peroxide in dilute sulphuric acid.
22. Apparatus according to any of claims 15 to 21, having a cathode in an' alkaline compartment separated from the membrane series by a cation exclusion membrane so that electricity is carried through the latter exclusively by hydroxyl ions.
23. Apparatus according to claim 22, in which the cathode is mild steel in a dilute solution of an alkali metal hydroxide.