Background of the Invention International patent application No PCT/AU00/01391 filed in the name of the applicant of the present invention relates to such an electrolyser and to a method of using that electrolyser for sub-fractionation and subsequent separation of fractions from highly complex protein/peptide mixtures, such as those found in total cell lysates, body fluids and tissue extracts in general.

That application discloses an electrolyser in the form of a series of chambers separated by isoelectric membranes with end chambers located either end of the series of chambers. Electrode means for applying an electric field across the series of chambers are provided. A means for agitating any liquid media within the chambers without recirculation of the liquid is also disclosed. The means for agitating is typically a magnetic stirrer.

The present invention is directed to improvements to the apparatus of the type disclosed in PCT/AU00/01391, the entire contents of which are incorporated herein by reference.

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.

Summary of the Invention In a first aspect, the present invention provides an apparatus for separating a mixture of components using isoelectric focusing in a liquid media, the apparatus comprising: a series of blocks defining chambers, the chambers being separable by isoelectric membranes of known pH; end chambers located at each end of the series of chambers;

at least two tie rods for use in clamping the blocks together to make the apparatus substantially liquid tight; and electrodes for use in applying an electric field across the series of chambers: characterised in that at least one of the tie rods is conducting and carries current from an electrode at one end of the apparatus to the opposite end of the apparatus.

Using one of the tie rods to carry current, allows power supply sockets for supplying power to the electrodes to be located at the same end of the apparatus.

This has the advantage that the apparatus is easier to use. By providing all the electrical connections on one end of the apparatus, the apparatus becomes suitable for use on a multi-use platform power supply and cooling system which provides electrical contacts along one side only of the platform.

The conducting tie rod will preferably be a steel rod, and is most preferably covered in an insulator along a substantial portion of its length.

It is preferred that there are four tie rods, although only one of the tie rods will typically be used to conduct electricity.

In a second aspect of the present invention, there is provided an apparatus for separating a mixture of components using isoelectric focusing in a liquid media comprising a series of blocks defining chambers, the chambers being separable by membranes and capable of being clamped together in a watertight arrangement, the apparatus including end blocks between which the series of chambers may be held by means of a plurality of tie rods extending through the blocks characterised in that a lever operated clamping mechanism is provided for tensioning the tie rods and clamping the chambers together between the end blocks in a liquid type fashion.

In a particularly preferred embodiment, one end of each of the tie rods is fixed with respect to one end block of the apparatus. The other end of each of the tie rods defines enlarged end portions. The other end block of the apparatus comprises a housing and a plate moveable relative to the housing.

The plate defines a series of keyhole shaped free apertures adapted to receive the ends of the tie rods and engage with the enlarged portions of the tie rods.

The clamping means is arranged to move the plate relative to the housing using a cam mechanism or the like.

In a particularly preferred embodiment, in cross-section the blocks and the plate are generally rectangular, typically square, in lateral cross-section.

The engagement of the ends of the tie rod to the plate is by means of a twist fitting with the tie rods being inserted with the plate being rotationally off-set relative to the apparatus and rotatable to align the cross-section of the plate with the blocks and engage the enlarged portions behind narrower portions of the keyhole shaped apertures.

The use of a lever makes the assembly of the apparatus much quicker than the traditional method which relies on wing nuts to tension the tie rods and also allows an increased pressure to be used and thus improves the sealing of the apparatus.

In a yet further aspect, the invention provides an apparatus for separating a mixture of components using isoelectric focusing in a liquid media, the apparatus comprising a series of blocks defining chambers, the chambers being open on front and rear faces of the blocks, the chambers being separable by membranes disposed between adjacent blocks, the blocks being capable of being clamped together in a watertight arrangement, characterised in that the membranes are retained in washers each of which defines a recess for receiving a membrane and locating the same in correct alignment with the chambers.

Typically, one face of each block defines a recess into which a corresponding annular protrusion locates and wherein an end wall of the recess defines a stepped portion and a front face of the annular protrusion defines a mating stepped portion the washer being stepped also and locating in use between the end wall and the front face of the annular protrusion The apparatus is typically designed to receive a set number of chamber blocks, typically seven, however, in some cases fewer chambers will be required.

Therefore, in related aspect, the present invention provides a dummy block defining a pair of spaced apart electrical contacts which are biased apart but electrically connected which is adapted to replace one of the chamber blocks in the apparatus while still allowing the passage of current along the chambers of the apparatus, in use.

The invention also provides a novel method of agitating fluid contained in chambers of the apparatus. In this aspect of the present invention, a well is provided beneath and open to the chamber in the block. The stirring means is

a magnetic disk and means for agitating the stirring means comprise a solenoid disposed beneath the magnetic disk, in operation. Typically this solenoid will be located in the platform which supplies any necessary cooling and power to the apparatus during use. The polarity of the solenoid is periodically reversed thereby causing movement of the magnetic plate.

Whilst a single solenoid may perform this function, a plurality of solenoids could be used.

In a particularly preferred embodiment, the magnetic plate is encased in a housing which is marginally smaller than the well in which the plate is located such that the magnetic plate is prevented from turning over but is free to move along the (vertical) axis of the well. Preferably, the top of the well is partly closed to prevent the magnetic plate from leaving the well.

Brief Description of the Drawings Specific embodiments of the invention will now be described, by way of the example only, and with reference to the accompanying drawings in which: Figure 1 is a perspective view of an apparatus embodying the present invention; Figure 2 is a perspective view of a chamber block which forms part of the apparatus shown in Figure 1; Figure 3 is an underneath plan view of two chamber blocks engaged together; Figure 4 is a section through lines IV-IV of Figure 3; Figures 5a, 5b, and 5c are a perspective view, front elevation and a section through a stepped washer which forms part of the apparatus of Figure 1.

Figure 6 is a perspective view of parts of an apparatus embodying the present invention illustrating internal features of one end block in particular; Figure 7 is an end view of the apparatus shown in Figure 1 with part of the housing of the end block of the apparatus removed; Figure 8 is an end view of a plate ; Figure 9 is a perspective view of an end plate illustrating connectors for a conducting tie rod; Figure 10 is a perspective view of a dummy block ; Figure 11 is a front view of the dummy block of Figure 10; Figure 12 is a cross section on lines XII-XII of Figure 11;

Figure 13 shows the apparatus of Figure 1 located on a platform with a top plate of the platform removed; and Figure 14 is a top plan view of the platform of Figure 13; Figure 15 is a top plan view of the apparatus of Figure 1 located on a complete platform; Figure 16 is a cross sectional view on XVI-XVI of Figure 15; Figure 17 is an enlarged view of part of Figure 16; and Figure 18 illustrates an alternative stirring mechanism for the apparatus.

Detailed Description of the Preferred Embodiment Referring to the drawings, Figure 1 shows an assembled multi- compartment electrolyser apparatus generally indicated at 10. The apparatus includes seven identical chamber blocks 12, clamped between end blocks 14, 16.

Figures 2 to 4 shows the chamber blocks 12 in more detail. Each chamber block is generally square in lateral/transverse cross section and is generally hollow so that the central area of the block defines a chamber 18 As is best seen in Figure 3 an annular ring 20 projects from one (front) end face 22 of the block. The opposite (rear) end face 24 defines a corresponding recess adapted to receive the projecting ring of an adjacent block. A through bore is defined when a plurality of blocks are engaged end to end as illustrated in Figure 3. Four narrower through bores 25 are defined near the corners of the end faces of each blocks. These bores 25 receive tie rods 40 best seen in Figures 6 and 7 which are used to align the bores together and link and compress the chambers together.

The front face 22 of each chamber block also defines two additional cylindrical protrusions 28 which are received in corresponding recesses located on the rear face of a contiguous block for ensuring accurate alignment of the blocks.

At the top 30 of each block there is a sample inlet in the form of an aperture 32 for sample entry into the chamber 18. The top of each block is closed with a lid 34. Each chamber also defines a generally cylindrical shallow well or recess 36 which is typically coaxial with the sample inlet 32. In use, the well receives a magnetic disk which functions as a stirrer when placed in a magnetic field generated by a solenoid whose polarity is periodically reversed. alternatively a standard bipolar magnetic stirrer may be used.

The cylindrical recess defined in the rear face is adapted to receive a washer and membrane assembly 38. As is best seen in Figure 5 the washer and membrane assembly comprises two stepped washers, which may be made of rubber or another suitable material and a circular membrane or septum 39 located and held between the two washers and which in use closes one end of the chamber 18. The fact that the washers are stepped holds the septa in position away from the walls of the chamber/block, improves sealing and makes assembly of the apparatus easier. It will be readily appreciated that when two blocks are juxtaposed as shown in Figure 4, the membranes will function as dividing walls and will separate one chamber 18 from the chamber 18 of the adjacent block. The septa between the various chambers are isoelectric, buffering membranes, cast onto a supportive glass fibres or other suitable material. An O-ring may be disposed outside the membrane/washers to provide additional sealing. Such membranes are flow tight and ensure proper pH control.

The multi-compartment electrolyser apparatus is assembled by placing septa/dividing walls between adjacent chambers, inserting a tie rod through the aligned holes in each chamber block and compressing the chamber blocks together between end blocks 14,16 in a manner which will be described in more detail below.

The clamping mechanism will now be described in more detail. One end of each of the four tie rods 40 is embedded in one of the end blocks 16 of the apparatus, spaced apart and in the correct orientation for simultaneous insertion through the bores 25 of one of the chamber blocks 12.

The other end block 14 of the apparatus is best described with reference to Figures 7 to 9. It includes a generally square plate 42 which is movable relative to the housing 44 of the end block which housing in use, engages against an adjacent cylinder block 12. For clarity, Figure 7 shows part of the housing removed. The end block 42 defines a keyhole shaped aperture 43 in each corner of the block so that there are four apertures in total. The keyhole shaped apertures have a relatively wider part 46 and a relatively narrower part 48 (refer to Figure 8). Each of the tie rods has an enlarged portion 50 at the end distal from block 16. The enlarged portion 50 is small enough to pass through the wider part 46 of the keyhole apertures 43 but is too large to pass through the narrower part 48 of those keyhole apertures 43. The keyhole apertures are aligned such that the narrower parts 48 of the keyhole apertures

are located on diagonals"D"passing through the corners of the plate and the wider portion of the keyhole are all rotated through an angle"a"relative to the diagonals (refer to Figure 8). This enables a bayonet type latching of the plate to the tie rods in which the plate 42 is first aligned such that it is off set by angle a relative to the cross section of the chamber blocks 14 to allow the tie rods to pass through the keyhole apertures and then turned through an angle a so that the orientation of the square plate 42 is aligned with that of the chamber blocks 12. In that orientation, the tie rods are locked behind the plate as the enlarged portions 50 cannot pass through the narrower part 48 of the keyhole apertures.

A lever 60 is linked to the plate 42 by means of a metal rod and a camming means or other suitable mechanism is provided such that releasing the lever allows the plate to move generally towards the chamber blocks 12 whereas closing the lever into the position shown in Figure 7 tends to pull the plate away from the chamber blocks 12 thus tensioning the tie rods and compressing the chambers between the end block 16 and the housing 44 of end block 14.

Figure 6 illustrates a flexible springy connector 70 which is in electrical contact with one of the tie rods 40A which is a conducting electrode. The connector is connected to a positive terminal of a power supply and in use current is passed down the tie rod to a positive electrode (not shown). Figure 9 shows the connector 72 for the negative electrode (not shown) located in the end block 14. This allows both positive and negative electrical connections to be at the one end of the electrolyser in one socket 76-see Figure 1.

A further novel feature of the invention is the use of dummy blocks 80 illustrated in Figures 10 to 12. The dummy blocks are substantially the same shape and size as the chamber blocks and define four bores 25 for receiving the tie rods. However, instead of defining a chamber, the dummy blocks define a contact plate 82 and a contact pin 84 which are biased apart by means of an electrically conducting spring means 86. If electrophoretic separation is to be carried out using fewer than seven chambers, one dummy block is inserted in the apparatus to replace each of the"missing"chamber blocks.

Figures 13 to 17 illustrate the apparatus of the present invention disposed on a power supply/platform 100. Figure 13 shows the top plate 102 of the platform removed so that magnets 106 mounted on belt 107 driven rotatable turntables 104 can be seen. In use, as the magnets rotate, bipolar magnetic stirrers located in the wells of the chambers of the blocks of the electrolyser rotate to stir the contents of the chamber. The platform 100

includes an aluminium top plate 102, a Peltier 108 for cooling the plate and a fan (not shown) for cooling the Peltier.

The invention provides an alternative novel stirring mechanism which is illustrated in Figure 18. A solenoid 120 is disposed underneath the well 36 of a block which as can be seen in the Figure, has a top entrance 36a which is narrower than the bore of the well 36. Inside the well, is located a stirrer means 130 comprising a magnetic plate 132 which is encased in a plastic coating 134 or the like which defines an annular cylindrical portion 136 around its extreme and which prevents the stirrer from over-turning in the well. Periodic reversal of the polarity of the solenoid 120 causes the stirrer means 130 to move up and down inside the well and therefore stirs the contents of the chamber.

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.