Abstract:
An electrolyser for separating a mixture of components using isoelectric focusing in a liquid media comprises a series of blocks defining chambers. The chambers are separable by isoelectric membranes of known pH. End chambers are located at each end of the series of chambers. The blocks are clamped together using four tie rods to make the apparatus substantially liquid tight. An electrodes is provided in each end chamber for use in applying an electric field across the series of chambers. 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. A lever operated clamping mechanism is provided for tensioning the tie rods and clamping the chambers together between the end blocks in a liquid tight fashion.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention relates to a improved apparatus for electrophoresis and in particular to an apparatus of the type commonly referred to as an “electrolyser”.  
       BACKGROUND OF THE INVENTION  
       [0002]     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.  
         [0003]     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.  
         [0004]     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.  
         [0005]     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  
       [0006]     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.        
 
         [0012]     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.  
         [0013]     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.  
         [0014]     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.  
         [0015]     It is preferred that there are four tie rods, although only one of the tie rods will typically be used to conduct electricity.  
         [0016]     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.  
         [0017]     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 damping means is arranged to move the plate relative to the housing using a cam mechanism or the like.  
         [0018]     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.  
         [0019]     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.  
         [0020]     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.  
         [0021]     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.  
         [0022]     The apparatus is typically designed to receive a set number of chamber blocks, typically seven, however, in some cases fewer chambers will be required.  
         [0023]     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.  
         [0024]     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.  
         [0025]     Whilst a single solenoid may perform this function, a plurality of solenoids could be used.  
         [0026]     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  
       [0027]     Specific embodiments of the invention will now be described, by way of the example only, and with reference to the accompanying drawings in which:  
         [0028]      FIG. 1  is a perspective view of an apparatus embodying the present invention;  
         [0029]      FIG. 2  is a perspective view of a chamber block which forms part of the apparatus shown in  FIG. 1 ;  
         [0030]      FIG. 3  is an underneath plan view of two chamber blocks engaged together;  
         [0031]      FIG. 4  is a section through lines IV-IV of  FIG. 3 ;  
         [0032]      FIGS. 5   a ,  5   b , and  5   c  are a perspective view, front elevation and a section through a stepped washer which forms part of the apparatus of  FIG. 1 .  
         [0033]      FIG. 6  is a perspective view of parts of an apparatus embodying the present invention illustrating internal features of one end block in particular;  
         [0034]      FIG. 7  is an end view of the apparatus shown in  FIG. 1  with part of the housing of the end block of the apparatus removed;  
         [0035]      FIG. 8  is an end view of a plate;  
         [0036]      FIG. 9  is a perspective view of an end plate illustrating connectors for a conducting tie rod;  
         [0037]      FIG. 10  is a perspective view of a dummy block;  
         [0038]      FIG. 11  is a front view of the dummy block of  FIG. 10 ;  
         [0039]      FIG. 12  is a cross section on lines XII-XII of  FIG. 11 ;  
         [0040]      FIG. 13  shows the apparatus of  FIG. 1  located on a platform with a top plate of the platform removed; and  
         [0041]      FIG. 14  is a top plan view of the platform of  FIG. 13 ;  
         [0042]      FIG. 15  is a top plan view of the apparatus of  FIG. 1  located on a complete platform;  
         [0043]      FIG. 16  is a cross sectional view on XVI-XVI of  FIG. 15 ;  
         [0044]      FIG. 17  is an enlarged view of part of  FIG. 16 ; and  
         [0045]      FIG. 18  illustrates an alternative stirring mechanism for the apparatus. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0046]     Referring to the drawings,  FIG. 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 .  
         [0047]     FIGS.  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  FIG. 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  FIG. 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  FIGS. 6 and 7  which are used to align the bores together and link and compress the chambers together.  
         [0048]     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.  
         [0049]     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.  
         [0050]     The cylindrical recess defined in the rear face is adapted to receive a washer and membrane assembly  38 . As is best seen in  FIG. 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  FIG. 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.  
         [0051]     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.  
         [0052]     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 .  
         [0053]     The other end block  14  of the apparatus is best described with reference to FIGS.  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,  FIG. 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  FIG. 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 “α” relative to the diagonals (refer to  FIG. 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 α 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 α 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  FIG. 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 .  
         [0054]      FIG. 6  illustrates a flexible springy connector  70  which is in electrical contact with one of the tie rods  40 A 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).  FIG. 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  FIG. 1 .  
         [0055]     A further novel feature of the invention is the use of dummy blocks  80  illustrated in FIGS.  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.  
         [0056]     FIGS.  13  to  17  illustrate the apparatus of the present invention disposed on a power supply/platform  100 .  FIG. 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.  
         [0057]     The invention provides an alternative novel stirring mechanism which is illustrated in  FIG. 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  36   a  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.  
         [0058]     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.