Abstract:
The current invention relates to an apparatus for performing a centrifugal field-flow fractionation. In particular, in the apparatus according to the invention, radial rotary shaft seals are used for sealing the rotating parts of the shaft for carrying out the centrifugal field-flow fractionation and the stationary parts. Furthermore, a separate line is introduced for reducing the pressure differential across these seals. In such way, the driving force driving sample fluid across the seals is reduced, thereby reducing leakage.

Description:
TECHNICAL AREA 
       [0001]    The invention relates to an apparatus for performing a centrifugal field-flow fractionation according to the preamble of claim  1  as well as to a method for performing a centrifugal field-flow fractionation. 
         [0002]    Field-Flow Fractionation (FFF) is a family of unique separation techniques, comprising various different sub-techniques. All FFF techniques utilize the same basic separation principle, but employ different separation fields. Depending on the separation field, the technique is called Flow FFF, Sedimentation FFF, Thermal FFF, etc. FFF is providing fast, gentle and high resolution separations of particulate matter from 1 nm up to 100 μm in liquid media. The sample is separated inside a longitudinal, open flow channel without the presence of any packing or stationary phase inside the channel. The FFF apparatus are typically arranged so that the fluid inside the channel forms a laminar flow with a parabolic stream profile. 
         [0003]    The different force fields used, such as liquid flow, centrifugal force, temperature gradient or gravity, are applied perpendicularly to the main flow which transports the sample along the length of the channel. Under the influence of these force fields and counteracting diffusion of the particles, different equilibrium layer heights are formed by the different particle size fractions. Smaller particles, with stronger diffusion, are located higher in the channel in fast stream lines and elute first. Bigger particles with a lower diffusion coefficient are located in slow stream lines and elute later. 
         [0004]    Centrifugal FFF has been an important member of the FFF family of techniques since its invention in 1974 by Giddings et al. The first commercial Centrifugal FFF in the 1980s was a system based on a Dupont Sorval Ultracentrifuge, the Model SF3-1000 Sedimentation Field Flow Fractionator. In the 1990s the S-101 Sedimentation FFF was launched. Since 2001 the model CF1000 was provided, which was followed up in 2010 by the new CF2000 Series for nanoparticle separation and characterization. 
         [0005]    In centrifugal FFF the separation force is generated by rotating the entire longitudinal, ring-shaped flow channel. As the main flow carries the sample particles along the length of the channel they are affected by the centrifugal field which is generated by the rotation. The larger/heavier particles are more strongly forced towards the radially outer channel wall than the smaller and lighter particles which stay away from the radially outer channel wall. As a result, smaller particles are located in the area of faster stream lines and thus will be eluted first out of the channel, followed by larger particles which are located in the region of slower streamlines. The separation in Centrifugal FFF is based on particle mass (size and density) and because of this allows a very high resolution separation of particles showing only 5% difference in size. 
         [0006]    As centrifugal FFF offers a high resolution separation of particles, detectors coupled to the channel are used for further characterization and quantization. Typical detection principles for centrifugal FFF are UV, Dynamic Light Scattering and Static Light Scattering to yield concentration, particle size and elemental distribution. 
         [0007]    Although a well-designed system in theory, prior attempts at designing a centrifugal FFF apparatus have failed in practice. 
       BACKGROUND ART 
       [0008]    One such attempt is disclosed in U.S. Pat. No. 4,448,679, on which the preamble of the independent claims of this application is based. This document relates to a centrifugal FFF apparatus in which a liquid to be fractionated is led into a channel present between an inner ring and an outer bowl-shaped structure of a rotor which can be rotated by a motor. During rotation, the liquid to be fractionated is fractionated by the action of the centrifugal force as well as the flow of the liquid through the channel. 
         [0009]    A recurring problem with the prior art has been to reduce the leakage of sample fluid introduced into the field-flow fractionation apparatus where the leaks frequently occur at the inlet of the sample fluid into the apparatus: as the rotor rotates whereas the fluid supply is generally stationary, there is a need for a tight seal of the connection between the rotating and the stationary parts of the FFF apparatus. This is particularly challenging due to the need to prevent a leak occurring at an interface between a stationary and a moving part, which is more difficult than just sealing off two stationary parts. 
       DISCLOSURE OF THE INVENTION 
       [0010]    The FFF apparatus according to the invention as laid out in claim  1  solves the problem of being able to transfer minimum amounts of liquid through a rotating seal without leakage. The key idea is to avoid axial seals and to instead employ sets of radial seals. Furthermore, and importantly, another key idea is that the pressure differential across each radial seal is minimized which avoids leakage through the seals: there is as a consequence less pressure which could drive a sample liquid across the radial seals, thereby reducing the amount of sample liquid crossing the seal. This is particularly important in radial seals contacting rotating objects as are frequently used in an FFF apparatus as in such an application, a large fraction of the seal contacts the rotating object. Hence, there is a large area available where leaks could occur. 
         [0011]    According to claim  1 , an apparatus for performing a centrifugal field-flow fractionation comprises a shaft having a central axis and conduits along the central axis for sample fluid to pass therethrough. The shaft has an upstream shaft end for the sample fluid to enter into and a downstream shaft end for the sample fluid to exit from the shaft. The shaft carries a rotor between the upstream and the downstream shaft end and is rotatably supported in bearing blocks at both shaft ends so that the rotor and the shaft are rotatable about the axis. The shaft does not have to be a single piece but may consist of several components, which generally makes servicing the shaft easier as in the case of failure of the shaft, only one component needs to be repaired or replaced. 
         [0012]    There is a field-flow fractionation channel disposed on the rotor. The field-flow fractionation channel is configured to let the sample fluid pass therethrough and has an upstream channel end for the sample fluid to enter into the channel and a downstream channel end for the sample fluid to exit from the channel. The upstream channel end is connectable in a fluid tight manner to a conduit at the upstream shaft end and a downstream shaft end is connectable in a fluid-tight manner to a conduit at the downstream shaft end. 
         [0013]    Further, there is an upstream end cap which fits over the upstream shaft end and can be attached to the bearing block at the upstream shaft end and a downstream end cap which fits over the downstream shaft end and can be attached to the bearing block at the downstream shaft end. 
         [0014]    The upstream end cap and the downstream end cap each have a terminal for connection to a line for the sample fluid. The upstream end cap and the downstream end cap each have a further terminal for connection to a line for flushing fluid. The upstream end cap and the downstream end cap each have a first recess and a second recess, each of the first recesses being for accommodation of a first radial rotary shaft seal for sealing the end cap against the shaft and each of the second recesses being for accommodation of a second radial rotary shaft seal for sealing the end cap against the shaft. 
         [0015]    In other words, the radial rotary shaft seals are disposed about an axis of rotation of the rotor and are provided to tightly seal the end cap against the shaft. The second radial rotary shaft seal seals between the sample fluid and the flushing fluid whereas the first shaft seal seals between the flushing fluid and the exterior (or potentially another fluid). 
         [0016]    Furthermore, according to the invention, each of the second recesses on one side of its second radial rotary shaft seal is in fluid communication with the terminal for connection to the sample fluid line. On the other side of its second radial rotary shaft seal, each of the second recesses is in fluid communication with the terminal for connection to the flushing fluid line, and each of the first recesses on one side of its first radial shaft seal is in fluid communication for connection to the flushing fluid line so that both sides of the second radial rotary shafts are subjected to fluids. 
         [0017]    By means of having the connection for introducing the sample fluid into the shaft and by having the possibility of introducing the flushing fluid into the recess, it becomes possible to minimize the pressure differential across the seals which seal the rotating parts against the non-rotating parts. Thus, it becomes possible to reduce the driving force which would otherwise drive the sample fluid through the seal and out of the conduit leading to the FFF channel. In this way, it is avoided to have sample fluid leaking out from the apparatus. 
         [0018]    Preferred embodiments of the apparatus according to the invention as described in claim  1  are detailed in claims  2  to  9 . 
         [0019]    Preferably, the pressure differential across the second radial rotary shaft seal is determined by the difference in pressure of the sample fluid and the flushing fluid at a respective end cap, and the flushing fluid is pressurized so as to control the pressure differential. Here, the pressure differential arises from about 4 to 7 bar sample fluid and about 200 mbar flushing pressure, which is larger than expected. In such way, it becomes possible to minimize the pressure differential, so as to avoid any leakage of sample fluid. 
         [0020]    It is preferred that the terminals for connection to the line for the sample fluid are aligned with the central axis when the end caps are attached. Thus, it becomes easier to use a flexible hose or a conduit for introducing the sample fluid into the apparatus as an off-axis terminal would not allow for an easy introduction thereof. Also, this avoids adding an extra variation of the pressure to the sample fluid which would otherwise occur if a terminal was used which is off-axis with respect to the axis of rotation: in such a case, a rotation would lead to a sinusoidal variation in the sample fluid pressure, which would negatively affect the quality of the FFF performed. Thus, by the preferred design, the quality of the outcome of the centrifugal FFF as well as the tightness of the seal is improved. 
         [0021]    It is furthermore preferred that at least one of the conduits along said central axis of the shaft has a diameter sufficiently large for the sample fluid line to extend through the terminal and into the conduit. In this way, the sample fluid line can be easily introduced into the shaft. Thereby, the sample fluid is released from the sample fluid line further inside the apparatus, which in turn means that there is less of a risk of sample fluid leaking out of the apparatus. 
         [0022]    It is preferred that the sample fluid line is inserted into the conduit. As an advantage, this reduces the risk of leakage occurring in a manner similar to the previous mechanism. 
         [0023]    Preferentially, the second recesses are axially remote from the bearing block and the first recesses are axially closer to the bearing block. This allows for an easier design of the end cap as well as for an easier handling of the apparatus: if the first recess was axially more remote from the bearing block than the second recess, it would be necessary to have a more complicated pattern of connecting the fluid lines. Thus, setting up the apparatus would become more difficult. Also, it becomes easier to replace the seals. 
         [0024]    It is additionally preferred that the first and/or second radial rotary shaft seals are free of spiral grooves or helices. In such a way, the rotary shaft seals have less of a tendency to leak. Also, wear of the seals during use is reduced as radial rotary shaft seals which are free of spiral grooves or helices have good wearing properties, which ensures that they keep their advantageous sealing properties during use. 
         [0025]    Additionally, it is preferable that each of the first recesses on the other side of its first radial rotary shaft seal is in fluid communication with a further terminal for connection to a drain fluid line or the like. This leads to a further potential for improvement in leakage tightness as the pressure differential across these two lines can also be minimized. 
         [0026]    Preferably, the upstream end cap and the downstream end cap are of the same design. This makes producing the field-flow fractionation channel more affordable as only one type of end cap is needed. 
         [0027]    The problem is also solved by the method according to claim  10 . According to this claim, the method for performing a centrifugal FFF comprises the following steps: 
         [0028]    A flow of a sample fluid is provided to an upstream terminal of an apparatus for performing a centrifugal field-flow fractionation. The flow of sample fluid is passed through a field-flow fractionation channel. The flow of sample fluid is conveyed away from the downstream terminal of the apparatus. A flow of a flushing fluid is provided to an upstream terminal of the apparatus. A flow of flushing fluid is provided to a downstream terminal of the apparatus. The flow of sample fluid is sealed from the flow of flushing fluid at the upstream terminal by means of a radial seal, and the flow of sample fluid is sealed from the flow of flushing fluid in a downstream terminal by means of a radial seal. 
         [0029]    Again, the use of the radial seals together with there being two fluids connected by them avoids any leakage of sample fluid from the apparatus. The mechanism is essentially the same as described in the above. 
         [0030]    Preferred embodiments of the method according to claim  10  are described in claims  11  and  12 . 
         [0031]    It is preferred that the method additionally comprises the step of pressurizing the flushing fluids so as to reduce the difference in pressures of the flushing fluid and of the sample fluid at the upstream terminal and the difference in pressures of the flushing fluid and of the sample fluid at the downstream terminal. As an advantage, this leads to lower leakage as there is even less of a pressure differential across a radial seal which would otherwise drive the sample fluid out of the apparatus, thereby leading to leakage. 
         [0032]    Preferably, there are the additional steps of providing the flow of sample fluid to the upstream terminal of the apparatus for performing a centrifugal field-flow fractionation and of conveying the flow of sample fluid away from the downstream terminal of the apparatus comprising the introducing of a sample fluid line into a conduit in a shaft of the apparatus. In particular, the conduit shall extend along a central axis of the shaft. 
         [0033]    As an advantage, this leads to a reduced leakage as the sample fluid line can be introduced further into the shaft which avoids the potential of leakage compared to the case where it was only introduced into the apparatus to a lesser extent. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0034]      FIG. 1 : shows an assembled centrifugal field-flow fractionation apparatus according to the invention. 
           [0035]      FIG. 2 : shows a cross-sectional view of the seal and bearing structure of the field-flow fractionation apparatus of  FIG. 1 . 
           [0036]      FIG. 3   a : shows a plan view of the end cap of  FIG. 2 . 
           [0037]      FIG. 3   b : shows a cross-sectional view of the end cap of  FIG. 2 . 
           [0038]      FIGS. 4   a - c : show the spacers used in the apparatus according to  FIG. 1 . 
           [0039]      FIGS. 5   a +b: show the foil used in the apparatus according to  FIG. 1   
           [0040]      FIGS. 6   a +b: show the holder of the FFF channel of  FIG. 1 , and 
           [0041]      FIGS. 6   d - f : display the wedge for holding the holder of  FIG. 1  in place. 
       
    
    
     DETAILED DESCRIPTION 
       [0042]    A preferred way of carrying out the invention will now be described with reference to the accompanying drawings. 
         [0043]      FIG. 1  shows an assembled centrifugal field-flow fractionation apparatus  10  according to the invention. It should be noted that end caps  28   a/b , which will be described in more detail further below, are missing in this Figure but will be attached before operation of the FFF apparatus. The drawing shows a rotor  10   a  which can be rotated about a shaft  61  (shown in more detail in  FIG. 2 ). The shaft, in turn, comprises several individual pieces. The rotor  10   a  is driven by a DC electric motor (not shown). The rotor  10   a  has a generally bowl-shaped structure, with a hub  10   b  as a radially inner part and a rim  11  as a radially outer part. The hub is essentially disc-shaped, with a circular outer circumference. The outer rim  11  is annular, with a rectangular cross-section, and extends circumferentially around the hub  10   b  as well as axially beyond the hub  10   b . Thus, the overall shape of the rotor  10   a , when viewed perpendicularly to the plane of the hub  10   b  and in the direction of the shaft  61 , is circular. Within the hub  10   b  and adjacent to the outer rim  11 , there is an attachment mechanism, such as a threaded hole (not shown) for insertion of a bolt and attachment of the fractional field flow channel, as will be apparent from the below. The rim and the hub can be made of one unitary piece, or of separate elements which are later assembled. 
         [0044]    On an inner circumferential surface of an outer rim  11  of rotor  10   a , a holder  12  is attached to the rotor  10   a . A structure  13 , which is described below, constitutes the fractional field flow channel. 
         [0045]    The holder  12  has a generally annular shape with an interrupted segment  92  and, so as to reduce its weight, yet provide the mechanical strength needed, is made of an aluminium manganese zinc copper alloy. When the structure  13  is assembled with the holder  12  and the rotor  10   a , a wedge  18  is inserted into the interrupted segment  92 . The holder  12  further comprises two holes  32 . These holes can be used for extracting wedge  18  from its inserted position. Further holes  34  are present in the holder  12  at a position radially opposite the interrupted segment  92 . These holes  34  are balancing bores the location and volume of which are ideally determined by means of dynamic balancing. They prevent an imbalance occurring during operation of the FFF apparatus. 
         [0046]    Outside the rotor  10   a , a separate covering element  20  in the form of an inverted U is provided. It extends around a periphery of the rotor  10   a  and prevents a person from accidentally touching the spinning rotor  10   a . This element  20  is made of a strip of material, preferably metal, and preferably has an extension along the axial direction of the rotor-carrying shaft  61  beyond both the front and the rear of the rotor  10   a  as seen along the axial direction of the shaft  61 . 
         [0047]    The rotor  10   a  is carried by shaft  61  which passes into end caps  28   a  and  28   b  (in short referred to as  28   a/b ) ( FIGS. 2 and 3 ). In the centre of these end caps  28   a/b , connecting pieces  48   a/b  ( FIG. 1 ) for letting the sample fluid pass into or out of the centrifugal field-flow fractionation apparatus is provided. These connecting means  48   a/b  can preferably be a hollow tube. The terminal  40   a/b  itself takes the form of a threaded bore, with the bore extending through the end cap (with the threads not extending all the way). 
         [0048]    Connecting pieces  48   a/b  are connected to another means  31   a/b  for letting fluid pass into or out of the centrifugal field-flow fractionation channel  13 . The means  31   a/b  for letting fluid pass into and out of the channel  13  is disposed on an inner circumference of the holder  12  adjacent the interrupted segment  92  for accommodating the wedge  18 . In the interest of avoiding a multiplicity of different parts, it can be embodied in the form of tubing with standard attachments. 
         [0049]    The shaft  61  passes through and is rotatably supported in an opening disposed in a bearing block  24   a/b , and extends further into end cap  28   a/b . At least one of the bearing blocks  24   a/b  consists of two individual elements. The bearing blocks  24   a/b  are themselves supported on a base  26 . Preferably, the position of the bearing blocks relative to one another can be adjusted, for instance by having one of them, such as bearing block  24   b , displaceably connected to the base  26 , as schematically indicated in  FIG. 2 . The other bearing block  24   a  is fixed in its location relative to the base  26 . 
         [0050]    Alternatively, both blocks could be adjustable, or the position adjustment could be achieved through different means, such as a split base. 
         [0051]      FIG. 2  shows in more detail the support structure for supporting the shaft  61  as well as the structure inside the end caps  28   a/b . It can be seen that the two bearing blocks  24   a/b  are supported on the base  26 , where block  24   a  is fixedly supported by the base  26  whereas block  24   b  is displaceably supported (the direction of the displacement being indicated by an arrow) on the base  26 . On each of the bearing blocks  24   a/b , end caps  28   a/b  are fixed by suitable means such as screws or bolts or the like. These end caps  28   a/b  each generally have the shape of a cup. At the base end of each of these “cups”, two terminals  40   a/b  and  44   a/b  are provided, which are each penetrating the cup and take the shape of threaded holes. These terminals  40   a/b ,  44   a/b  each have a diameter which decreases when moving in the axial direction of the shaft from the outside towards the hub (at  33  in  FIG. 2 ), i.e. from the closed to the open end of the end caps  28   a/b . The terminals are designed for introducing fluid lines such as flexible conduits such as hoses or flexible tubing. Each terminal  40   a/b  is arranged along the central axis of the respective end cap  28   a/b . The end caps are placed so that the central axes of their openings coincide with the axis of rotation of the shaft  61 . Axially offset towards the rim of the caps  28   a/b , where the caps are attached to the supports  24   a/b , there are disposed additional terminals  42   a/b  extending along a generally radial direction. These terminals extend into bores through the thickness of the rim of the end cap  28   a/b . They are, again, designed for an exchange of liquid between the interior and the exterior of the cap, in this case for introducing a drain fluid line and draining fluid. 
         [0052]    In the assembled apparatus, the end caps  28   a/b  are arranged such that the central axes of their openings are aligned with the central axis of an opening in the respective bearing block  24   a/b . The opening of the bearing block  24   a/b  serves to accommodate roller bearings which support the central element  32  of shaft  61 . 
         [0053]    Roller bearings  34   a ′/b′ consist of inner and outer raceways  34   a/b  and  35   a/b , respectively, and a plurality of roller elements  35   a ′/b′, respectively, disposed between them. The raceways  34   a/b  and  35   a/b  are made of steel whereas the roller elements  35   a ′/b′ are made from ceramics. The roller elements  35   a ′/b′ all have a spherical shape. In the present embodiment, both roller bearings  34   a/b  are single row deep grooved roller bearings. Although not recommended for this application, they are standard parts and can be ordered from suppliers such as INA/FAG, NTN, SKF or others. The roller elements  35   a ′/b′ are made of a material which differs from that of the inner and outer raceways  34   a/b  and  35   a/b  so as to reduce friction and avoid lubrication during use. Whilst the roller bearings  34   a/b  may be provided with a minimal amount of oil as a lubricant upon assembly of the FFF apparatus, such as one or two drops per bearing, they are not lubricated during operation, even after prolonged use. 
         [0054]    Each roller bearing  34   a ′/b′ is each fixed both with respect to the shaft  61  and its support in bearing block  24   a/b  so that there is no axial clearance. Upon assembly of the FFF apparatus, possible axial clearance can be eliminated by displacing one of the bearing blocks relative to the other, or by accordingly adjusting a split base, if present, or by other suitable means known in the art. 
         [0055]    As was stated in the above, the shaft  61  comprises several individual components, namely, those denoted by reference signs  32 ,  48   a/b ,  50   a/b , and  52   a/b . The shaft  61  has the overall shape of a generally rotationally symmetric longitudinal element with an axis of symmetry running approximately along its longitudinal direction. A central shaft element  32  is arranged at the longitudinal centre of shaft. It consists of a sleeve having a hollow inner portion  60  arranged along the axis of the shaft. At about the longitudinal centre region of the shaft portion  32 , the hollow inner portion  60  is preferably constricted, i.e. the wall of the hollow inner portion  60  becomes thicker, which ensures a lower weight and higher stability of the shaft  61 . At this location, however, the shaft could also be solid. At the radially outer periphery at a position corresponding to the preferably constricted or possibly solid portion, the shaft has a circumferential flange  33  with means such as threaded bores for attaching the rotor  10   a . This also corresponds to approximately midway axially between the supports  24   a  and  24   b . This particular position is chosen in accordance with the shape of the rotor so that it has the highest stability, which means that it will more likely withstand the forces due to the spinning rotor  10   a , and will generate the lowest imbalance when the rotor is spinning. Further, the hollow inner portion  60  is open to an exterior at a portion  62  connecting the hollow inner portion  60  of the central shaft element  32  to an exterior. 
         [0056]    At both longitudinal ends of the central shaft portion  32 , first connecting pieces  52   a/b  are provided in a cylindrical recess in central shaft portion  32 . The first connecting pieces  52   a/b  take the form of a cylindrical plate with a recess portion. The recess is generally cylindrical but exhibits an irregular cylinder surface in that the cylinder surface has a plurality of segments which are enlarged in diameter and accordingly provide a passage for fluid. On the radial outside, the recess portions leave a ring-shaped web which is preferably provided with a thread. In the assembled state, the cylindrical recess portions are directed to the outside, away from the shaft element  32 . The recess portions do not penetrate through the first connecting pieces  52   a/b . Thus, the recesses are only present at one axial side of the first connecting piece  52   a/b.    
         [0057]    Placed on and along the axis of rotation of these cylindrical plates, terminals  46   a/b  are provided in the form of threaded bores. In the assembled disposition of the first connecting pieces  52   a/b , these bores have a diameter which decreases when moving through the terminals to the outside, away from the shaft element  32 . At one side of the first connecting piece  52   a/b , the terminal  46   a/b  opens up into the cylindrical recess portion with a diameter larger than that of the terminals. It is evident from  FIG. 2  that the terminals  46   a  and  46   b  are axially aligned with a bore that is located axially outside of the terminals and provided in second connecting pieces  48   a/b . At least one of the first connecting pieces  52   a/b  is connected to the other side of the plate at one off-centre portion of the first connecting piece  52   a  (i.e. not connecting piece  52   b ) by means of a further terminal  47   a  taking the form of a threaded bore. However, in the interest of economical manufacturing, the first connecting pieces  52   a  and  52   b  could both exhibit the off-centre connection by means of a further terminal. In this manner, there would be only one type of connecting piece. If the further terminal  47   a  is not in use in one of the first connecting pieces  52   a  or  52   b , it could be blocked by a blind plug. 
         [0058]    Second connecting pieces  48   a/b  are assembled with ring-shaped elements  49   a/b , preferably by means of a thread as shown in  FIG. 2 , and the resulting sub-sub-assembly is inserted, for instance press-fitted, into a recess provided in the third connecting pieces  50   a/b . As shown in  FIG. 2 , however, the irregular cylinder surface of recess portion in third connecting pieces  50   a/b  leaves areas of fluid communication. 
         [0059]    The result is a sub-assembly of elements  48   a/b ,  49   a/b  and  50   a/b . The elements of this sub-assembly are so designed and arranged that their end faces which, in the assembled state of the sub-assembly, are oriented towards the cylindrical recess in the first connecting piece  52   a/b , are flush. 
         [0060]    Third connecting pieces  50   a/b  preferably carry a thread on the outside. If they do, the sub-assembly is screwed into the recessed portions of first connecting pieces  52   a/b . In the absence of a thread, the sub-assembly would otherwise be fitted into place, for instance by press-fitting or gluing. 
         [0061]    Alternatively, the recess portion of first connecting piece  52   a/b  could exhibit the shape of a ring instead of a cylinder. In this case, elements  49   a/b  would be of one piece with first connecting piece  52   a/b  and both, second connecting piece  48   a/b  and third connecting piece  50   a/b  could simply be screwed into first connecting piece  52   a/b . Fluid communication would then be provided through a radial gap between element  49   a/b  and third connecting piece  50   a/b . The end faces of the second and third connecting pieces could also be axially offset relative to one another. 
         [0062]    The resulting assembly of elements  48   a/b ,  49   a/b ,  50   a/b  and  52   a/b  is then inserted into and retained in the central shaft portion  32  by means of screws, or bolts, as shown in  FIG. 2  for first connecting piece  52   b  to the right-hand side. It should be noted that all connecting pieces  48   a/b ,  50   a/b  and  52   a/b  are aligned with the axis of the shaft  61  they constitute. 
         [0063]    The second connecting piece  48   a/b  takes the form of a cylinder with a passage connecting the centres of its end faces, one of the end faces being aligned with terminal  46   a/b  of first connecting piece  52   a/b  and the other with terminal  40   a/b  of the end cap  28   a/b , and acts as a tube. The passage is preferably a central conduit  54   a/b  which is aligned with both terminal  46   a/b  of first connecting piece  52   a/b  and terminal  40   a/b  of the end cap  28   a/b . The second connecting piece  48   a/b  is preferably recessed on the outside surface so as to provide a fluid chamber. The axial length of the second connecting piece  48   a/b  is greater than the axial length of the third connecting pieces  50   a/b . Third connecting pieces  50   a/b , in turn, have an axial length greater than the web of cylindrical recess portions in first connecting pieces  52   a/b . As a result, the radially innermost connecting pieces  48   a/b  project furthest in the axial direction, and the third connecting pieces  50   a/b  which are located radially further outside, project less. The amount of the axial projection is sufficient to present a sealing surface for seal rings  38   a/b  and  36   a/b.    
         [0064]    The first seal rings  38   a/b  are located radially outside of third connecting pieces  50   a/b , and are sealingly disposed between these first connecting pieces  50   a/b  and the neighbouring wall of end caps  28   a/b . They are accommodated in recesses  39   a/b  of the end cap. The first seal rings  38   a/b  surround first connecting pieces  50   a/b  in a fluid-tight manner. Similarly, second seal rings  36   a/b  are located radially outside of second connecting pieces  48   a/b , accommodated in recesses  37   a/b  of the end cap and are sealingly disposed between these second connecting pieces  48   a/b  and the wall of end caps  28   a/b . The seal rings  38   a/b  and  36   a/b  are rotary shaft seals which are free of spiral grooves or helices. 
         [0065]    The end cap  28   a/b  preferably lies flush against bearing block  24   a/b  and retained in place so that there is a fluid tight connection between them. Tightness against leakage of fluid could, however, also be obtained through other means known in the art. 
         [0066]      FIG. 3   a  shows a plan view and  FIG. 3   b  a cross-sectional view of end cap  28   a/b  along line B-B in  FIG. 3   a . It is apparent that terminal  40   a/b  extends through the end wall of end cap  28   a/b  along the central axis of the essentially rotationally symmetric end cap  28   a/b . Additionally, in  FIG. 3   a , three through-holes  45 , preferably countersunk bores, are shown. They serve for connecting the end cap  28   a/b  to the respective bearing block  24   a/b  and are meant for the insertion of a screw or any other suitable fastening means.  FIG. 3   a  also shows terminal  44   a/b.    
         [0067]    Particularly  FIG. 3   b  shows second recess  37   a/b  and first recess  39   a/b  in the end cap  28   a/b , both the second  37   a/b  and the first recess  39   a/b  having an essentially cylindrical shape that is coaxial with the bore of terminal  40   a/b . The radial dimension, i.e. the diameter of second recess  37   a/b  is smaller than that of first recess  39   a/b . Both recesses  37   a/b  and  39   a/b  are immediately adjacent to one another, with the first recess  39   a/b  being located axially further towards the opening of the “cup” of cup-shaped end cap  28   a/b , i.e. the second recess  37   a/b  is axially remote from the bearing block  24   a/b  whereas the first recess  39   a/b  is axially closer to the bearing block  24   a/b    
         [0068]    Still axially further towards the opening of the cup, there is a further (third) recess  64   a/b  which takes the shape of a cylinder with a circularly shaped portion disposed around it such that its radially outermost surface is curved like the segment of a torus. The radial dimension of the third recess  64   a/b  is even bigger than that of the second  37   a/b  and first recess  39   a/b . The third recess  64   a/b  is preferably also arranged coaxially with the second  37   a/b  and first  39   a/b  recess but other arrangements are possible. The bore of terminal  42   a/b  opens into third recess  64   a/b , as may be taken from  FIG. 3   a.    
         [0069]      FIGS. 3   a  and  3   b  further show two through holes  43   a/b , which are partially threaded. They open into recess  37   a/b  and serve so as to be able to push seal ring  38   a/b  out of recess  37   a/b.    
         [0070]    End cap  28   a/b  is dimensioned relative to the assembly of elements  48   a/b ,  49   a/b ,  50   a/b  and  52   a/b  so that, when end cap  28   a/b  carrying sealing rings  36   a/b  and  38   a/b  is placed over the assembly, there are axial gaps between the axially outer end face of second connecting piece  48   a/b  and the inner, axially outermost surface of recess  37   a/b  of end cap  28   a/b  as well as between the axially outer end face of third connecting piece  50   a/b  and the inner, axial end face of recess  39   a/b  of end cap  28   a/b . In the assembled state of the FFF apparatus, the latter gap is thus located between the two sealing rings  36   a/b  and  38   a/b.    
         [0071]    Terminal  44   a/b  is in fluid communication with, and preferably opens straight into, the axial gap between the first and second seal rings  36   a/b  and  38   a/b  and second and third connecting pieces  48   a/b  and  50   a/b . Terminal  42   a/b  leads to the gap between seal rings  38   a/b  and the fluid-tight interface between the end cap  28   a/b  and the bearing block  24   a/b , i.e. each of the first recesses  39   a/b  on the other side of its first radial rotary shaft seal  38   a/b  is in fluid communication with terminal  42   a/b . Terminal  47   a  leads to the enlarged portions of the cylinder surface of the recess in third connecting piece  50   a/b  or to the radial clearance between connecting piece  52   a/b  and connecting piece  50   a/b . Terminals  40   a/b  and conduits  54   a/b  are designed such that their diameters are sufficiently large to allow for a sample fluid line such as standard flexible tubing to extend through the terminals  40   a/b  and into the conduits  54   a/b . The bore of terminals  46   a/b , conversely, does not permit tubing to pass. In the present embodiment, the upstream and the downstream end cap  28   a/b  both have the same design. 
         [0072]      FIGS. 4   a  to  c  show strips for use in defining the FFF channel  13 . 
         [0073]    The strips are stacked onto each other between the holder  12  and, radially outside of the stack, the rim  11 . In the stack, an inner spacer  70  lies adjacent a channel-defining foil  78  ( FIG. 5 ) which, in turn, lies adjacent an outer spacer  66 . Optionally, a compensation strip  74  is placed between outer spacer  66  and rim  11 . 
         [0074]      FIG. 4   a  shows the outer spacer  66  to be used between the foil  78  ( FIG. 5 ) defining the channel  13  and the rim  11  of the rotor  10   a . Outer spacer  66  is made of a Mylar polyester foil, i.e. a foil made of biaxially oriented polyethylene terephthalate, or a metal sheet and, at least on the side facing foil  78 , has an average surface roughness of 1.5&lt;Ra&lt;1.7, and preferably Ra=1.6, the surface roughness being measured in accordance with DIN EN ISO 4287:2010-07 as the arithmetic average of absolute values, for instance by means of a laser scanning type camera and providing the roughness in μm. This way of defining the surface roughness is used throughout the whole of this present description unless otherwise stated. 
         [0075]    Four holes  68  are disposed on corners of the otherwise generally rectangular strip  66 . The thickness of strip  66  is significantly less than 1 mm and preferably app. 250 μm, and its surface facing foil  78  is completely scratch-free so as to aid in sealing the channel  13 . Strip  66  is checked for scratches by visual inspection, i.e. by an optical measurement. 
         [0076]      FIG. 4   b  shows an inner spacer  70  which is disposed between the holder  12  and the foil  78  defining the channel  13 . Again, similarly to spacer  66 , it has a generally rectangular form, with holes  72  disposed at the four corners of the strip. Whilst the axial, or transverse distance between the holes  68 ,  72  at one longitudinal end of the spacers  66  and  70  is identical, the circumferential, or longitudinal distance between holes  72  of inner spacer  70  is shorter than the corresponding distance between holes  68  of outer spacer  66 . The difference in circumferential distance is adapted to the inner diameter of the rim  11  so that holes  68  and  72  coincide once spacers  66 ,  70  with foil  78  placed therebetween assume the radius of curvature defined by the diameter of rim  11 . The inner spacer  70  can be made of the same materials as the other spacer  66 . 
         [0077]    Further, holes  71  are arranged on the longitudinal axis of inner spacer  70 . As illustrated, these holes  71  have a circular cross-section; however, any other cross-section can be used. They serve for letting a sample fluid enter and exit the FFF channel  13 . At least on the side facing foil  78 , the surface roughness of spacer  70  is 1.5&lt;Ra&lt;1.7, and preferably Ra=1.6. In the illustrated embodiment, spacer  70  is made of stainless steel of grade 1.4310 for use in springs. The spacer  70  has a thickness of significantly less than 1 mm and preferably app. 250 μm. 
         [0078]      FIG. 4   c  shows the compensation strip  74 . The compensation strip  74  is optionally disposed immediately between the rim  11  and the outer spacer  66 . Similar to the other strips in  FIGS. 4   a  and  4   b , it has four holes  76  disposed at the respective corners of the otherwise rectangular strip  74 , placed so that their location coincides with that of holes  68 ,  72  once compensation strip assumes the radius of curvature defined by the inner diameter of rim  11 . Compensation strip  74  is also made of stainless steel of grade 1.4310, with a thickness of less than 1 mm and preferably app. 250 μm. 
         [0079]      FIG. 5  shows the foil  78  for defining the field-flow fractionation channel  13 . The foil  78  has a generally rectangular form with four holes  82  being disposed at the corners of the foil  78 . They are placed so that their location coincides with that of holes  68 ,  72 ,  76  once foil  78  is placed in the stack adjacent rim  11 . In conjunction, these holes serve to align the elements of the stack on the holder  12 . So as to assist in compressing the stack, the holes can also be oblong, with the longitudinal direction of the holes running in the circumferential direction of the rim  11  and the holder  12 . 
         [0080]    Foil  78  has a thickness which is adapted to the fluids to be fractionated. In the illustrated embodiment, the thickness is app. 250 μm, but it can also have a thickness such as app. 100 μm to app. 800 μm. The foil  78  is made of Polytetrafluoroethylene (PTFE) which is more commonly known as “Teflon”, a brand name of E.I. du Pont de Nemours and Company. However, any other material such as a fluoroelastomer can be used as long as it is a self-sealing material, i.e. a material which automatically forms a good seal against leakage, and as long as it is resilient. The surface roughness of both faces of the foil is 1.5&lt;Ra&lt;1.7, and preferably Ra=1.6. The surface on both faces has to be scratch-free to aid in sealing. 
         [0081]    In the central part of the foil  78 , there is provided a recess  80  penetrating the foil. The recess defines the entire geometry of the FFF channel  13 , i.e. thickness, length, width, diverging and converging sections. Recess  80  is disposed completely inside foil  78 , i.e. the material of foil  78  completely surrounds it. The holes  82  are not connected to the recess  80  defining the channel  13 . The shape of the recess  80  can be described as a longitudinally stretched hexagon, with slots  86  ( FIG. 5   b ) longitudinally extending away from the farthest corners of the hexagon. The remaining four corners are disposed on the corner of what can be described as a rectangle. Alternatively, the shape could be described as a longitudinal rectangle with a triangle each joined to the short sides of the rectangle, and the apexes of the triangles terminating in the slots  86 . At each end of the slots  86 , there is an opening  87  in the form of a segment of a circle, with the diameter of the circle defining the opening  87  being at least equal to the width of the slots  86 , and preferably being larger than the width of the slots  86 . In an assembled apparatus, these openings  87  are aligned with holes  71  of inner spacer  70 , with holes  71  being larger than openings  87 . 
         [0082]      FIGS. 6   a  to  c  show the holder  12  in detail.  FIG. 6   a  shows a side, or axial view of the holder  12 . The holder  12  is formed of an aluminium zinc manganese copper alloy which has been hard coated. Its outside surface is smooth, with the surface roughness on the outside being 0.35&lt;Ra&lt;0.45, preferably being app. Ra=0.4. It consists of a generally rectangular strip which has been bent into the shape of an annulus or circle having an interrupted segment  92 . Interrupted segment  92  serves to accommodate wedge  18  ( FIGS. 6   d ,  6   e ,  6   f ). 
         [0083]    As can be seen from the centre of the annulus or circle, the end faces  91  of the holder  12  adjacent the interrupted segment  92  subtend an angle α of app. 10°, preferably 10°±0.05°, which is also the angle the surfaces of the end faces  91  of holder  12  subtend when viewed along the axis of the annulus. At least one recess, and preferably one or more holes  34 , is disposed opposite the interrupted segment  92  of the holder  12 , in the illustrated embodiment five bores  34 . By having one or more recesses in this position, it is avoided that the field-flow fractionation apparatus is out of balance during operation. Any number of holes can be used as long as their number and design is such that the reduction in imbalance is achieved. 
         [0084]    Holes  90  are disposed at both sides of the interrupted segment  92 . They serve for bolts, screws, pins or other suitable means for connecting the holes  68 ,  72 ,  76 ,  72  in the foil  78  and in the spacers  68 ,  72 ,  76  to the radially peripheral surface of holder  12 . Axial bores  32  can be disposed in the holder  12 , as illustrated, for helping in removing wedge  18 , as will be discussed further below. 
         [0085]    Threaded through holes  88  are placed circumferentially spaced further away from the interrupted segment  92  than holes  90  and  32 . Through holes  88  serve to let a fluid flow enter into and exit from the FFF channel  13  when the apparatus is in use. These holes  88  thus extend through the thickness of the holder  12 . One hole  88  next to each side of the interrupted segment  92  would be sufficient for a particular type of channel  13 . In the illustrated embodiment, however, two holes are provided next to each side so as to be able to use the holder with two channel types of different length. Naturally, the number of holes could also be larger than two. 
         [0086]    In use, one hole  88  on one side of interrupted segment  92  is aligned with one hole  71  in order to allow sample fluid to enter into the channel  13 . One hole  88  on the other side of interrupted segment  92  is aligned with hole  71  at the opposite end of channel  13  in order to allow sample fluid to exit from the channel  13 . 
         [0087]      FIG. 6   b  shows a radial view of holder  12  when looking at interrupted segment  92  from radially outside of the holder  12 . It becomes apparent that the interrupted segment  92  is also tapered along the axial direction of holder  12 . Once the FFF apparatus is assembled, the interrupted segment  92  becomes narrower due to the taper when moving in the direction of hub  10   b . This is also the depth direction of threaded holes  32  away from the axially peripheral surface and into the body of holder  12 . The angle of taper β subtended by the end faces of interrupted segment  92  in this direction is app. 8° and preferably 8°±0.05°. The surface roughness of the end faces  91  defining the interrupted segment  92  is 0.35&lt;Ra&lt;0.45, and preferably Ra=0.4. 
         [0088]      FIG. 6   c  shows another view of the interrupted segment  92  when viewed along the axial direction and is essentially a close-up view of the corresponding part of  FIG. 6   a.    
         [0089]      FIGS. 6   d  to  f  show a wedge  18  to be inserted into interrupted segment  92 . The wedge  18  is configured to be inserted and wedged into the interrupted segment  92  of the holder so as to force the holder  12  and spacers  68 ,  72 ,  76  and foil  78  defining the FFF channel  13  against the inner circumference of the rim  11  of rotor  10   a.    
         [0090]    Wedge  18  is made of an aluminium zinc manganese copper alloy which has been hard coated by a polytetrafluoroethylene (PTFE) coating. The coating preferably has a thickness of between 20 to 25 μm. Also preferably, the surface quality of the wedge is as noted above with respect to holder  12 . 
         [0091]    The wedge  18  has a pair of generally opposed surfaces  94 . Each surface has the shape of an oblong rectangle, the longer edges of the rectangles being essentially parallel to the axial direction and the shorter edges being essentially parallel to the radial direction of the FFF apparatus. The surfaces  94  of the pair are inclined with respect to one another in two directions. 
         [0092]    Firstly, they are inclined so that in the inserted position of the wedge  18  and in an axial view, both surfaces  94  extend along radial beams with an angular offset approximately corresponding to the angular circumferential dimension of interrupted segment of the holder  12 . Their angle of inclination is thus such that they fit tightly with the surfaces defining the end faces  91  of holder  12 . In particular, in the currently preferred embodiments, the angle of inclination γ with respect to one another is app. 8° and preferably 8°±0.05°. 
         [0093]    Secondly, the wedge  18  is designed such that the surfaces  94  are inclined with respect to one another so that they subtend an angle δ when viewed along the axial direction of the assembled FFF apparatus. The angle δ is designed such that the wedge  18  circumferentially displaces the holder  12  by a predetermined amount when it is axially advanced into the interrupted segment. This can appropriately be effected with the assistance of a tool. In this particular preferred embodiment, the angle of axial taper subtended by the end faces  94  is app. 10° and preferably δ=10°±0.05°. 
         [0094]    Along the direction of the wedge  18  which is parallel to the longer edges of the rectangular surfaces  94 , i.e. parallel to the axial direction of the rotor  10   a  when the wedge  18  is inserted into the interrupted segment  92 , there is a throughhole in the form of a stepped bore  16  ( FIG. 6   f ). The diameters of the stepped bore  16  increase when moving from the end of the bore opening in the smaller end face of the wedge  18  to the end of the bore opening in the larger end face of the wedge  18 , “large” and “small” in this context referring to the overall area of the respective end faces. The bore  16  also contains a thread  96  for introducing a screw by means of which the wedge can be removed from the corresponding bore in the rotor  10   a . In the present embodiment, the wedge  18  has an axial length which is slightly less than the axial dimension of holder  12 . 
       BEST MODE OF OPERATING THE APPARATUS 
       [0095]    In the following, the currently preferred way of operating the previously described apparatus  10  will be described, starting from the assembly of the apparatus. 
         [0096]    First, the hub  10   b  is attached to flange  33  of the shaft. Then, or in parallel, the inner spacer  70 , then the foil  78 , followed by the outer spacer  68  and optionally the compensation strip  74  are stacked on the holder  12  such that the holes  82 ,  68 ,  72 ,  76 , and  90  are aligned with one another. This will serve to automatically align holes  88 ,  71  and openings  87  with one another to let a sample fluid enter into and exit from the recess  80  defining the FFF-channel  13 . The resulting stack is then connected by suitable means such as bolts. Then, holder  12  together with the assembled FFF channel  13  is inserted into the interior of the rim  11  of the rotor  10   a.    
         [0097]    Wedge  18  is inserted into interrupted segment  92  of holder  12  by means of a suitable tool such as a gear puller or extractor which is applied to the surface of the hub facing away from the holder, reaches through the stepped bore  16  of the wedge and acts on the end face  95  of the wedge. The act of wedging the wedge  18  into the interrupted segment  92  can then be effected by a screwing action of the gear puller whilst leaving the thread  96  in the bore  16  of the wedge  18  and the thread in the corresponding hole of the hub  10   b  unused. Otherwise, due to the high forces needed, wedging the wedge  18  into the interrupted segment  92  might damage or destroy these threads. It is the general idea to use the tool to apply this force between the axial end face  95  of the wedge  18  and a corresponding surface of the hub  10   b  of the rotor  10   a  such that the wedge  18  is wedged into the interrupted segment  92 , thereby wedging apart holder  12  and forcing the channel  13  against rim  11 . This is done at such a position that the bore  16  of the wedge  18  is aligned with the corresponding bore in the hub  10   b  to enable a screw, a bolt or other suitable means to attaching the wedge  18  to the hub  10   b  and secure the wedge in its wedged position. 
         [0098]    Once the wedge  18  has been wedged into the interrupted segment  92 , it is secured to the rotor by means of a screw inserted through hole  16  and screwed into the corresponding threads of the bore in the rotor  10   a . However, it should be noted that the screw is under normal circumstances of operation not needed. The wedge  18  and the interrupted segment  92  are designed so that, in normal operation, the wedge will not be displaced. The function of the screw is simply a safety precaution in case the FFF apparatus experiences unusual shocks which may cause the wedge to be dislocated. Also, if the wedge was not held in place, a problem might arise when turning on the FFF apparatus: initially, when the holder  12  is not firmly pressed against rim  11  due to the centrifugal force, there would only be a small force holding the wedge  18  inside the interrupted segment  92 . Thus, the forces during turning on might act as to dislodge the wedge  18 . As it is, at least at this stage, not firmly secured inside the interrupted segment  92 , it might as a consequence be ejected from the apparatus, thereby potentially hitting bystanders and equipment. 
         [0099]    The first, second and third connecting means as well as the end caps are assembled as described above. 
         [0100]    After the rotor has been assembled in this manner, means for supplying and extracting field-flow fractionation liquid (sample fluid) to and from the shaft  61  are connected by suitable means, such as fitting flexible tubing by means of standard tubing adapters so that the tubing extends from terminal  46   a  to one of holes  88  and from the other of holes  88  to terminal  46   b . This establishes a connection between the shaft  61  and the field-flow fractionation channel  13  defined by the foil  78 . 
         [0101]    Afterwards, suitable means, such as flexible tubing, for supplying field-flow fractionation liquid (sample fluid) to and from the rotor are applied. The flexible tubing is introduced through terminal  40   a  into conduit  54   a  for introducing the fluid to be fractionated. Further flexible tubing is introduced through terminal  40   b  into conduit  54   a  for removing the fractionated fluid. 
         [0102]    Here, it should be noted that the means such as the flexible tubing extends into the conduits  54   a/b  until close to, but not up to contact with the first connecting pieces  52   a/b.    
         [0103]    Flushing fluid connection lines are connected to terminals  44   a/b  and drain fluid lines are connected to terminals  42   a/b.    
         [0104]    Terminals  47   a/b  are closed by plugging them with a blind terminal connector, i.e. one which does not permit fluid to pass. Flushing fluid is supplied through terminals  44   a/b . It fills the space between the two seal rings in the end cap, i.e. the space downstream of the second seal ring  36   a/b  and upstream of the first seal ring  38   a/b . The pressure of the flushing fluid is controlled so that its pressure corresponds to the pressure of the sample fluid at the respective terminal  40   a/b . In this manner, the pressure difference across the second seal rings  36   a/b  is minimized, preferably to less than 0.05 bar, and even minimal loss of sample fluid is prevented. It has turned out to be particularly advantageous if the pressure differential across the second seal rings is minimized by having 4 to 7 bar pressure of the sample fluid and about 200 mbar pressure of the flushing fluid and that by means of controlling the flushing pressure, the pressure difference across the first seal ring  38   a/b  is controlled, thereby sealing the flow of sample fluid. Since the first seal rings  38   a/b  seal a liquid from a gaseous space, fluid is more likely to leak there than at the second seal rings  36   a/b  which seal to liquid-filled spaces from each other. Possible excess flushing fluid will thus leak across the first seal rings  38   a/b  and is then drained through terminals  42   a/b.    
         [0105]    Alternatively, terminal  47   a  and/or terminal  47   b  could be connected to a discharge line for the flushing fluid. If terminal  47   a/b  is connected in this manner, the flushing fluid is supplied to terminals  44   a/b . The space “downstream” of the seal rings  36   a/b  is filled, as in the previously-described alternative, but here the flushing fluid is permitted to flow past the second seal ring  36   a/b  and to leave the space through the one or both of terminals  47   a  and  47   b  which is connected to a discharge line. This alternative facilitates operation of the FFF apparatus since control of the flushing fluid pressure is less intricate. 
         [0106]    In the thus assembled apparatus  10 , the motor is made to turn the shaft  61  about its axis. The motor is, as was mentioned previously, a DC electric motor. It is supplied by an external AC to DC power supply or by another type of power supply, e.g. a battery. The AC to DC power supply is suitable for use in the country the apparatus is to be used in that it is capable of converting that country&#39;s AC mains voltage and frequency into the correct DC power for use in the motor driving the FFF apparatus. Also, sample fluid and flushing fluid are led into and conducted out of the FFF apparatus at the respective inlets and outlets. The sample fluid thus passes through the field flow fractionation channel  13 .