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(11) | EP 0 104 664 A2 |
| (12) | EUROPEAN PATENT APPLICATION |
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| (54) | Replaceable field flow fractionation channel |
| (57) Replaceable channel insert is provided for a sedimentation field flow fractionation
channel. |
BACKGROUND OF THE INVENTION
[0001] Field flow fractionation is a versatile technique for the high resolution separation of a wide variety of particulates suspended in a fluid medium. The particulates include macromolecules in the 105 to the 1013 molecular weight (0.001 to 1 µm) range, colloids, particles, micelles, organelles and the like. The technique is more explicitly described in U.S. Patent No. 3,449,938, issued June 17, 1969 to John C. Giddings and U.S. Patent No. 3,523,610, issued August 11, 1970 to Edward M. Purcell and Howard C. Berg.
[0002] In the case of sedimentation field flow fractionation (SFFF), use is made of a centrifuge to establish a force field for separating the particulates. For this purpose a long, thin annular belt-like channel is made to rotate about its axis in a centrifuge. The resultant centrifugal force causes components of higher density than the mobile phase flowing in the channel to sediment toward the outer wall of the channel. For equal particulate density, because of higher diffusion rates, smaller particulates will accumulate in a thicker layer against the outer wall than will larger particulates. On the average, therefore, larger particulates are forced closer to the outer wall.
[0003] If now the mobile phase or solvent is fed continuously from one end of the channel, it carries the sample components through the channel for later detection at the outlet of the channel. Because of the shape of the laminar velocity profile of the solvent within the channel and the placement of particulates in that profile, solvent flow causes smaller particulates to elute first, followed by a continuous elution of components in the order of ascending particulate mass.
[0004] In order to reduce the separation times required using this technique, it is necessary to make the channels relatively thin. This creates many problems in that the walls of the channel must have a microscopically smooth finish to prevent the particulates from sticking to the walls or being trapped in crevices of the same height as the particulate distributed. Unfortunately, in the construction of such a thin belt-like channel for use in a centrifuge, the microfinish cannot be easily obtained or maintained. Another problem is that, even though the walls of the channel have a microfinish, the particulates in many instances tend to adhere to the walls of the channel. This problem is noted by Purcell et al. at column 4, line 43 who suggest it is necessary to select for the surface of the channel wall a material to which the particle will not be adsorbed.
[0005] .A copending European patent application filed September 29, 1983, Serial No. and entitled "Film Insert for Sedimentation Field Flow Fractionation Channel" by Donald R. Johnson (IP-0307) describes positioning a replaceable, thin film to cover the outer wall of the channel. This facilitates cleaning the channel and simplifies the manufacture of the outer channel wall--it need no longer have a micro finish. If the film is resilient and extended through the interface between the channel rings, leakage from the channel is reduced. The particular film may be selected according to the particulates to be separated, the objective being to prevent the particles from adhering to the film surface of the outer wall. The film may be coated with a material or its surface may be modified using known techniques to reduce adhesion of the particulates to the outer support ring. Typically, the film may be a polymeric material that is flat, smooth, of a desired surface chemically to reduce particulate adhesion, resilient, insoluble in the solvents normally used, and noncorroding.
SUMMARY OF THE INVENTION
[0006] According to one aspect of this invention, \ an apparatus is constructed for separating particulates suspended in a fluid medium according to their effective masses. This apparatus includes a centrifuge bowl, means for rotating the bowl about its axis, means positioned in the bowl having an outer and inner, spaced apart, circumferential walls defining an annular slot, the bowl adapted to contain a liquid, tubing means for passing the fluid medium circumferentially through the slot, and means for introducing said particulates into said medium for passage through said slot, the improvement wherein:
a flat, flexible, elongated, closed channel is positioned in the slot and is in communication with the tubing means for passage of the fluid medium and particulates through the channel, said slot being in liquid communication with the bowl, thereby to compensate for fluid pressure within the channel.
[0007] In one embodiment of the invention, the replaceable channel is formed of a smooth film material laminated together at its edges. Preferably, however, the channel is a laminate of three strips of a plastic film; the middle strip has its mid-portion removed and hence defines the length and width of the channel. The outside strips define the inner and outer walls of the channel and, of course, the thickness of the middle strip, the thickness of the channel. Inlet and outlet plastic tubes are attached to either end of the channel through the inner channel wall. These tubes extend through the inner ring.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Further advantages and features of this invention will become apparent upon the following description wherein:
FIG. 1 is an exploded pictorial representation of mating split rings that may be used to form a slot adapted to receive a replaceable sedimentation field flow fractionation channel;
FIG. 2 is a fragmentary cross sectional view of the slot of FIG. 1 formed in accordance with this invention;
FIG. 3 is a fragmentary cross-sectional elevation view of an alternative continuous ring slot adapted to receive the replaceable channel insert of this invention ;
FIG. 4 is a plan view of the ring slot of FIG. 3; and
FIG. 5 is a plan view, partially cut away of the replaceable channel insert of FIGS. 1 and 3.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0009] Typical sedimentation field flow fractionation apparatus with which this invention may find use are described in U.S. Patent 4,283,276 issued August 11, 1981 to John Wallace Grant, U.S. Patent 4,353,795 scheduled to issue October 12, 1982, copending application Serial No. 326,156, filed November 30, 1981 and entitled "Apparatus and Method for Sedimentation Field Flow Fractionation" by Dilks et al. (IP-0304) and copending application Serial No. 326,157, filed November 30, 1981 and entitled "Sedimentation Field Flow Fractionation Channel and Method" by Dilks et al. (IP-0313).
[0010] As is described in the Grant patent, split ring, SFFF channels 10 (FIGS. 1 and 2) are constructed to have what is illutrated for simplicity as an outer ring 12. Actually the ring 12 is in the form of a bowl type centrifuge rotor having a constant inner radius and functions to support an inner ring 13. The bowl rotor 12 may be driven directly by a centrifuge drive 14 acting through a linkage depicted by the cashed line 16. The centrifuge bowl rotor 12 has a cylindrical inner surface formed thereon to provide the outer channel wall. The inner ring 13 is split, i.e., its longitudinal circumference is divided or separated to have a gap 18 with the longitudinal ends 20 of the inner ring 13 slightly tapered to facilitate the use of wedges 22. The wedges 22 retain the inner ring sufficiently expanded to maintain contact with the rotor 12 at all times even when stopped. The radially outer wall 24 of the inner ring 13 and the radially inner wall 26 of the outer ring 56 are formed to define the channel slot 10.
[0011] , Normally these walls are formed as by polishing, for example, or by coating the surfaces with a suitable material to have a microfinish. This smooth finish tends to reduce'the possibility that particles will stick to the walls or become entrapped in small crevices or depressions and also insures that the expected sample retention takes place. In accordance with this invention, the split ring assembly of Grant is used to form a slot 10 in which a replaceable, flat, flexible, elongated, closed channel insert 11 may be positioned. By forming the replaceable channel of a plastic of the type described in the Johnson application, the tendency of the particulates to stick is reduced without the need for forming the channel with a microfinish or coating the channel walls as described by Purcell et al.
[0012] Thus a groove 24 may be formed in the outer wall of the inner ring 13 form the annular slot 10 between lands 28. No effort is made to provide the lands with a seal because. as is described below, liquid in the bowl is allowed to fill the slot 10 and surround the channel insert 11. Additionally, the upper edge of the inner ring may formed with n radial, outwardly extending flange 34, as is seen most clearly in FIG. 2, such that the inner ring may rest upon and be supported by a step in the bowl rotor 12 against axially downward displacement. Inlet and outlet conduits 36, typically stainless steel, communicate via fittings 38 with the ends of the channel insert 11 through the inner ring. As is known, fluids containing particulate samples are passed through these conduits via a suitable rotating seal (not shown). The outlet conduit is coupled to a detector (not shown). As noted, the thickness of an SFFF channel is relatively small, typically being in the order of 0.1 cm or less.. The dimensions of the channel, both width and thickness must be very precisely maintained. The actual thickness is selected according to the separations to be performed as is known.
[0013] The channel insert 11 typically is placed over the inner ring, the ring compressed (the wedges 22 being removed) and placed within the bowl rotor 12 and the inner ring 13 allowed to expand, thus forming the closed slot 10 or housing for the replaceable channel insert. The wedges are then reinserted to stabilize the inner ring 13 as described by Grant. This film, which may be of any suitable material, provides the smooth surface for the channel walls without the need for the normal polishing that would normally be required of the channel walls.
[0014] The films that may be used, as is described in the Johnson application, should be flat, have a smooth surface and the desired surface chemical characteristics, be flexible, insoluble in the mobile phase used, noncorroding, and desirably colored for ease of handling. Among the films that may be used are cellulose, polyethylene terephthalate, polytetrafluoroethylene, aromatic polyimide, polypropylene, polyvinyl ncetates, and polyvinyl propionate polyesters. These tend to provide hydrophobic surfaces. If hydrophilic surfaces are desired, the above noted materials in many cases can be treated or coated. Film thickness typically should exceed 0.005 cm. Thinner films than this have a tendency to wrinkle, which is highly undesirable - for this purpose. Films up to .02 cm thickness have been used successfully. Films significantly in excess of this thickness may be used but tend to be unnecessarily wasteful of the film material and can require excessively large channel slots 10.
[0015] The replaceable channel or channel insert 11 may be formed by folding a strip of plastic along its length and bonding or sealing the edges. Suitable inlet and outlet tubes 30 providing access to the interior of the channel 11 are bonded at either end of the channel insert on the radially interior side when positioned in the slot 10. Preferably, however, the channel 11 is a laminate of three strips 40, 41, 42 (FIG. 5) of the plastic film used, i.e., a bottom strip 40, a middle strip 41 with its elongated center 44 removed to define the length and width of the channel and a top strip 42. The strips may be bonded as heat sealed as desired. The top and bottom strips 40 and 42 define the inner and outer walls of the channel with the channel thickness determined by the thickness of the middle strip 41. Tubes 30 are introduced through the inner wall 42 at either end of the center 44 and bonded to provide fluid ingress and egress to the channel.
[0016] In use, a replaceable channel insert 11 of suitable dimensions is selected, positioned in the slot 10 and its tubes 30 introduced through the inner ring ports to the fittings 38 which join the tubes 30 to the tubes 36. The inner ring is compressed and fitted in the outer ring 12 so that the slot 10 defines and provides structural strength and support for the channel insert 11, i.e., the replaceable channel in effect serves as a liner for the slot 10. The bowl is filled with a liquid having a density comparable to that of the fluid medium to be passed through the channel 11. This equalizes the pressure about the channel during centrifugation and prevents rupture of the channel insert during operation.
[0017] In SFFF separations, samples of particulates in suspension are introduced into a mobile phase which carries the particulates through the channel insert 11. The particulate under test will determine the mobile phase and the type of film that is used to form the channel.
[0018] As described by the Johnson application, adhesion to the channel walls can be avoided by properly selecting or coating or modifying the surface of a film. Adhesion usually occurs where the surface charge of the particulate and the film's surface polarity are opposite. It also occurs where there is covalent bonding, chemical adsorption, or van der Waals forces. Ideally the channel should "look like" the mobile phase, i.e., be of the same polarity.
[0019] It also is important that the mobile phase density be less than the particulate density and that the mobile phase wets the particulates. If the particulates are hydrophobic, the mobile phase should be nonaqueous. If the particulates are hydrophilic, the mobile phase should be aqueous. Although of secondary importance, it is desirable that the mobile phase wets the channel walls.
[0020] In the event a hydrophilic surface is desired, many of the above noted polymeric films which are hydrophobic, may be surface modified. This typically is accomplished by forming OH or carboxyl groups on the surface as by chemically bonding desired molecules to reactive sites on the surface. Alternatively the hydrocarbons may be oxidized by a corona discharge, the fluorocarbons may be reduced by sodium in ammonia, esters, amides and imides may be hydrolyzed. Cellulose and polyvinyl alcohol have hydrophilic surfaces.
[0021] The replaceable channel insert 11 of this invention finds use with any double ring or other type SFFF channel which can be opened for cleaning and the like and which can be submerged in a liquid. For example, it may be used in the SFFF rotors described in the copending Dilks and Yau and bilks, Yau and Kirkland applications mentioned above. These rotors are formed with a continuous inner ring 54. The inner ring described by the Dilks et al. application is modified to facilitate insertion of the replaceable channel insert in the slot. As seen in FIG. 3, an inner ring 54 is positioned in the bowl of a centrifuge rotor 60. The upper portion of the ring's outer radial surface is cut away to form a slot or recess 58 leaving a bottom land 59. A second spacer ring 82 is formed and sized to fit over the outer surface of the inner ring 54. This permits the channel insert 11 (FIG. 5) to be fitted in the slot 58. Radial slots 68 are formed in the upper portion of the inner ring to permit the inlet and outlet and, if desired, sample inlet tubes 30 to slide into position for connection to fittings 78 which are connected to the tubes 36. Dummy fittings 78' may be inserted on the opposite side of the ring 54 for balancing.
[0022] As described by Dilks and Yau, the inner ring 54 is formed of an appropriate engineering plastic such as Delrinl& acetal resin or Noryl@ polyphenylene oxide polymer. This inner ring 54 is inserted into the bowl of a bowl-type centrifuge rotor, such as a zonal rotor 60, such that the land 59 and spacer ring 82 which defines the recess or slot 58 contacts the inside peripheral surface 63 of the rotor 60. Due to its inherent elasticity, this plastic inner ring 54 is designed to grow, as will be described below, with the expansion of the zonal rotor 60 as the centrifugal force field is increased.
[0023] The rotor 60 is filled with a liquid. This reduces the pressure differences between the fluid medium in the channel 10 and that outside of the channel, thus reducing leakage through the seal at the interface between the inner ring and the rotor bowl. Stress on the plastic inner ring is also reduced because it is surrounded by the liquid. The liquid for filling the rotor bowl and the fluid medium for the channel preferably are selected to have densities that are about equal although fluids with densities in the range of 0.6 to 1.2 g/ml may be used.
[0024] This rotor 60 is adapted to be driven in a conventional manner (not shown) to rotate about the axis 62. The inner ring 54 is seated in the bottom of the bowl of the zonal rotor 60 which rotates about the axis 62. The rotor GO has a cover 70 that fastens onto the bowl. A rotating seal (not shown) permits the passage of the fluid medium and sample, if desired, to and from the inner ring 54 via conduits 30 which connect to fasteners 38. The rotating seal may be of any conventional design used to couple fluids to and from rotating bodies such as zonal rotors. The rotating seal described in U. S. Patent 4,375,871 is suitable. Alternatively, the rotating seal described in U.S..Patent 4,357,235 (Serial No. 125,854 filed February29, 1980 enti.tled "Drive for Rotating Seal" by Charles Heritage Dilks, Jr.) may be used. Whatever the rotating seal used, the conduits 36 (FIG. 3) transmit the fluids from the rotating seal through a hollow drive shaft 72 for the rotating seal 28 (FIG. 5) into the rotor 60 and thence to the inner ring 54. The drive shaft 72 is secured to the rotor cover 70 and the rotor cover 70 to the rotor 60.
[0025] The channel slot 10 is defined, as described above, by the circumferential recess 58 in the inner ring 54 and the inner wall of the rotor 60 (FIG. 3). The inner ring 54 and spacer ring 82 are both formed of a suitable engineering plastic that is chemically inert, strong, and yet resilient such as the two materials mentioned above. Alternatively, the rings may be formed of materials such as polytetrafluoroethylene, polyethylene, polyurethane or nylon. One of the main criteria used for selecting the particular engineering plastic for use with high force fields is that its effective density ϕ to tensile modulus E ratio generally should exceed the effective density φ to tensile modulus E ratio of the material forming the rotor 60. The effective density φ of the plastic is the actual density of the plastic minus the density of the bowl filling liquid.
[0026] In this manner, as the bowl rotor expands under the influence of centrifugal force, the inner and spacer rings 54, 82 expand outwardly a like or slightly greater amount to maintain contact between the land 59 and spacer ring 82 with the inner surface 63 of the rotor 60 and thus to provide a housing for the replaceable channel 11. The zonal rotor bowl 60, as is typically used in centrifuges, is formed of titanium, stainless steel or aluminum. Titanium is preferred. Preferably, the materials of the rotor bowl and inner ring are selected as described above such that the thickness of the channel is maintained throughout operation within + 2%. This reduces separation errors.
1. In an apparatus for separating particulates suspended in a fluid medium according
to their effective masses, said apparatus having a centrifuge bowl, means for rotating
the bowl about its axis, means positioned in the bowl having an outer and inner spaced
apart, circumferential walls defining an annular slot, tubing means for passing the
fluid medium circumferentially through the slot, the bowl adapted to contain a liquid,
and means for introducing said particulates into said medium for passage through said
slot, the bowl adapted to contain a liquid, the improvement wherein:
a flat, flexible, elongated, closed channel is positioned in the slot and is in communication with the tubing means for passage of the fluid medium and particulates through the channel, said slot being in liquid communication with the bowl, thereby to compensate for fluid pressure within the channel.
2. The apparatus of claim 1 wherein said slot inner wall is defined by an inner ring
having an outer wall the middle portion of which defines a circumferential groove
of constant depth.
3. The apparatus of claim 1 wherein said slot inner wall is defined by an inner ring
separated at one point along its circumference and whose middle portion defines a
circumferential groove of constant depth.
4. The apparatus of claim 1, 2 or 3 wherein the channel is formed of a smooth film
material laminated together at its edges.
5. The apparatus of claim 1, 2 or 3 wherein the film is coated with a material that
reduces adhesion of the particulates to the outer support ring.
6. The apparatus of claim 1, 2 or 3 wherein said film has a surface modified chemically
to reduce the adhesion of the particulates to the outer support ring.
7. The apparatus of claim 1, 2, or 3 wherein the channel is a laminate of three sheets
of a filmlike material with the middle portion of the middle sheet removed to define
the length and width of the channel.
8. The apparatus of claim 1 wherein said inner ring comprises a first ring with a
bottom flange defining the lower side of the groove and a second ring insertable into
the slot to define the upper side of the groove.