US3664593A - Gel fractionator - Google Patents

Gel fractionator Download PDF

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Publication number
US3664593A
US3664593A US24640A US3664593DA US3664593A US 3664593 A US3664593 A US 3664593A US 24640 A US24640 A US 24640A US 3664593D A US3664593D A US 3664593DA US 3664593 A US3664593 A US 3664593A
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Prior art keywords
gel
column
cylinder
screen
advancing
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Expired - Lifetime
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US24640A
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English (en)
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Warren E Gilson
Robert E Gilson
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Individual
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Individual
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

Definitions

  • a gel fractionator includes a cylinder for holding a column of gel, together with a piston assembly for advancing the column through the cylinder toward a fractionating apparatus.
  • the piston assembly includes an air release valve operable from the exterior of the cylinder for releasing trapped air from above the gel column.
  • the fractionating assembly includes a cutting screen engaged by the advancing column for severing the advancing column in a generally axial direction into subdivisions or segments. A rotating wire beneath the screen severs the subdivisions of gel as the gel advances.
  • the wire is mounted across the mouth of a funnel which receives the severed gel particles, and the funnel includes structure for directing a stream of liquid at the underside of the screen for flushing the severed particles.
  • the fractionating operation is carried out by repeatedly advancing the column a predetermined distance, pausing, and carrying out several discrete dlushing operations to remove all severed particles.
  • the present invention relates to improvements in an apparatus and in a method for fractionating a column of gel.
  • a gel fractionator is a device for dividing a cohesive column of gel material into a series of individual samples or fractions. Such devices are useful in connection with known analytical procedures wherein a material to be analyzed, such as for example a protein or other macromolecular mixture, is dispersed by electrophoresis or the like within a column of a suitable gel material, such as a polyacrylamide gel;v The column is divided into sequential individual fractions during the fractionating process, and the fractions are individually analyzed as by determination of radio-activity in ⁇ a scintillation counter or the like.
  • Important objects of the present invention are to provide an improved fractionating method and apparatus;
  • a gel fractionator constructed in accordance with the principles of the present invention includes a cylinder for holding a column of gel and a piston assembly for advancing the gel in the cylinder toward a fractionating assembly including first cutting means for subdividing the column into axially oriented subdivisions or ex tensions and second cutting means for severing the subdivisions or extensions.
  • first cutting means for subdividing the column into axially oriented subdivisions or ex tensions
  • second cutting means for severing the subdivisions or extensions.
  • the cross section of the gel column is not substantially reduced.
  • Means are provided for ushing the severed gel particles from the region of the cutting means.
  • the gel is advanced a predetermined distance and then stopped during a delay period in which the cutting oepration is completed. Following the delay period, several separate sequential iiush operations are carried out fully to Wash the severed gel particles from the cutting region.
  • FIG. 1 is a fragmentary, broken, partly sectional and partly diagrammatic view of a gel fractionator constructed in accordance with the present invention.
  • FIG. 2 is a greatly enlarged, fragmentary, partly sectional, exploded vieW of one portion of the apparatus of FIG. l.
  • the gel fractionator 10 includes a cylinder 12 held in a relatively fixed position by a support structure 14 and adapted to contain a unitary, cohesive column of gel to be diwided into individual samples or fractions.
  • a relatively movable piston assembly generally designated as 16 is moved by a piston driving structure generally designated as 18 thereby to move the gel column within the cylinder 12.
  • the piston assembly 16 includes a novel air release valve generally designated as 20 for permitting the release of air trapped between the column and the piston assembly 16.
  • the column of gel is moved by the piston assembly 16 toward a novel fractionating assembly generally designated as 22 and serving to divide the advancing gel column into fractions which are discharged from an outlet 24.
  • the fractionator 101 may be mounted directly on the carriage structure of a fraction collector, in which case the fractions may be deposited directly from the outlet 24 into a series of vials or containers (not shown) disposed successively beneath the outlet 24.
  • the cylinder 12 ⁇ comprises a glass cylinder, and if desired, may constitute the same cylinder in which the gel column and sample are subjected to a preceding electrophoresis operation. This arrangement avoids the necessity of transferring the column of gel from a different cylinder in order to carry out the fractionating operation.
  • the cylinder 12 is held in a relatively xed position by the support structure 14 including a lower holder 26 and an upper holder 28.
  • -Lower holder 26 includes a neck portion 30 loosely received in an opening 32 in a lower mounting bracket 34, while the upper holder is threaded into an opening 36 in an upper bracket 38.
  • a support rod 40 is fixed to each of the brackets 34 and 38.
  • the lower holder 26 In order to mount the cylinder 12 holding a column of gel and a dispersed sample, the lower holder 26 is first positioned as illustrated on the lower bracket 34. The column 12 is then slipped through the opening 36 and into a recess 42 in the lower holder 26. The upper holder 28 is then threaded into the opening 36, the upper end of the cylinder l12 being received in a recess 44. The upper holder 28 is threaded into the upper bracket 38 until the cylinder 12 is held 'firmly in position.
  • the piston assembly 16 is inserted.
  • the piston driving structure 18 is moved upwardly as viewed in FIG. 1 to its uppermost position to provide clearance for insertion of the piston assembly 16 through an opening 46 in the upper holder 28 and into the top of the cylinder 12.
  • the piston assembly 16 includes the novel air release valve for releasing air that may be trapped in the cylinder 12 between the upper end of the gel column and the lower end of the piston assembly.
  • an inner piston rod 48 received within an outer piston sleeve 50.
  • the air release valve 20 is made up of an enlarged piston head member 52 of the rod 48, a resilient sealing gasket 54, and the lowermost end or edge 56 of the outer piston sleeve 50.
  • the valve 20 assumes its illustrated position wherein the gasket 54 is held in sealing relation between the edge S6 and the head S2. Since the upper surface of the head 52 is sloped or cone-shaped, the gasket 54 is also forced outwardly into sealing relation against the inner wall of the cylinder 12. In this position, air and gel is prevented from leaking or extruding upwardly past the piston assembly 16. lHowever, the release valve 20 can be opened from the exterior of the cylinder 12 in order to permit the release of trapped air.
  • the upper end of the piston sleeve 50 is received in a recess 58 in a piston cap element 60, while the piston rodl 48 extends outwardly of the cap 60 through a smaller diameter recess 62.
  • a spring 64 held in compression between the top of the cap 60 and the underside of a push button or enlargement 66 carried at the top of the rod 48 normally squeezes the gasket 54 between the edge 56 and the head 52.
  • the push button 66 When the head 52 engages the top of the gel column and the trapped air is fully vented, the push button 66 is released and is returned by the spring 64 to the position wherein the air release valve 20 is fully closed by the force applied to the gasket 54 between the edge 56 and the head 52.
  • the sealing force is increased when a driving force is applied to the piston sleeve 50 during the fractionating process.
  • the piston driving structure 18 serves to force the piston assembly 16 into the cylinder 12 thereby to move the gel column toward the fractionating assembly 22 and outlet 24.
  • a drive plate 68 including a recess 70 receiving .the cap 60 and including a shoulder 72 for applying a force by way of the cap 60 to the piston sleeve 50.
  • a reduced diameter recess 74 freely receives the push button 66 and the spring 64.
  • a driving force is applied to the drive plate 68 by means of a threaded drive shaft 80 held against axial movement and extending through an opening 82 in the plate.
  • the shaft 80 threadedly engages drive nut 84 held on the plate 68 by means of a suitable holding bracket 86.
  • Drive shaft 80 is rotated by means of a suitable driving connection indicated by a broken line to a suitable drive motor 88 illustrated schematically in FIG. l. Rotation of the shaft 80 causes movement of the drive nut 84 axially along the shaft and concurrent movement of the plate 68 along the slide bars 76 and 78.
  • the fractionating assembly 72 carries out a novel fractionating operation on the column of gel advanced toward the assembly 22 by the piston assembly 16 operating within the cylinder 12.
  • the cross sectional area occupied by the advancing column of gel is not greatly restricted in carrying out the fractionating operation and excessive extrusion of the gel and the resulting zone distortion are avoided.
  • the advancing column of gel is first subdivided by severing the gel in substantially axial directions to produce a plurality of generally axially oriented portions or extensions of the gel column. The subdivisions are then severed from the gel column by cutting in a substantially radial direction. The severed gel segments are ush'ed from the cutting area and are delivered through the outlet 24.
  • the lower holder 26 includes the recess 42 for receiving the lowermost end of the cylinder 12, which recess 42 also defines a shoulder 90 upon which is mounted a first cutting structure 92 arranged substantially normal -to the axis of the cylinder 12 and thus substantially normal to the path of advancement of the gel column.
  • the cutting member 92 comprises a screen which, for example, may be an 80 mesh screen. The screen 92 is held against the shoulder 90 by means of a gasket 94 sandwiched between the screen and the lower end of the cylinder 12.
  • a gel severing and flushing unit Disposed directly beneath the screen 92 in a reduced diameter recess 96 of the lower holder 26 is a gel severing and flushing unit generally designated as 98 and serving both to sever the gel subdivisions produced by advancing the gel through the screen 92 and to flush the severed subdivisions from the region of the screen 92.
  • the unit 98 includes a body portion 100 mounted for rotation in the recess 96 together with a stem portion 102 extending through a further recess 104 in the stern portion 30 in the lower holder 26.
  • the unit 98 includes an axially oriented central passageway 106 communicating the outlet 24 with the region of the screen 92.
  • the uppermost end of the passageway 106 comprises a cone-shaped funnel por-tion 107 having a mouth located beneath the screen 92 for receiving severed portions of the gel column.
  • the second cutting means Supported across the mouth of the funnel portion of the unit 98 is a second cutting means movable through a plane parallel to and just below the screen 92.
  • the second cutting means comprises a wire 108, the ends of which are attached to the mouth of the funnel 107 as by inserting the ends of the wire into notches 110 (FIG. 2).
  • Movement of the wire 108 is caused by rotating the gel severing and flushing unit 98.
  • the stem portion 102 carries a drive gear 112 driven by a drive pinion 114 mounted on a rotatable shaft 116.
  • the shaft 116 is drivingly coupled for rotation by a suitable drive motor 118.
  • Operation of the motor 118 causes rotation of the stem 102 and thus of the unit 98 and accordingly causes the wire 108 to rotate beneath the screen 92 and thereby carry out a cutting operation on the advancing gel subdivisions.
  • the unit 98 is rotated continuously throughout the fractionating operation at about twenty-live revolutions per minute. Continuous rotation, however, is not necessary to the method of the invention.
  • the fractionating assembly 22 includes novel provision for flushing the segments from the region of the screen 92.
  • a suitable liquid pressurized by a pump 119 ows through a conduit 120 to a passage 122 in the lower holder 26 communicating with the recess 96.
  • the outer peripheral wall of the body portion 100 of the unit 98 includes an annular recess 124 (FIG. 2) communicating with passage 122.
  • the fluid When pressurized lluid is channeled from the pump 119 to the recess 124, the fluid is directed by a passage 126 extending through the body 100 from the recess 124 to the surface of the cone 107 and toward the underside of -the screen 92.
  • the pressure of the uid and the diameter of the passage 126 are chosen so that the fluid squirts upwardly against the screen.
  • the unit 98 is rotated, the fluid is directed over the entire cutting area ⁇ thereby efficiently and completely to carry away severed gel particles.
  • the fractionation of gel with the apparatus is carried out in a novel sequence of steps assuring minimal zone distortion.
  • the first step is to advance the piston assembly through 16 through a predetermined increment of distance, which may for example be one millimeter. This causes the gel column to be advanced into engagement with the first cutting means, i.e. the screen 92.
  • the screen 92 severs the advancing gel in an axial direction to produce gel subdivisions or worm-like extensions of the gel column.
  • a pause of several seconds duration is provided in order to allow the gel fully to ooze through the screen.
  • the unit 98 may be continuously rotating in order that the wire 108 severs the subdivided gel extensions. Alternatively, if desired, the unit 98 may be operated only brielly near the end of the pause.
  • a dlushing operation is carried out.
  • several distinct flushing operations are provided by providing a pulse of iluid through the passage 126, pausing briefly, and applying a subsequent pulse of fluid. It has been found that three or four distinct flushing operations during rotation of the unit 98 produce very complete removal of gel particles from the fractionating apparatus 22. The particles and the liquid drop through the passage 106 and out of the outlet 24 and into the container or vial. Prior to repeating the process a next container is located beneath the vial.
  • a gel fractionator comprising a cylinder for holding a column of gel having a cross-sectional area, a piston means slidable within the cylinder for moving the column axially in the cylinder, first cutting means positioned in the gel path and engaged by the advancing column for subdividing the gel into a plurality of axially oriented sections having substantially the same combined total cross-sectional area as the cross-sectional area of said column of gel, and second cutting means movable across the gel path for severing end portions from said axially oriented sections.
  • said first cutting means comprising a wire mesh screen arranged in a plane intersecting the gel path.
  • said first cutting means comprising a wire mesh screen arranged generally parallel to said plane of movement.
  • Gel fractionating apparatus comprising a cylinder for holding a gel column; a screen disposed at one end of said cylinder, piston means for forcing the column through said screen thereby to subdivide the column, cutting means movable in a plane substantially parallel with said screen for severing the gel column divisions, means lfor washing the severed subdivisions from the screen, and means for collecting the severed subdivisions.
  • said collecting means comprising a funnel disposed beneath said screen.
  • said cutting means comprising a wire extending across the mouth of said funnel, and means for rotating said funnel.
  • said Washing means comprising an opening through the wall of said funnel directed at said screen.
  • a gel fractionator comprising a cylinder for holding a column of gel, a piston means slidable within the cylinder for moving the column axially in the cylinder, first cutting means positioned in the gel path and engaged by the advancing column for subdividing the gel into a plurality of axially oriented sections, and second cutting means movable across the gel path for severing end portions from said axially oriented sections, said piston means nicluding valve means for releasing air from between said piston means and the column, and 'valve operating means disposed externally of said cylinder.
  • a piston assembly for forcing a column of gel material through a cylinder comprising an inner member including an enlarged head, an outer sleeve surrounding said inner member, said head and sleeve having diameters smaller than the inside cylinder diameter, a resilient gasket sandwiched between said head and the end of said sleeve, resilient means forcing said head and said sleeve toward one another and against said gasket and forcing said gasket into se'aling relation against said cylinder wall, and said inner member being movable against the force of said resilient means to free said gasket and permit the release of air past said head.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
US24640A 1970-04-01 1970-04-01 Gel fractionator Expired - Lifetime US3664593A (en)

Applications Claiming Priority (1)

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US2464070A 1970-04-01 1970-04-01

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FR (1) FR2092400A5 (en:Method)
GB (1) GB1349482A (en:Method)

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CN113390807B (zh) * 2021-06-16 2022-12-02 桂林理工大学 一种模拟低碳化学品燃烧气体光谱测试系统

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FR2092400A5 (en:Method) 1972-01-21
GB1349482A (en) 1974-04-03

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