|Publication number||US3862029 A|
|Publication date||Jan 21, 1975|
|Filing date||Oct 1, 1973|
|Priority date||Oct 1, 1973|
|Publication number||US 3862029 A, US 3862029A, US-A-3862029, US3862029 A, US3862029A|
|Inventors||Joyce John E|
|Original Assignee||Joyce John E|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (8), Referenced by (4), Classifications (11)|
|External Links: USPTO, USPTO Assignment, Espacenet|
[ Jan. 21, 1975 Primary Examiner-Frank W. Lutter Assistant Examiner-Ralph J. Hill  ABSTRACT A density gradient fractionator sometimes referred to as a pycnograph has a chamber to receive a volume of the gradient, light density end uppermost and a volume of a sample as a surface layer so that its particulate contents that will settle therefrom in response to the earth's gravity can sediment into the gradient. The fractionator has piston means reciprocable in the chamber and having a discharge tube connected thereto with its lower end extending through and slidably sealed in the bottom of the chamber and with its upper end open to function as an overflow for the chamber so that as the piston means is advanced, the gradient and its sedimented zones are forced into the DENSITY GRADIENT FRACTIONATOR Inventor: John E. Joyce, 22 Nelson Rd., South Weymouth, Mass. 02190 Filed: Oct. 1, 1973 Appl. No.: 402,589
US. 209/172, 209/173, 209/208, 209/495, 73/614 lnt. B03b 3/46 Field of Search 209/172, 208, 209, 463, 209/465, 483, 1725, 173, 490, 494, 495; 73/32, 61 R, 6l.4
References Cited UNITED STATES PATENTS United States Patent Joyce 27 Claims, 6 Drawing Figures discharge tube, light density end first. The piston means includes a piston with an upper chamber for the sample provided by a recess in its upper surface. the piston having an axial port and the piston means including a valve in control of the port and connected to the piston for movement relative thereto for a short distance to provide opened and closed positions, movement of the valve thereafter resulting in movement of the piston in a corresponding direction. Means are provided to stir the gradient in a transverse plane in any zone in which particles may be trapped against settling or rising to the level in the gradient equal to their density.
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929923 9 s //070072 SO 992 22 2 t 00 .W .W t a 22 m m N m m T m n m A m S m m C m S we u N u. H n m m P m I n6 7 m P mT 9 6 mmn n A E m m m R m m C a 6 m3. 0 6 w Maflmh e S c B M] CHa CEWT M U .l haur N r Zl MLCDDGKJ F 2 0 7 T E h 28 09993 A mnr 255 67 P7 a 99999999 9 T y ll l l l l ll 1 N/O D.0 500 95927 G4 on l 1 l0 6 m VM 93577383 00 e 40 42564 O4 t. ,l FA m 745 7237 3 Cl 27628084 5 h ,3 9 T ll22233 2 u C PATENTED JAN 21 ms SHEET 10F 3 FIG! "illllllilllilllllllllllllllllllll Illlllllllllllllllllllllllllll SHEET 2 OF 3 PATENTED JAN 2 1 I975 PATENTEI] JAN 2 1 I975 SHEET 3 OF 3 DENSITY GRADIENT FRACTIONATOR BACKGROUND OF THE INVENTION In various research programs, it is required that the different particles contained in samples be separated and counted. Where the size of such matter varies substantially from a minimum theoretical size in the neighborhood of twenty microns to much larger and clearly visible particles it is difficult to meet that requirement accurately and without requiring an inordinate amount of a technicians time.
By way of one example, a program exists that requires the monitoring of many areas in which marine life breeds in order to predict population trends at a later time in certain harvest areas. In each such breeding area, samples of uniform volume are regularly taken that are presumed to contain representative algae, plankton, fish eggs, and larvae. The value of the program obviously requires that the number of each type of such marine life in each sample be accurately determined. At the present time, such determination is effected by first manually sorting the different types of marine life and then counting the sorted populations. This procedure is unsatisfactory both because of the time required of the technicians and because it usually results in a material and unpredictable error.
THE PRESENT INVENTION The objective of the present invention is to provide means to effect the separation of diverse particles contained in samples when the size range of the particles is substantial, with the smallest particles being at least collectively visible, the separation being effected by the sedimentation of the particles in a suitable liquid density gradient in response to the earths gravity into visibly distinguishable zones.
It is of course well known that particles can be sepa-' rated in accordance with their densities by introducing a sample containing them into a centrifugal field in which there is a suitable liquid density gradient. This procedure is unsatisfactory, however, when the size of the particulate matter is as varied as it is, for example, with the types of marine life to be found in the monitored areas.
In accordance with the invention, the above generally stated objective is attained with a density gradient fractionator having a vertical chamber for the gradient with its light density end on top and for a predetermined volume of a sample as a layer on the surface of the gradient. Piston means are reciprocable in the chamber after sedimentation has occurred and a discharge tube is connected thereto with its upper end open and providing an overflow in the upper end of the chamber and its lower end extending through and slidably seated in a port in the bottom of the chamber.
With such a fractionator, after sedimentation of the particles in response to the earths gravity, the piston is advanced into the chamber and as it advances, the gradient with its sedimented zones is forced into said discharge tube, the lighter density end first.
Another objective of the invention is to provide the fractionator with an upper chamber for the sample. The piston means includes a piston having an axial port effecting communication between the sample and gradient chambers and a valve which, when opened, permits the sample flow onto the gradient and, when closed, permits the piston to function as such, in forcing the gradient and its sedimented zones into the discharge tube.
Another objective of the invention is to utilize the valve as the means by which the piston is reciprocated, an objective attained by connecting the valve thereto so that after its short movement between its open and closed positions the piston is moved thereby in corresponding directions. The means by which piston reciprocation is to be effected is connected to the valve and such means may be of various types depending on the degree of accuracy that is wanted in separating the sedimented zones.
Another objective of the invention is to provide a density gradient fractionator having a sample chamber placed in communication with the gradient chamber by valve means with the bottom of the sample chamber in the plane of the surface of the gradient when freely infused in the gradient chamber.
Yet another objective of the invention is to minimize disturbance of the sedimented zones while being successively forced into the delivery tube, an objective attained by forming the piston with its undersurface upwardly and inwardly inclined towards the upper end of the delivery tube. The attainment of this objective is also assisted by providing means enabling the advance of the piston to be accurately controlled with its travel at a sufficiently slow rate to enable even the narrowest of desired zones to be precisely removed.
A further objective of the invention is to provide for the delivery of the gradient into its chamber, light density first, an objective attained by providing an inlet in its bottom that includes an annular upwardly opening portion with the bottom upwardly inclined at the outlet thereof.
Due to the large amount of plankton in a typical marine life sample, a barrier results in which heavier organisms are or may be caught mechanically in the intermediate plankton zone and thus prevented from settling to the level equal to their density to which they would normally settle. Lighter particles may also be entrapped in the plankton zone. Particles caught in the wrong zone may be freed by stirring the gradient and an important objective of the invention is to provide means to sweep through iso-dense planes in the gradient and cause convective disturbances which are effective only in a horizontal plane as the buoyant forces restrict fluid from mixing vertically but freeing the entrapped particles so that they may rise or settle to the level appropriate for their density.
BRIEF DESCRIPTION OF THE DRAWINGS In the drawings, preferred embodiments of a density gradient fractionator in accordance with the invention are illustrated.
FIG. I is a side elevation of the fractionator;
FIG. 2 is a vertical section on an increase in scale, taken centrally of the chambers of the fractionator and through the drive to show the gearing of FIG. 1;
FIG. 3 is a section taken approximately along the indicated line 33 of FIG. 2;
FIG. 4 is a section taken approximately along the indicated line 4-4 of FIG. 2;
FIG. 5 is a fragmentary vertical section showing the attachment of a fraction-receiving container to the lower end of the delivery tube; and
FIG. 6 is a perspective view of the fractionator with different zones illustrated and with the means for stirring the gradient in selected iso-dense planes shown in an operative position.
THE PREFERRED EMBODIMENT OF THE INVENTION The density gradient fractionator illustrated by the drawings consists of a transparent, tubular gradient chamber 10 closed at its bottom end by a base assembly generally indicated at 11 and at its upper end by a piston assembly, generally indicated at 12 and having an axial delivery tube 13 extending downwardly through the chamber 10.
The base assembly 11 consists of an annular base member 14 having an internal annular shoulder 15 in its upper surface into which the lower end of the chamber 10 is seated and sealed as at 16. The upper surface of the base member 14 is upwardly and outwardly inclined. The lower surface of the base member 14 has an annular shoulder 17 seated on and anchored to the inturned flange 18 of a holder 19 into which the base assembly 11 fits and which is bolted to a suitable stand 20. The upper part of the stand 20 has a pair of transversely spaced mounts 21 between which the chamber 10 fits and to which the ends of holding straps 22 that extend around the upper part of the chamber 10 are secured.
The base member 14 has an axial bore 23 with its lower end counterbored to provide a seat 24 to which the flange 25 ofa closure 26 is detachably secured. The closure 26 has an axial bore 27 through which the de livery tube 13 slidably extends and in which suitable seals 28 therefor are incorporated. The closure 26 is dimensioned to be a sufficiently free fit so that it and the bore 23 define an upwardly opening annular passageway with a seal 29 below a port 30 extending radially through the base member 14 and provided with a fitting 31 to enable flexible tubing to be detachably attached thereto to enable the gradient to be introduced into the chamber 10, light end first, until the chamber 10 gradient is fully infused therein. Should the volume of the gradient be insufficient, cushioning liquid is added to the heavy density end thereof.
Turning now to the piston assembly 12, it will be noted that it includes a piston 32 having a recess 33 serving as a sample chamber in its upper surface with its lower part downwardly and inwardly inclined and an upwardly and inwardly inclined recess 34 in its lower surface with the recesses interconnected by an axial bore 35. The upper end of the bore 35 is counterbored to provide a seat 36 for a valve member 37 having a downwardly opening axial socket 38 in which the upper end of the delivery tube 13 is connected by a spacer 39, see FIG. 3, with its upper extremity spaced from the upper end of the socket 38 and substantially in the plane of the bottom part of the sample chamber 33 when the valve member 37 is seated. In practice the gradient is introduced, light density end first, into the chamber 10 with the valve member 37 seated until the light end of the gradient starts to flow through the tube 13.
A serrated upper end wall 40 is connected to the piston 32 by screws 41 extending through tubular spacers 42 with one screw 41 serving as a pivot for a.lock 43. The valve member 37 has its stem 44 connected to a rack 45 extending through a hole 66 in the end wall 40,
with the connection providing a shoulder 47 engageable by the lock 43 when swung inwardly to bring its curved end 43A into its position shown in FIG. 4.
The rack drive 48 is shown as consisting of a pinion 49 meshing with the teeth of the rack 45 and mounted on a shaft 50 rotatably supported within a housing 51 mounted on the stand 20. A hand wheel 52 is fast on a shaft 53 extending into and rotatably supported within the housing 51 at right angles to the shaft 50 and provided with a worm 54 meshing with a worm gear 55 fast on the shaft 50. In practice the gearing and the handle provide a reduction ratio of 48:] a reduction which enables the operator to accurately control the advance of the piston assembly 12. While the rack drive may be motor driven, the use of manually operated means is preferred.
The exposed lower end of the delivery tube 13 has, see FIG. 5, an annular groove 56 for a keeper 57 detachably attaching a disc 58 to the tube 13. The undersurface of the disc 58 has holders 59 spaced and arranged to slidably receive and support the flange 60 of a fraction-receiving container 61.
In the preferred use of the density gradient fractionator and with the piston 32 in its upper position and the valve member 37 held in its seated position by the lock 43, a suitable concentrationdensity gradient is pumped by conventional means, not shown, into the chamber 10 through its inlet port 30, light density end first. The gradient is fully infused when the light density end thereof starts to flow outwardly through the delivery tube 13. If the volume of the gradient is inadequate for that purpose, a cushioning liquid is added, as previously stated, to the heavy density end of the gradient. The inlet is then shut as by applying a clamp to the tubing from the gradient pump, not shown.
Since the valve member 37 of the piston assembly 12 was initially in its seated position, the sample can be introduced into its chamber 33 either before or after the infusion of the gradient chamber. In either case, with the gradient chamber 10 infused and a desired volume of the sample in the sample chamber 33, the valve member 37 is then raised by the handwheel drive in a smooth manner to its fully opened position thus enabling the sample to flow onto the upper surface of the gradient. It will be noted, as previously stated, that the upper end of the outflow tube 13 is, when the valve member 37 is fully seated, so positioned that the upper level of the gradient is substantially in the plane of the bottom of the sample chamber 33 thus preventing any material disturbance of the gradient when the valve member 37 is opened. With the valve member thus opened the piston assembly 12 is raised by the handwheel drive a sufficient distance in a smooth manner to cause the sample to flow downward through the axial bore 35 into the annular recess 34 and is thus now in contact with and exposed to the entire area of the gradient chamber.
The fractionator is provided with stirring means generally indicated at 62 best seen in FIG. 6. The stirrer 62 is desirably formed from a length of stainless steel wire in the approximate range of 0.06 to 0.09 inches in diameter to provide a shank 63 of substantial length, an angularly disposed handle 64, and a substantially parallel stirring end 65 that is curved with its radius closely approximating the inside diameter of the chamber 10. The piston 32 has a shank-receiving bore 66 close to its periphery and in vertical alignment with one of the notches 40A of the end wall 40 of the piston assembly 12.
The liquid column defined by the two interconnected liquid volumes (i.e. sample and fluid density gradient) is allowed to set for a sufficient predetermined interval, in practice, one hour, to ensure that sedimentation has occurred under the influence of the earths gravity. After such sedimentation, common species of organisms originally present in the sample are grouped in zones in the form of horizontal, visible layers with the positions of the common species being at the depth where their density equals that of the density gradient. It will be appreciated, accordingly, that all components of the sample whose density is encompassed by the density range of the gradient will be distributed in such zones throughout the gradient. It should be noted that, in practice, immediately after the sample has been released from its chamber, the chamber, the valve, and its seat should be brushed to ensure that no components of the sample remain on their surface.
When the sample is a typical marine life sample, the particles contained therein may include fish eggs or particles of the same density, larvae, and plankton of an intermediate density and usually in an amount that is relatively large.
In FIG. 6, the zones are shown in which organisms of the same density are grouped as visible layers with the fish eggs layer or zone indicated at 67, that of the plankton at 68, and that of the larvae at 69.
Because of the fact that the amount of plankton is usually relatively large, the layer 68 may and usually does form a barrier to the heavier particles or organisms whenever they become mechanically caught and thus prevented from reaching the level of the density gradient equal to their density to which they would ordinarily settle; Lighter particles may also be entrapped in the intermediate zone 68.
It is because of such entrapments that the stirring means 62 is provided. In use the stirring end 65 is moved to the appropriate level with the stirring end against or close to the wall of the chamber 10. The operator then turns the handle 64 first in one direction and then the other to cause the stirring end 65 to sweep through the gradient in the selected horizontal plane. If desired or necessary, other levels may be similarly swept to ensure the release of particles trapped at the wrong level.
While the entrapped particles may be thus freed to settle or float to the correct levels and thus ensure accuracy in the separation, such sweeping of horizontal zones causes convective disturbances that are substantially confined to the swept plane because buoyant forces restrict movement of the gradient vertically.
The fractionating of the gradient is then effected to enable each of the species of identical densities to be separately recovered, each specie to be recovered in a fraction-receiving container 61 detachably attached to the bottom end of the discharge tube 13. With the valve member 37 returned to its seated closed position, a fraction may be removed by carefully advancing the piston means 12 downwardly into the gradient chamber with the tube 13 being correspondingly advanced through the bottom of the gradient chamber 10. Liquid at the upper end thereof is forced into the upper end of said tube then to flow by gravity into the fractionreceiving container 61 attached thereto. As the different zones are visible, the operator can determine to what extend the piston means must be advanced to effect the discharge of each iso-dense zone or layer with the other zones or layers remaining at rest. As the delivery tube 13 quickly drains after each incremental advance of the piston means, the fractions are received substantially without one being contaminated by the others. Zones whose width correspond to less than 1 percent the inside diameter of the gradient chamber 10 can be thus received and such a zone represents about one-half of one percent of the gradient volume normally contained therein.
It will be appreciated that contributing to accuracy in operation is the relationship of the upper end of the delivery tube to the bottom of the sample chamber and the upwardly and inwardly inclined undersurface of the piston. It should also be noted that the piston need not be sealed to be liquid tight since the delivery tube provides adequate pressure relief.
In order that substantially all of the gradient may be removed through the discharge tube 13 it is preferred that the bottom of the chamber 10 have an insert seated therein. The insert 70 has an axial bore 71 through which the discharge tube 13 slidably extends, its upper surface upwardly and inwardly inclined to complement the piston surface 34, and its bottom surface formed with an inner downwardly and inwardly cavity 72 to fit on the upper end of the closure 26, and an outer upwardly inclined surface 73 with spacers 74 holding the insert 70 so that it and the surface of the annular base member 14 provide an annular, everwidening outlet end of the gradient inlet.
1. A density gradient fractionator comprising a vertical tubular chamber closed at its bottom and dimensioned to receive a predetermined volume of the gradient and a predetermined volume of a sample, with the light density end of the gradient on top, with sedimentation to occur therein in response to the earths gravity, piston means reciprocable in said chamber, and a discharge tube connected to said piston means and extending through and slidably sealed with respect to the bottom of the chamber with its upper end open providing an overflow in the upper end of the chamber whereby the advance of the piston means, after sedimentation, forces the gradient and sedimented zones into said discharge tube, the lighter density end first.
2. The density gradient fractionator of claim l and means to detachably attach a fraction-receiving container to the lower exposed end of the delivery tube.
3. The density gradient fractionator of claim 2 and a drive operable to advance the piston means into the chamber.
4. The density gradient fractionator of claim 3 in which the drive includes a gear train and a driving member, the gear train providing a substantial reduction in the travel of the piston means in relation to movement of the driving member.
5. The density gradient fractionator of claim 4 in which the driving member is a handwheel.
6. The density gradient fractionator of claim 1 in which the piston means includes a piston having an axial port and a valve operable to open and close that port and the fractionator also has a chamber for the sample above the gradient chamber and placed in communication therewith by said axial port when said valve is open, said piston being operable as such when said valve is closed.
7. The density gradient fractionator of claim 6 and means to reciprocate said piston means and including an actuating member connected to said valve and said piston means to enable said valve to be moved between port-opening and port-closing positions relative to said piston before initiating movement of said piston in corresponding directions in said gradient chamber.
8. The density gradient fractionator of claim 7 in which the discharge tube is connected to the valve.
9. The density gradient fractionator of claim 7 in which the axial port of the piston includes an annular seat against which the valve seats and thereby establishes a driving connection by which the piston is advanced into the gradient chamber.
10. The density gradient fractionator of claim 9 in which the valve has a downwardly opening socket into which the upper end of the discharging tube extends, and the discharge tube is connected to the valve to move therewith.
11. The density gradient fractionator of claim 10 in which the undersurface of the piston means is upwardly and inwardly tapered.
12. The density gradient fractionator of claim 11 in which the bottom of the gradient chamber is upwardly and inwardly tapered to complement the undersurface of the piston.
13. The density gradient fractionator of claim 10 in which the upper surface of the piston includes a portion that is the bottom of the upper chamber and tapers downwardly and inwardly to the axial piston port.
14. The density gradient fractionator of claim 13 in which the upper open end of the discharge tube and the lowermost part of the upper chamber are substantially in the same plane when the valve is seated.
15. The density gradient fractionator of claim 10 in which the upper surface of the piston has a recess that is the upper chamber and the bottom of which tapers downwardly and inwardly to the axial piston port.
16. The density gradient fractionator of claim 15 in which the piston means includes a cover plate connected to the upper surface of the piston in vertically spaced relation thereto and having a port through which the actuator extends, the actuator has a shoulder, and the piston means includes a lock engageable with the shoulder of the actuator when the actuator is positioned to seat the valve then to prevent the unseating thereof.
17. The density gradient fractionator of claim 16 in which the shoulder on the actuator provides a positive drive of the piston means away from the chamber with the valve unseated allowing the upper and lower recesses of the piston means to be in communication with each other during said movement with the lock consequentially disengaged.
18. The density gradient fractionator of claim 6 and lock means positionable when the valve is in its portclosing position to prevent its movement towards its port-opening position.
19. The density gradient fractionator of claim 1 in which the undersurface of the piston means tapers upwardly and inwardly towards the overflow end of the discharge tube.
20. The density gradient fractionator of claim 1 in which the gradient chamber has an inlet in its bottom for the introduction of the gradient, less dense end first.
21. The density gradient fractionator of claim 20 in which the inlet for the gradient includes an annular portion surrounding the discharge tube and opening upwardly into the chamber.
22. The density gradient fractionator of claim 21 in which the bottom of the chamber is upwardly and in wardly inclined and the undersurface of the piston is downwardly and outwardly inclined substantially as the complement thereof.
23. The density gradient fractionator of claim 1 in which the fractionator includes a sample chamber above the gradient chamber and valve means operable to place the sample chamber in communication with the gradient chamber, the bottom level of the sample chamber being substantially in the same plane as said overflow.
24. The density gradient fractionator of claim 1 and vertically adjustable stirring means rotatably supported within the chamber including a transversely extending stirring portion operable to sweep in a transverse plane, a selected zone of the gradient into which particles have settled that may include particles of at least one other density that belong in a different gradient level.
25. The density gradient fractionator of claim 24 in which the thickness of the stirring portion is in the approximate range of from 0.06 to 0.09 inches.
26. The density gradient fractionator of claim 24 in which the stirring means includes a shank at the lower end of which the stirring portion is located and which extends upwardly through the piston adjacent the periphery thereof at the upper end of which includes a manipulator handle.
27. The density gradient fractionator of claim 26 in which the stirring portion is arcuate with a radius closely approximating the inside diameter of the chamber.
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|U.S. Classification||209/172, 209/208, 209/173, 209/495|
|International Classification||G01N15/04, B03B5/28, B03B5/36|
|Cooperative Classification||B03B5/36, G01N15/04|
|European Classification||B03B5/36, G01N15/04|