|Publication number||US3925207 A|
|Publication date||Dec 9, 1975|
|Filing date||Apr 2, 1975|
|Priority date||Apr 17, 1974|
|Also published as||DE2418509A1, DE2418509B2, DE2418509C3|
|Publication number||US 3925207 A, US 3925207A, US-A-3925207, US3925207 A, US3925207A|
|Inventors||Scriba Peter C|
|Original Assignee||Sartorius Membranfilter Gmbh|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (4), Referenced by (25), Classifications (9)|
|External Links: USPTO, USPTO Assignment, Espacenet|
United States Patent Scriba Dec. 9, 1975  SEMI-AUTOMATIC CHROMATOGRAPHIC 3,504,799 4/l970 Ogle 210/198 C SEPARATIO APPARATUS 3,649,203 3/l972 Schneider 2l0/l98 C 3,70l,609 l0/l972 Bailey 210/198 C Peter C. Scriba, Munich, Germany Sartorius-Membranfilter GmbH, Gottingen, Germany Filed: Apr. 2, 1975 Appl. No.: 564,465
Foreign Application Priority Data Apr. l7, I974 Germany 2418509 US. Cl. 210/138; 2lO/l98 C Int. Cl. B01D 15/08 Field of Search 2l0/l38, 198 C; 55/67,
References Cited UNITED STATES PATENTS ll/l964 Perry 55/l97 Primary ExaminerJohn Adee Attorney, Agent, or Firm-Browdy and Neimark ABS I'RACT The invention relates to an apparatus for the simultaneous semi-automatic column chromatographic separation of n sample solutions which are each separated into at least two fractions in the column with the aid of wash and/or buffer solutions. The apparatus comprises a proportionating pump with n channels, which are each connected upstream to a suction needle and downstream to a column of a group of n chromato graphic columns. A fraction collector comprising n collection flasks is arranged beneath the group of columns.
12 Claims, 4 Drawing Figures US. Patent Dec. 9, 1975 Sheet 1 of 3 3,925,207
U.S. Patent Dec. 9, 1975 Sheet 2 of3 3,925,207
U.S. Patent Dec. 9, 1975 Sheet 3 of3 3,925,207
SEMI-AUTOMATIC CHROMATOGRAPI-IIC SEPARATION APPARATUS The invention relates to an apparatus for the simultaneous semi-automatic column chromatographic separation of :1 sample solutions which are each separated into at least two fractions in the column with the aid of wash and/or buffer solutions. The apparatus comprises a proportionating pump with n channels, which are each connected upstream to a suction needle and downstream to a column of a group of n chromatographic columns. A fraction collector comprising n collection flasks is arranged beneath the group of columns.
It is an object of the invention to provide an apparatus of the above type, which can be operated quickly and simply with a minimum demand on personnel, also with a large number of sample solutions to be examined.
As solution to this object, an apparatus of the above type is provided by a carrier arm carrying the n suction needles, which is displaceable relative to a surface for supporting supply flasks for the solutions, not only in the axial direction of the suction needles but also at right angles thereto into predetermined positions, a carrier plate on which collection flasks can be placed and which is displaceable under the column group at right angles to the axis of the columns into predetermined working positions for the collection flasks, and a time control arrangement with adjustable time elements which co-ordinates the displacements of the carrier arm, the pump and the carrier plate.
A significant advantage of the invention consists in that the apparatus, once the sample solutions as well as the wash and buffer solutions have been prepared and arranged in their provided positions on the carrier surfaces and the apparatus set in action, the column chromatographic separation proceeds automatically without the work of personnel being required.
A further advantage of the invention consists in that a large number of sample solutions, namely 20 or 30 sample solutions and more, can be chromatographically separated at the same time.
Furthermore an advantage of the invention consists in that the same person who set the apparatus into operation can already examine the first fractions obtained by the process while the remaining fractions are still being produced on their own by the apparatus. This is particularly meaningful in separation by column chromatography of sample solutions in which the measurable radioactivity of individual fractions is important and the half-life period of the active elements is short. In this case the measurement of the radioactivity can be begun with the first fraction as soon as this has been fully produced and production of the next has been commenced.
The carrier arm is preferably slideable along its length, whilst the carrier arm and/or the carrier surface is adjustable in height.
In a particularly preferred embodiment, however, the carrier arm is rotatable and the turning axis of the carrier arm is adjustable in height whilst the drive for the carrier arm is advantageously a motor which can be arrested in predetermined positions by brakes. In accordance with an improvement of the invention, this motor is a step-switch motor whilst in another preferred improvement of the invention the motor is an 2 asynchromatic motor and the brake is a DC. current brake.
Further, the drive for the carrier ann advantageously comprises a contact which is displaced with displacement of the carrier ann, which is alloted to adjustable stationary contacts by means of which the predetermined drive positions of the carrier arm can be set. In this construction, it is preferred that the movable contact is arranged on a ring, whilst the stationary contacts are arranged outwardly of the ring about its circumference. Advantageously, a rest is provided for the carrier arm for each position into which it may be driven.
In this embodiment, the sample flasks with the different solutions can be arranged in predetennined positions on the carrier surface. In operation the carrier arm is automatically elevated by at least the length of the suction needles and is turned into the necessary position in which it is again lowered and the suction needles carried on the arm dip into the sample flask standing in that position. With the next step the arm is again elevated, turned into the next predetermined position and lowered in that position. Advantageously the suction needles are adjustable in height.
In an advantageous development, the carrier arm has openings for receiving the suction needles, whereby each suction needle is removable from its particular opening and is advantageously flexibly located.
In accordance with a feature, supply containers for the wash and buffer solutions are arranged relative to the carrier surface for the sample flasks so that each flask is connectible with the particular sample flask in accordance with the syphon principle.
The proportionating pump is preferably a tube pump which advantageously comprises at least 25 channels. In this manner it is possible to process just as high a number of different samples at the same time.
Preferably each collector device carrying the collection flasks comprises at least one member which couples with a complementary member on the carrier plate when placing the collector device and which fixes the situation of the collector device for this particular predetermined position on the carrier plate. Additionally each collector device advantageously comprises one carrier tube at each corner which are, apart from one, all blocked to provide a carrier surface, whilst the carrier plate in the predetermined positions for the collector device which is provided with insert openings for its collector tubes, is provided with a protruding pin to fit into the insert opening formed by the open bore.
In this embodiment each collector device can only be arranged in a very particular manner, namely in such a manner that the protruding pin is inserted into the op en bore. Hereby, it is avoided in a simple manner that a collection flask is erroneously allocated to the incorrect chromatographic column.
An advantageous development is provided by having the carrier plate quadratic in form and mounted as a turntable, whilst each collector device for the collection flasks may be placed in one quadrant of the carrier plate.
In this embodiment an optimal employment'of space is achieved.
Advantageously a disc is connected to the turning axis of the turntable, which provides four rest positions displaced at while this disc is provided with at least one pivotally arranged rest element which is biased by a spring with a rest nose against the circumference of the disc. Advantageously, however, two opposing pivotable rest elements are provided in which the rest noses are biased against the periphery of the disc by a common spring. Preferably a conventional spring arrangement is provided; however the rest elements with their rest noses could be lifted out of the rest positions of the disc by electromagnets.
In this embodiment it is possible in a simple manner to arrest the carrier plate exactly at the end of a turn of 90 in one position in which the collector device with each of the collection flasks arranged in the collector device finds itself exactly beneath the allocated chromatographic column.
Advantageously, the chromatographic columns are arranged in a common block, which is preferably a block which may be kept at the same temperature by means of a thermostat. In this construction according to one feature, a closure valve is arranged on the bottom of the block, which is adjustable between an open position in which the outlet openings of the columns are open and a closed position in which the outlet openings are closed. Preferably the closure valve is a plate which is provided with a number of silicone seals corresponding to the number of the chromatographic columns.
The proportionating pump is preferably reversible in its direction of rotation. In the automatic operation of the apparatus, the proportionating pump can then at the beginning of the position changes of the carrier plate for the collector devices be reversed for a short time to suck in the drops suspended at the openings of the chromatographic columns and thus prevent a possible mixing of the individual fractions. At the same time, the timing elements of the time control arrangement can be switched off during this position change.
Preferably, the proportionating pump, the fraction collector and the carrier arm form a common housing by which the carrier surfaces are presented and within which the drive for the carrier arm and the carrier plate is located. A contemplated advantageous feature is that the housing forms a pedestal in its central portion for carrying the proportionating pump, to one side of which pedestal is a step forming the carrier surface for the sample flasks and on the opposite side of the pedestal a lower set plateau for the fraction collector, a closure plate extending from the pedestal of the housing on the side opposite to the carrier surface being provided.
This embodiment is compact and can in relation to its weight of about 60 kg, without pump and without control portion, be relatively easily transported and rearranged.
The timing elements of the time control arrangement are advantageously clocks which can be set between the time zero and a maximum time. In this construction it is preferred that the chromatographic system and the time control arrangement are contained in separate housings which are electrically connected to one another by a cable.
This construction of the time control arrangement makes a very broad pre-selection possible for the timing of individual time steps of an operation, whilst it is at the same time possible after the selection of the particular time step eliminations, to eliminate such by setting the provided clock at zero.
Furthermore, an apparatus can be advantageously provided in which a substitute block with chromatographic columns can be connected up.
The apparatus in accordance with the invention is advantageously employed in the hormone analysis of medical solutions and preferably in the analysis of the thyroid gland hormone Tri-iodide Thyronin (T and Thyroxin (T The invention is described by way of example below with reference to the drawings; in these there is schematically shown:
FIGS. 1 and 2 a side view and plan view respectively of an apparatus for column chromatographic separation of sample solutions, which serve to explain the process,
FIG. 3 a side view of a practical embodiment of an apparatus in accordance with the invention for semiautomatic column chromatographic separation, and
FIG. 4 a side view of the carrier arm, in which this is in section in the area of a suction needle to expose the spring mounting of the suction needle.
The principal process of simultaneous column chromatographic separation of sample solutions is now described in conjunction with a Tri-iodide Thyronin (T in vitro test of the thyriod gland function diagnosis with reference to the FIGS. 1 and 2.
In accordance with FIGS. 1 and 2, a supply flask l for a buffer solution and an incubation flask 8 (FIG. 2) with inserted test tubes 2 for taking up of the sample solutions is arranged upstream from a roller pump 10. A suction needle 11 is shown dipped into the test tube 2 shown in FIG. 1, which is arranged on the end of a tube 9 of a group of tubes which is upstream of the roller pump 10.
A thermostatically controllable block 5, with inserted chromatographic columns 3, is arranged downstream from the roller pump 10. The columns 3 are arranged vertically in the block 5 although for clarity they are shown in the plane of the drawing in FIG. 2. A closure lid 4 is arranged at the top end of the block 5 which extends over the total breadth and length of the block. Each column 3 is connected to the roller pump 10 through the closure lid 4 by the downstream end of a tube 9.
Beneath the column 3 containing block 5, a drain 7 and groups of fraction tubes 6 is arranged which reach over the total breadth of the block 5, whereby the three test tubes 6 arranged behind each other from left to right are allocated to one column 3.
The columns 3 are Sephadex columns which contain Dextran gel.
In carrying out a T test, samples and an elution solution is sucked from the supply flask l and the tubes 2 one after the other and pumped in a closed system over as many Sephadex columns 3 as there are test tubes 2 filled with sample solutions. In the T test, for example, 0.4 ml of an incubation mixture consisting of a serum sample, radio active marked solution T and Barbital buffer is sucked up and brought onto the Sephadex columns arranged in the thermostatically controlled block 5 for gel filtration. Elution is then effected for 10 minutes with Barbital buffer. After the separation of the incubation mixture in the Sephadex columns into three fractions, the T "J bonded to the Dextran gel. is eluted by washing with bovine and human serum. The eluate in the three fractions, i.e. collected in the three fraction test tubes 6 arranged behind each other in the FIGS. 1 and 2 can then be directly evaluated in a gramma-ray measurement location.
The drain 7 is provided for collection of the buffer solution which is then pumped through the Sephadex columns, which is disposed of as rinse solution.
An apparatus in accordance with the invention is now described in more detail with reference to FIG. 3.
In accordance with FIG. 3, a housing 46 is provided which forms an elevated platform 48 in its middle portion, on which a roller pump 38 is arranged.
On the upstream side of the pump 38, i.e. in the left hand portion of FIG. 3, the housing 46 forms a step whose surface provides carrier surface 26 on which the supply flasks 28 for the samples, wash and buffer solutions can be placed.
A carrier arm 24 is connected to the housing 46 on the upstream side of the roller pump 38 and has a turning axis 40 which carries the carrier arm 24 at a predetermined distance above the carrier surface 26. Suction needles 22 are inserted in the carrier arm 24 which, in the representation of FIG. 3 are dipped into the supply flasks 28, whereby the suction needles, which are advantageously spring mounted, are displaced upwardly l to 2 millimeters relative to the carrier arm when pressed onto the bottom of the supply flasks 28. It is thus achieved that the sample solution is completely sucked off.
The carrier arm 24 with its turning axle 40 is adjustable in height relative to the carrier surface 26 and is tumable about the turning axle 40 into predetermined angular positions. The setting of the height is obtained with a lifter, which is at least as long as the suction needles 22 so that their bottom ends in the top position of the lifter of the carrier arm are out of the supply flasks 28 or out of such a flask in which it was located. The lifter is slightly larger than the height of supply flasks 28. The motors required for the height adjustment and turning of the carrier arm 24 are arranged in the housing 46 beneath the carrier surface 26.
The predetermined angular positions of the carrier arm 24 are clearly marked on the carrier surface 26 of the housing 46 so that the supply flasks 28 and the tubes with the sample solution containers can be placed exactly into their provided positions in only one direction on'the carrier surface 26, whereby the supply flask for the sample solution can only be arranged in one predetermined position.
Each suction needle 22 is connected at its top end to the upstream end of a tube 42 of the roller pump 38.
On the downstream side of the roller pump 38 there is arranged a thermostatic controllable block 36 containing chromatographic columns and a fraction collector 44. The block 36 stands on a carrier plate 50 which extends from the pedestal 48 of the housing 46 on the side opposite to the carrier surface 26. The top end of each column is connected to the downstream end of a tube 42 of the roller pump 38. The fraction collector 44 is so arranged beneath the carrier plate 50 that there is still space between its upper end and the underside of the carrier plate 50.
The fraction collector 44 comprises a carrier plate 30 which is connected to a turning axle 54 which is located in the housing 46 beneath the plateau 52.
Furthermore, the fraction collector 44 comprises carrier devices 32 into which the collector flasks 34 for the fractions may be inserted. Carrier devices for two types of collection flasks are thus contemplated.
The carrier plate 30 of the fraction collector 44 is made in the form of a turntable tumable on its turning axle 54 by means of a motor arranged in the housing 46.
The carrier plate 30 is formed approximately quadratically in the same way as the carrier device 32, whereby one carrier device 32 can always be placed on one quadrant of the carrier plate 30.
In operation, the carrier plate 30 may be arrested in four displaced positions at 90 relative to one another, whereby a carrier device 32 is arranged exactly beneath the thermostatically controllable block 36 and a collection flask 34 in this carrier device 32 is beneath each lower end of a column in each of these four positions.
The motors for setting the height of the axle 40 of the carrier arm 24, for the turning of the carrier arm 24, for the drive of the roller pump 38 and for the turning of the fraction collector 44 are co-ordinatively controlled by a common time controlled arrangement. The time control arrangement has adjustable time elements to each of which a time can be allocated. Both the portion of the apparatus which is arranged upstream of the pump and that portion which is arranged downstream is allocated to its own group of timing elements which proceed one after the other, whereby the time elements allocated to the upstream group and the time elements allocated to the downstream group can function independently of one another. The time elements of one group can be connected one after the other so that the next following time element is set into operation only after the time has passed which was set for a time element. It is thus possible to pre-select at will the time for the individual steps in carrying out the semi-automatic column chromatographic separation.
A plate 55 is arranged at the bottom of the block 36 as closure valve, which has as many silicone seals as chromatographic columns contained in the block 36. The drive of the plate is effected over a member which is mounted on a rotatable eccentric. The eccentric is secured to an axis which extends from the housing 46 and is provided with a control knob. By turning the knob the plate 55 is displaced and the chromatographic columns are thus closed or opened.
In accordance with FIG. 4 the suction needles 22 in the carrier arm 24 are spring supported. Each suction needle 22 is inserted for this purpose in a sheath 56. The sheath 56 forms a broadened annular flange 58 which is slidably displaceable in the bore 60 provided in the carrier arm for the suction needle 22. Further, a compression spring 62 is arranged in the bore 60 which on the one hand presses against the annular flange 58 of the sheath 56 and on the other hand against the annular shoulder 64 provided in the bore 60. In accordance with FIG. 4, the annular shoulder 64 is situated at the top end of the bore 60 in the upper end position of the carrier arm 24, whilst in the rest position of the suction needle 22, the annular flange 58 is situated at the bottom end of the bore 60 with its bottom surface about level with the bottom side of the carrier arm 24. Some sort of suitable security can be provided which prevents that the sheath 56 of the suction needle 22 falls downwardly out of the carrier arm 24. This can for example be achieved by a plate with openings whose diameter is smaller than the outer diameter of the annular flanges 58 of the sheath 56.
The compression springs 62 arranged in the bores 60 act on the sheaths 56 and thus the suction needles 22 are in their rest position. When the carrier arm 24 is lowered into a flask in operation and the bottom ends of the suction needles 22 reach the bottom of the flasks, the suction needles 22 are displaced upwardly with further lowering of the carrier arm against the action of the compression spring 62. The carrier arm 24 is in operation lowered so far that each suction needle is displaced l to 2 millimeters against the action of its allocated compression spring 62. It is thus ensured that the sample solution in the flask is completely sucked out.
I. An apparatus for the simultaneous semi-automatic chromatographic separation of n sample solutions into a least two fractions, which comprises n chromatographic columns, n flexible tubes each connected at one end to one inlet of the n chromatographic columns and connected at the other end to a hollow suction needle, a roller pump arranged to act on the n flexible tubes and thus pump sample solutions into which the suction needles may be dipped to the chromatographic columns, a collector device comprising n fraction collection flasks for each fraction, a carrier arm in which the n suction needles are mounted in parallel spaced relationship with their free ends extending from the carrier arm, the carrier arm being displaceable both in the direction of the length-wise axes of the needles as well as at right angles thereto into predetermined positions relative to a carrier surface for sample solution flasks, a carrier plate situated beneath the outlets of the chromatographic columns and onto which collector devices for carrying fraction collection flasks may be placed in predetermined positions, the carrier plate being rotatable in a plane which is at right angles to the length wise axes of the chromatographic columns, and a time control arrangement having adjustable time elements to co-ordinate and control displacement of the carrier arm, the carrier plate and pumping of the roller pump.
2. An apparatus according to claim 1, in which the carrier arm is an elongated member, and in which said carrier arm is additionally displaceable along the height of its axis of rotation.
3. An apparatus according to claim 1, in which the carrier arm is provided with bores in which the suction needles are removably mounted.
4. An apparatus according to claim 3, in which the suction needles are displaceable along their axes relative to the carrier arm, against a spring action acting between the carrier arm and the suction needles.
5. An apparatus according to claim 1, in which supply containers for wash and buffer solutions are provided, and in which said supply containers are arranged relative to the sample solution flasks so that the supply containers and sample solution flasks may be connected using the Syphon-principle.
6. An apparatus according to claim I, in which each collector device for carrying the fraction collection flasks is provided with at least one element which fits with a complimentary element provided on the carrier plate and thus fixes the collector device in its predetermined position on the carrier plate.
7. An apparatus according to claim 1, in which the carrier plate is mounted to function as a turntable, and in which the carrier plate is quadratic in form, each quadrant being adapted to receive a collector device.
8. An apparatus according to claim 1, in which the roller pump is reversible in direction.
9. An apparatus according to claim I, in which there is provided a common housing for housing drive means for driving the carrier arm and carrier plate, and in which a surface of said housing forms the carrier surface for carrying sample solution flasks.
10. An apparatus according to claim 9, in which the housing is shaped to form a central pedestral for carrying the roller pump, the carrier surface for carrying sample solution flasks then being to one side of the platform and a deeper set plateau being provided to the other side of the platform over which the carrier plate is mounted, and in which a closure plate is provided for closing off access to fraction collector flasks, the closure plate extending sidewardly from the pedestal on the side of the plateau.
11. An apparatus in accordance with claim 1 further including a column block in which said n chromatographic columns are disposed.
12. An apparatus according to claim 11, in which the column block for receiving the chromatographic columns is provided with temperature control means for maintaining the chromatographic columns at a constant temperature.
at s a
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|U.S. Classification||210/138, 210/198.2|
|International Classification||G01N30/24, G01N30/46, G01N30/00|
|Cooperative Classification||G01N30/24, G01N30/466|
|European Classification||G01N30/24, G01N30/46E|