U.S. patent number 3,925,207 [Application Number 05/564,465] was granted by the patent office on 1975-12-09 for semi-automatic chromatographic separation apparatus.
This patent grant is currently assigned to Sartorius-Membranfilter GmbH. Invention is credited to Peter C. Scriba.
United States Patent |
3,925,207 |
Scriba |
December 9, 1975 |
Semi-automatic chromatographic separation apparatus
Abstract
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 chromatographic columns. A fraction
collector comprising n collection flasks is arranged beneath the
group of columns.
Inventors: |
Scriba; Peter C. (Munich,
DT) |
Assignee: |
Sartorius-Membranfilter GmbH
(Gottingen, DT)
|
Family
ID: |
5913131 |
Appl.
No.: |
05/564,465 |
Filed: |
April 2, 1975 |
Foreign Application Priority Data
|
|
|
|
|
Apr 17, 1974 [DT] |
|
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2418509 |
|
Current U.S.
Class: |
210/138;
210/198.2 |
Current CPC
Class: |
G01N
30/466 (20130101); G01N 30/24 (20130101) |
Current International
Class: |
G01N
30/24 (20060101); G01N 30/46 (20060101); G01N
30/00 (20060101); B01D 015/08 () |
Field of
Search: |
;210/138,198C
;55/67,197,386 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Adee; John
Attorney, Agent or Firm: Browdy and Neimark
Claims
I claim:
1. 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 1, 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 1, 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.
Description
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 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 asynchromatic motor and the brake is a
D.C. current brake.
Further, the drive for the carrier arm advantageously comprises a
contact which is displaced with displacement of the carrier arm,
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 predetermined 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 open 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
90.degree. 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.degree. 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.sub.3) and Thyroxin (T.sub.4).
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 semi-automatic 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.sub.3) -- 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 1 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.sub.3 test, samples and an elution solution is
sucked from the supply flask 1 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.sub.3
test, for example, 0.4 ml of an incubation mixture consisting of a
serum sample, radio active marked solution T.sub.3 .sup.125 J 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.sub.3 .sup.125 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 20 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 1 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 turnable 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 turnable 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.degree. 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 1 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.
* * * * *