WO2003076054A1

WO2003076054A1 - Concentration methods and devices

Info

Publication number: WO2003076054A1
Application number: PCT/EP2003/002692
Authority: WO
Inventors: Owe Salven; Jill Simon
Original assignee: Amersham Biosciences Ab
Priority date: 2002-03-14
Filing date: 2003-03-14
Publication date: 2003-09-18
Also published as: AU2003219056A1; GB0205947D0

Abstract

A sample concentrating device comprises a hollow body (3) having a waste chamber (10) and a concentration chamber (20) communicating with said waste chamber (10) through a concentrating membrane (21), wherein said concentration chamber (20) has a pressurised gas inlet (23) connectable via valve (27) to a supply of pressurised gas (29), and said waste chamber (10) has a pressure relief opening (45) able to be opened to ambient pressure, wherein the concentration chamber (20) has a diluted sample inlet (31) connectable to an inlet valve (35) for inputting a suspension of a sample diluted in a liquid into said concentration chamber (20) and a concentrated sample outlet (39) connectable to an outlet valve (35) for outputting the contents of said concentration chamber (20). The valves (27, 35) are adapted to be operated by control means such as a computer (49).

Description

Concentration Methods and Devices

Field of the Invention

The present invention relates to concentrating devices and methods of the type mentioned in the preambles of the independent claims for performing concentration or dialysis of liquids.

Prior Art

Concentrators are devices used to concentrate liquid samples by removing unwanted liquid and small particles from the suspensions. An example of a concentrator is the Vivacell 100 ™ from Sartorius AG, Goettingen, Germany, similar to a device described in WO98/26859. This device has a concentrator body in which the suspension to be concentrated is placed. The concentrator body has a cut-off membrane in its floor which allows molecules below the cutoff size to exit the concentrator body. The concentrator body is attached on top of a waste body which collects the waste liquid that passes through the cut-off membrane. In order to speed up the passage of the waste through the membrane the concentrator body is provided with an airtight lid which has an inlet able to be connected to a supply of pressurised gas. This gas can be injected into the concentrator body, thereby causing a pressure differential to occur across the membrane. This pressure differential increases the flow rate of waste molecules through the cut-off membrane. The concentrator may also be spun in a centrifuge to increase the force pushing the waste through the membrane. Once the volume of the sample in the concentrator body has been reduced to the desired extent, the lid is removed from the concentrator body and an operator can then extract the concentrated sample, for example by pipetting or simply tipping the contents of the concentrator body into a suitable container. Such concentrators are designed to be filled and emptied manually and therefore carry the risk that operator error can lead to samples being spilt or lost. Additionally, it is time-consuming to manually fill and empty such concentrators.

Summary of the Invention

According to the present invention, at least some of the problems with the prior art are solved by means of a device having the features present in the characterising part of claim 1 and a method having the features mentioned in the characterising part of claim 5

Brief Description of the Figures Figure 1 shows a cross-section through a schematic lateral view of a first embodiment of a concentrating device in accordance with the present invention;

Figure 2 shows a cross-section along line 11-11 in figure 1 ;

Figure 3 shows a cross-section through a second embodiment of a concentrating device in accordance with the present invention;

Figure 4 shows a cross-section through a third embodiment of a concentrating device in accordance with the present invention containing a diluted sample; and,

Figure 5 shows a cross-section though the device of figure 4 after the sample shown in figure 4 has been concentrated.

Detailed Description of Embodiments Illustrating the Invention

Figure 1 shows a cross-section through a schematic lateral view of a first embodiment of a concentrating device 1 in accordance with the present invention. Figure 1 is not drawn to scale. Concentrating device 1 comprises a hollow body 3 with an upper end 5 joined to a lower end 7 by a wall 9. The space enclosed by the upper end 5, lower end 7 and wall 9 form a waste chamber 10. Preferably one or both of upper end 5 and lower end 7 are removable to allow access to the interior of body 3. Body 3 is preferably made of a fluid-tight, transparent material such as glass or plastic in order to prevent liquids leaking out of body 3 and to allow visual inspection of, and/or automatic detection of, the contents of body 3.

A concentrating unit 11 is positioned inside body 3. Concentrating unit 11 comprises a hollow concentrating body 13 with an upper end 15 joined to a lower end 17 by a wall 19. Concentrating body 13 is preferably made of a fluid-tight, transparent material such as glass or plastic in order to prevent undesirable leaking of fluids out of the concentrating body 13 and to allow visual inspection, and/or automatic detection, of the contents of the concenfrating body 13. The space enclosed by the upper end 15, lower end 17 and wall 19 form a concentration chamber 20. Part of the lower part of wall 19 and/or the lower end 17 is provided with one or more outlet ports 18 covered by a concenfrating cut-off membrane 21 with openings 22 adapted to the size of the molecules that it is intended to concentrate. Typically the openings 22 in the concenfrating membrane 21 are chosen so that the molecules of the liquid in which the sample to be concentrated is suspended are small enough to pass though the membrane but the sample molecules are too big to pass through the membrane openings. The upper end 15 of concenfrating body 13 is connectable, for example via a pipe 23 which passes through a hole 25 in upper end 5 of body 3, to a valve 27. Valve 27 is connectable to a supply of pressurised gas 29, e.g. air. The lower part of concenfrating body 13 is connectable via a combined inlet/outlet pipe 31 which passes through a hole 33 in lower end 7 of body 3 to a inlet means and outlet means such as a multi -position valve 35. Valve 35 can selectively connect inlet/outlet pipe 31 to an inlet pipe 37 or to an outlet pipe 39 or be closed. Inlet pipe 37 can be connected to a sample source (not shown), for example, the outlet of a liquid chromatography column or a reaction vessel or the like.

The lower end 7 of body 3 is provided with a waste opening 41 leading to a waste collection container 43 or waste disposal system such as a drain (not shown). The upper part of wall 9 is provided with an opening 45 though which the pressure inside body 3 can be equalised with ambient pressure. Opening 45 may be provided with a valve (not shown) to control the flow of air through it in order to prevent outside air contaminating the contents of body 3.

Preferably a liquid level detector 47 is provided at a level above the top edge of concenfrating membrane 21. Level detector 47 produces a warning signal when the level of the surface of liquid in the concenfrating body falls below the level detector and this signal can be used to indicate that the concentration has reached a certain level and, if necessary, to warn an operator that the surface of the membrane 21 is close to being exposed to the gas in the concenfrating body - which could cause samples on the exposed membrane to dry out.

Alternatively, in a preferred embodiment of the present invention, the level detector 47 may be connected to an automated control means such as computer 49 which is connectable to valve 27, 35 which are provided with actuating means e.g. electric motors. This permits the concenfrating operation to be controlled by the computer 49 when programmed appropriately. Additional level detectors 47', 47" may be provided at increasing heights from the bottom of the body 3 in order to permit the level of liquid in the body to be measured. This permits the degree of concentration of the sample to be measured. The uppermost level detector 47" can be used to detect when the concentration chamber is almost full and, by sending a signal to computer 49, it can initiate an automatic closure of the valve 35 in order to prevent the concentration chamber being overfilled.

A further level detector or liquid sensor 51 can be provided on inlet/outlet pipe 31 in order to detect the level of liquid in inlet outlet pipe 31. The signal from this detector 51 may be used to operate valve 35 to prevent gas from inside concenfrating body 13 entering outlet pipe 39. A method for concenfrating a sample using the concentration device in accordance with the present invention has the following steps, which preferably are controlled by control means 49: the sample, e.g. proteins suspended in a liquid, which is to be concentrated is inputted into concenfrating body 13 by being injected through inlet 37 into valve 35 which is set to communicate inlet 37 with inlet/outlet pipe 31 such that the sample to be concentrated flows into concentration chamber 20 in concenfrating body 13; when the desired volume of sample has been loaded into concentration chamber 20, valve 35 is closed so that no flow can take place through inlet/outlet pipe 31 ; valve 27 is opened and pressurised gas flows from the supply of pressurised gas into concentrating body 13. This causes an over pressure in concenfrating body 13 and a pressure difference to occur across concenfrating membrane 21. Molecules of the sample to be concentrated which are small enough to pass through the openings 22 in the concenfrating membrane 21, such as molecules of the liquid in which the sample is suspended, are pushed through the concenfrating membrane 21 into waste chamber 10, while sample molecules are retained in the concentration chamber 20; the molecules which are pushed through the concenfrating membrane 21 into waste chamber 10 flow out of the waste opening 41 for collection or disposal; once the sample has been concentrated by being reduced in volume by a sufficient amount, for example when the level of liquid in the concenfrating body fall below the level detector 47, the concentrated sample can be removed from the concenfrating body without being handled by an operator. This can be achieved by valve 27 being closed and valve 35 being opened in order to connect inlet/outlet pipe 31 to concentrated fluid outlet pipe 39. The residual overpressure in concentrating body 13 then causes the concentrated sample to be pushed out of concentration chamber 20 via inlet/outlet pipe 31 and valve 35 to concentrated fluid outlet pipe 39. Alternatively, valve 27 may be kept open and the full pressure from the pressurised gas supply used to push the concentrated sample out of the concentration chamber 20. Concentrated fluid outlet pipe 39 can lead to any suitable destination, for example an analytical device such as a mass spectrometer, chromatography column, electrophoresis gel, or a dispensing device such as a pipette or a storage container such as a test tube or bottle, etc.

Figure 2 illusfrates how the lower part of wall 19 is inclined and so that the bottom portion of the concentrating body is tapered. This portion is provided with two outlet ports 18 covered by a concenfrating cut-off membrane 21.

A concenfrating unit in accordance with the present invention may be used for dialysis or buffer exchanging e.g. for exchanging a buffer fluid X for a buffer fluid Y. A method for dialysis or buffer exchanging in accordance with the present invention comprises the following steps:

A volume V of buffer fluid or liquid being dialysed X containing the sample which is to be retained is introduced into concentration chamber 20 in concentration body 13 for example via inlet 37', valve 35 and inlet/outlet pipe 31 ; valve 35 is closed so that no flow can take place through inlet/outlet pipe 31 ; valve 27 is opened and pressurised gas flows from the supply of pressurised gas into concenfrating body 13. Molecules of the sample to be concentrated which are small enough to pass through the openings 22 in the concentrating membrane 21, such as molecules of the liquid X, are pushed through the concenfrating membrane 21 into waste chamber 10; once the sample has been concentrated by being reduced in volume by a sufficient amount e.g. to 0.03 V, the overpressure in concentration chamber 20 is removed e.g. by operating valve 27 so that concentration 20 is open to the atmosphere; a volume of liquid Y, e.g. W = 0.97 V, is introduced into concentration chamber 20 for example from inlet 37"via inlet/outlet pipe 31, so that the proportion of fluid X to fluid Y is at most 0.03 to 0.97; the above concenfrating and refilling procedure is repeated until the desired level of fluid exchange is achieved and the sample and liquid Y can be removed, possibly after concentrating to remove some of liquid Y, via inlet/outlet pipe 31, valve 35 and concentrated fluid outlet pipe 39.

A second embodiment of a concentration chamber in accordance with the present invention is shown in figure 3. In this embodiment there is a separate inlet pipe 31' and outlet pipe 31 ". Inlet 37' from a sample source and, optionally inlet 37" from a second source of fluid such as buffer fluid are connected to a valve 36 which is connected to inlet pipe 31 '. Outlet pipe 31 " is connected to an outlet valve 35 which is connectable to a concentrated fluid outlet pipe 39.

Figure 4 shows a third embodiment of the present invention in which the concentration chamber 20 is not surrounded by a hollow body that acts as a waste chamber but is intended to be positioned over a waste capture bowl 53 or other waste collection or disposal device such as a sink or drain into which fluid passing through concentrating 21 membrane can fall. A volume of liquid containing sample 55 which is to be concentrated is shown inside concentration chamber 20. Figure 5 shows the same embodiment of the present invention when the sample has been concentrated and unwanted liquid and sample matter 57 has been collected in waste capture bowl 53.

It is conceivable that any of the embodiments shown may be adapted by attaching the supply of pressurised fluid to the same inlet as used for inputting the fluid that is to be concentrated, or vice versa. Preferably the inlet and outlet pipes have small diameters in order to reduce the dead volume of the apparatus.

It is not necessary that the input means and output means shown in the embodiments include a valve. It is conceivable to use valveless input means such a syringe, in which case the diluted sample can be inputted by connecting a syringe containing the sample to the input pipe and injecting the sample into the concentration chamber by pushing the syringe plunger into the syringe body. Similarly, the concentrated sample can be outputted by connecting a syringe to the output pipe and withdrawing the syringe plunger from the syringe body. Additionally, the source of pressurised gas used to push the waste through the concentration membrane could be supplied directly by a simple pump, manually-or power-driven, actuated as necessary, instead of being supplied from a pressurised container.

Devices in accordance with the present invention may be used statically or in a centrifuge.

The above mentioned embodiments are intended to illustrate the present invention and are not intended to limit the scope of protection claimed by the following claims.

Claims

1. Concenfrating unit for increasing the concentration of a sample diluted in a volume of a liquid comprising a hollow body (3) having a concentration chamber (20) with at least one outlet port (18) provided with a concenfrating membrane (21), wherein said concentration chamber (20) has a pressurised fluid inlet (21) connectable to a supply of pressurised gas (29), characterised in that said concentration chamber (20) has a diluted sample inlet (37, 37') connectable to input means (35; 36) for inputting the volume of liquid containing said sample into said concentration chamber (20) and a concentrated sample outlet (39) connectable to output means (35) for outputting the contents of said concentration chamber (20).

2. Concenfrating unit in accordance with claim 1 characterised in that said input means is an inlet valve (35; 36) and said output means is an outlet valve (35), whereby said input means and output means are controllable by automatic control means (49).

3. Concenfrating unit in accordance with any of the previous claims characterised in that it comprises at least one level detector (47, 47', 47") for detecting the level of fluid in said concentration chamber (20).

4. Method of concentrating a sample comprising the steps of: a) inputting, via a valve (35; 36), a volume of liquid containing the sample which is to be concentrated into a concentration chamber (20) of a device in accordance with any of the previous claims; b) closing valve (35; 36) so that said volume of liquid cannot flow out of concentration chamber 20; c) opening valve (27) so pressurised gas flows from the supply of pressurised gas into concenfrating body (13), causing an over pressure in concenfrating body (13) and a pressure difference to occur across concenfrating membrane (21) so that molecules in the liquid and sample to be concentrated which are small enough to pass through the openings (22) in the concenfrating membrane (21), are pushed though the concenfrating membrane (21) in waste chamber (10) while larger molecules are retained in the concentration chamber (20); and, d) once the sample has been concentrated, opening an outlet valve (35) to connect concentration chamber 20 to outlet pipe (39), and outputting the concentrated sample.

5. Method of exchanging the buffer of a sample contained in a volume V of a first buffer X comprising the steps of: a) inputting, via a valve (35; 36), the sample and first buffer X into a concentration chamber (20) of a device in accordance with any of the previous claims; b) closing valve (35; 36) so that said sample cannot flow out of concentration chamber (20); c) opening valve (27) so pressurised gas flows from the supply of pressurised gas (29) into concentrating body (13), causing an overpressure in concenfrating body (13) and a pressure difference to occur across concenfrating membrane (21) so that first buffer molecules which are small enough to pass through the openings (22) in the concenfrating membrane (21) are pushed through the concenfrating membrane (21 ) in waste chamber (10) while molecules which are too large to pass said openings (22) are retained in the concentration chamber (20); d) once the sample and first buffer in concentration chamber (20) has been reduced in volume by a sufficient amount, releasing the overpressure in concentration chamber (20); e) opening valve (35, 36) to connect inlet pipe (31, 32) to a source of a second buffer Y and inputting a volume W of second buffer Y into concentration chamber (20); f) closing valve (33, 36) so that said sample cannot flow out of concentration chamber (20); g) opening valve (27) so pressurised gas flows from the supply of pressurised gas into concentrating body (13), causing an overpressure in concentrating body (13) and a pressure difference to occur across concenfrating membrane (21) so that first and second buffer molecules which are small enough to pass through the openings (22) in the concenfrating membrane (21) are pushed though the concenfrating membrane (21) in waste chamber (20) while larger molecules are retained in the concentration chamber (20); h) once the sample and first and second buffers in concentration chamber (20) have been reduced in volume by a sufficient amount, releasing the overpressure in concentration chamber (20), i) repeating stages e)-h) until the concentration of the first buffer has been reduced to a desired level, and optionally adding more of second buffer to the concentration chamber (20): and j) outputting the sample and second buffer from said concentration chamber (20) by opening valve (35) to connect the concentration chamber (20) to outlet pipe (39).

6. Method according to claim 5, characterised by the additional step of concenfrating said sample by passing some of said second buffer through the concenfrating membrane (21) after step I) and before step j).

7. Method according to claim 5 or 6 characterised in that some or all of the steps are controlled by an automated control means (49).