EP1244908A1

EP1244908A1 - Device and method for treating a sample by overpressured layer chromatography a stationary phase, under controlled forced flow

Info

Publication number: EP1244908A1
Application number: EP01903867A
Authority: EP
Inventors: Emil Mincsovics; László KECSK S; Ernö TYIH K; Barnabás TAPA
Original assignee: Bionisis SA
Current assignee: Bionisis SA
Priority date: 2000-01-04
Filing date: 2001-01-03
Publication date: 2002-10-02
Also published as: FR2803220A1; AU3183601A; FR2803220B1; CA2396189A1; CN1398349A; AU770513B2; JP2003529749A; CN1262837C; US20030019816A1; WO2001050123A1

Abstract

The invention concerns a device for overpressured layer chromatography treatment of a sample comprising a chamber (1) housing a stationary phase (2) provided in a first site (6) with a sample to be treated, means for externally pressurising (8) a top surface (5) of the stationary phase, means for dispensing (10) a mobile phase in a second site (7) of the stationary phase, a mobile phase supply intake (13), and an outlet (18) for discharging the mobile phase outside the chamber. The device further comprises means (23) for feeding the intake (13) with mobile phase, a sensor (29) for measuring the pressure of the mobile phase upstream of the intake, a valve (31) arranged downstream of the outlet for varying the mobile phase flow rate at the chamber outlet, and a module (25) for controlling the valve on the basis of a comparison between the measured pressure and a selected threshold pressure, the valve (31) being open when the measured pressure is not less than the threshold pressure.

Description

Device and method for processing a sample by separation on a stationary phase. under controlled forced flow

The invention relates to the field of separation on a stationary phase of the constituents of a so-called “complex” sample, using a forced flow.

Separation under forced flow (better known by the acronym OPLC for OverPressured Layer Chromatography or Optimum Performance Layer Chromatography) is a technique used to separate the constituents of a sample, deposited in at least a first chosen location on a layer called " stationary phase ", using a carrier fluid, called" mobile phase ", undergoing external pressure. Trained by the mobile phase, the constituents migrate to the stationary phase in a known order depending on their retention.

To do this, certain treatment devices include a separation chamber, into which at least one stationary phase is introduced and which comprises external pressurization means, making it possible to apply an external pressure of selected intensity on an upper face of the phase stationary, distribution means, making it possible to deliver at least one mobile phase at at least one second chosen location of the stationary phase, at least one input for supplying these distribution means and at least one output allowing the evacuation of the mobile phase .

The mobile phase, which is introduced into the separation chamber of this type of device, moves in the stationary phase according to a front called "alpha front". Under the effect of the pressure applied, the alpha front expels the air which is trapped in the stationary phase, the latter being initially dry. However, part of the air remains trapped in an area known as the “alpha area” located immediately behind the alpha front and preceding a completely wet area known as the “total humidity area”. As a result, the total humidity front is not linear, which affects the efficiency of the separation, the reproducibility of the results and the accuracy of the analyzes carried out simultaneously or consecutively. No known solution allows sufficiently precise control of the linearity of the fronts, and in particular that of the total humidity front.

The invention therefore aims to improve the situation.

To this end, it proposes a device of the type described in the introduction, and in which regulating means are provided making it possible to control the pressure of the mobile phase upstream and / or downstream of the stationary phase, so that this pressure remains less than or equal to a limit pressure, linked to the external pressure chosen.

In what follows, the term “mobile phase” will be understood to mean any fluid making it possible to move the constituents of a sample over the stationary phase. It may therefore be a liquid, such as an eluent, or a gas, such as air, making it possible to expel a solvent previously introduced into the separation chamber.

These regulation means preferably comprise at least one valve placed downstream from the first mobile phase outlet and arranged to vary the flow rate of the mobile phase at the outlet of the chamber between a zero value and a maximum value.

More preferably still, the regulating means further comprise means making it possible to measure the pressure of the mobile phase upstream of the first inlet and / or downstream of the first outlet, as well as a module arranged to control the valve in function a comparison between the measured pressure and the limit pressure linked to the chosen external pressure (or more briefly, limit pressure), the valve being open to let out the mobile phase when the measured pressure becomes greater than or equal to the limit pressure. This limit pressure is by definition lower than the external pressure.

In this way, the air which is initially trapped in the stationary phase is gradually compressed in front of the alpha front until the measured pressure reaches the chosen limit value. Pressure ^'therefore gradually increases in the entire mobile phase, in the alpha region, so that the edge of the migration velocity alpha and the width of the area alpha decrease, leading to a quasi-linearity of the front total humidity. Two operating modes can advantageously be envisaged for the device according to the invention. In a first mode called "offline", or "infusion", the stationary phase is extracted from the chamber, after separation of the constituents of the sample, then placed in an external analyzer, for the purpose of identification and / or quantification of the components, for example by densitometry or video scanning or even radiometric scanning.

In this mode where the sample is preferably placed on the stationary phase before the supply in mobile phase, the control module jointly controls the valve, the supply means and the external pressurization means so that the supply in mobile phase is carried out for the entire duration of the separation by keeping the valve closed, that is until the measured pressure of the mobile phase reaches the limit pressure (or threshold pressure). Then, the mobile phase supply is interrupted, then the valve is opened and the external pressure is released.

In a second mode called "online" or "infusion-transfusion", the separate constituents on the stationary phase are identified and / or quantified using analyzers, on this stationary phase and / or online at the outlet of the bedroom. This will, for example, be analysis by ultraviolet or visible detection or else by mass spectrometry. In this case of online analysis, the separate constituents are eluted by the mobile phase which leaves the chamber.

In this mode, the control module also jointly controls the valve, the supply means and the external pressurization means so that the mobile phase supply takes place throughout the duration of the separation and analysis by keeping the valve closed as long as the measured pressure of the mobile phase is lower than the limit pressure, then keeping it open.

Here, the sample can be placed on the stationary phase either before the mobile phase feed begins, or after the valve has been placed in the open position.

The device according to the invention may include other additional characteristics, taken separately or in combination, and in particular: - The chamber can be arranged so as to receive an extractable cassette comprising the stationary phase;

- The external pressurization means may include a flexible film housed opposite the upper face of the stationary phase and application means suitable for pressing the film against the stationary phase to apply the external pressure of selected intensity;

- The application means may include a fluid reservoir intended to generate the external pressure (or more briefly "external pressurizing fluid"), coupled to a supply circuit; but, other means can be envisaged, such as pneumatic or mechanical means;

- The external pressurization fluid reservoir and the mobile phase supply means can be housed in the same fluid supply unit;

the stationary phase may be provided with the sample to be treated before being introduced into the chamber, or else be provided with this sample once introduced into the chamber, via the same inlet or else via another inlet;

- The control module can be arranged so as to order, before the introduction of the sample, on the one hand, the valve to be placed in a state preventing the evacuation of the mobile phase and, on the other hand , to the supply means for supplying the stationary phase with a selected mobile phase volume, preferably, so that this volume corresponds to a measured mobile phase pressure substantially equal to the limit pressure.

The invention also relates to a process for treating the constituents of at least one sample by separation under forced flow. This process comprises the following steps: a) placing in a chamber at least one stationary phase suitable for receiving at least one first location chosen at least one sample to be treated, b) supplying in mobile phase at least one second location chosen from the phase stationary, while applying an external pressure of selected intensity to the upper face of this stationary phase and preventing the mobile phase from leaving the stationary phase, c) measure the pressure of the mobile phase upstream and / or downstream of the chamber, d) compare each pressure measured with a limit pressure, linked to the external pressure chosen, and, when the measured pressure becomes greater than or equal to the limit pressure, reduce the pressure of the mobile phase so that it remains less than or equal to this limit pressure.

Other characteristics and advantages of the invention will appear on examining the detailed description below, and the appended drawings, in which:

FIG. 1 schematically illustrates an example of a device according to the invention suitable for separation in infusion and / or infusion-transfusion mode, FIG. 2 is a cross-sectional view of the separation chamber of FIG. 1,

FIG. 3 is a variant of FIG. 1 in which the external pressurization means are not hydraulic but, for example, mechanical,

- Figures 4a and 4b are diagrams comparing the changes as a function of time of the alpha fronts and of total humidity and of the pressure measured for the modes of infusion (a) and infusion-transfusion (b), in the case of regulation on the pressure of the mobile phase measured upstream of the chamber,

- Figures 5a to 5d are variants of the devices of Figures 1 and 3.

The attached drawings are, for the most part, certain. Consequently, they can not only serve to complete the description, but also contribute to the definition of the invention if necessary.

In the detailed description which follows, reference will be made to a device for processing a complex sample by separation under forced flow (or OPLC). The term “treatment of a sample” is understood here to mean mainly the separation of the constituents which compose it, possibly coupled with one or more on-line and / or off-line analysis of these constituents.

The device illustrated in Figures 1 and 2 firstly comprises a separation chamber 1 adapted to receive a layer 2 (in English "sorbent layer") forming a stationary phase. This layer 2 is for example made up of powder or particles of silicate gel, alumina, magnesium silicate, talc based on inorganic components, cellulose, synthetic resin, polyamides based on organic components, or else derivatives or mixtures of some of these components on a support plate comprising a layer 2 with one or two stationary separation phases. However, it is clear that the material used and its surface condition (granularity, porosity, and the like) depend on the type of sample to be treated.

This layer 2 preferably rests on a rigid support 3 supported by the bottom of the lower part 4 of the chamber 1, and at a distance from it so that a cavity 12 is formed under the support 3. or the samples to be treated are placed in at least a first chosen location 6 on the upper face 5 of the layer 2, opposite the bottom 4. Furthermore, the mobile phase is introduced on this same upper face 5, in at least a second chosen location 7. As indicated above, the mobile phase is intended to cause the migration of the constituents of the sample.

The term "location" means both a localized location and an extended location of the straight line, curvilinear or circular type, or of any other chosen shape.

As will be seen below, the first 6 and second 7 places can be at least partially confused. A person skilled in the art knows how to choose the first and second places according to the type of treatment he wishes to perform. Thus, depending on the respective positions of the first (s) 6 and second (s) 7 locations the separation can be unidirectional or bidirectional, or circular, or even anti-circular. But all of this is well known to those skilled in the art, and is not the subject of the present invention.

A short distance above the upper face 5 of the layer 2 is placed a flexible waterproof film 8, for example of Teflon. As will be seen below, this film 8 is intended to apply an external pressure, uniform or not, on the upper face 5 of the stationary phase 2. In the example illustrated in FIG. 2, the film 8 comprises, opposite at least part of the second location 7, a first opening 9 for the sealed passage of the end of a tube 10 intended to distribute the mobile phase at the second location 7.

In a variant, layer 2 (or stationary phase) can be housed beforehand in a cassette arranged to be introduced into the chamber before treatment. The upper wall of this cassette can optionally include the flexible external pressurization film. In this variant, it is preferable that the sample is implanted in the stationary phase 2, before introduction of the cassette into the chamber 1. But this is not compulsory, in particular when the cassette does not include a flexible film.

If the sample is to be introduced onto layer 2 (or stationary phase), once this has been placed in chamber 1, the film 8 comprises, opposite at least part of the first location 6, a second opening for the sealed passage of the end of a tube intended for the introduction of the sample at the first location 6. Of course, when the first 6 and second 7 locations are at least partially combined, a single opening may be sufficient for the introduction of the mobile phase and the sample.

The upper part of the chamber 1 is closed by a wall 11 placed, in the example illustrated in FIGS. 1 and 2, slightly above the film 8. This upper wall 11 comprises a first inlet 13 for the sealed passage of the end of the tube 10 for distributing the mobile phase. The lower wall 4 of the chamber comprises a second inlet 14 for the sealed passage of the end of a tube 15 for supplying external pressurization fluid. This external pressurization fluid (or pressure transfer fluid) can be a gas or, as in the example described below, a liquid such as oil.

The external pressurization fluid preferably circulates in a closed circuit, the upstream supply part of which is formed by the tube 15 and the downstream part is formed by a tube 19 of which a first end 20 opens into the cavity 12 of the chamber 1 by a sealed opening 21 formed, for example, in its lower wall 4, while the opposite end 22 of this tube 19 opens into a reservoir of external pressurization fluid 27. This reservoir 27 is coupled to a first micro-pump which is controlled by the control module 25 of the device and, preferably, housed in a unit 23 for supplying fluid. Also preferably, the tube 15 for supplying external pressurization fluid is provided with a pressure sensor 32 which delivers its pressure measurement to the control module 25. Thus, the control module 25 can act on the first micro- pump to fix, as required, the flow rate of supply of external pressurizing fluid, and consequently the external pressure applied to the layer 2. It is also possible to provide on the tube 20, between the opening 21 and the reservoir 27, a valve 34.

When the external pressurization fluid circulates, it exerts a substantially vertical external pressure on the underside of the support 3, which leads to lifting it and consequently pressing the film 8 and the stationary phase 2 against each other according to an external pressure of selected intensity.

As is the case in the example of FIG. 3, other means of external pressurization of the stationary phase 2 can be envisaged, such as for example mechanical, or pneumatic means, or the like. This can avoid, in some cases, using an external circuit for supplying external pressurizing fluid.

In the example illustrated in FIGS. 1 and 2, the chamber comprises, at at least a third chosen location 16, localized or extended, a zone for collecting the mobile phase having served for the separation of the constituents of the sample. This third location can be located on the stationary phase 2, as illustrated in FIG. 2, or else on the periphery thereof. In the first case, the film 8 comprises a second opening 17 for the sealed passage of the end of a tube 24 for collecting the mobile phase, and the chamber 2 comprises a sealed outlet 18 allowing the passage of this end of the tube 24. In the second case, the second opening is not useful, and therefore only the outlet 18 is required.

The tube 10 which feeds layer 2 in mobile phase has one end

26 connected to a second micro-pump coupled to one or more mobile phase reservoir (s) 28, for establishing a continuous gradient or stepwise, and also controlled by the control module 25. Preferably, the second micro-pump is also housed in unit 23.

Of course, in the example of FIG. 3, the fluid supply unit 23 is only used for mobile phase supply, and therefore it only has one micro-pump.

According to the invention, a pressure sensor 29 is provided upstream from the mobile phase inlet 13 of the chamber 1, or a pressure sensor 30 downstream from the mobile phase outlet 18 from this chamber 1.

However, as illustrated in FIGS. 1 and 3, it is also possible to provide a first pressure sensor 29 upstream of the mobile phase inlet 13 of the chamber 1 and a second pressure sensor 30 downstream of the outlet of mobile phase 18 of this chamber 1. This latter solution with two sensors is particularly advantageous because it improves the precision of the control of the device. The first sensor 29 is arranged so as to carry out its pressure measurement on the mobile phase which circulates in the supply tube 10, while the second sensor 30 is arranged so as to carry out its pressure measurement on the mobile phase which circulates in the collection tube 24. The first sensor 29 therefore delivers an upstream pressure measurement PI to the control module 25, while the second sensor 30 delivers a downstream pressure measurement PO to said control module 25.

Furthermore, the invention also provides a valve 31 arranged to control the flow of the mobile phase downstream of the mobile phase outlet 18. This valve is therefore installed on the collection tube 24, preferably downstream of the second sensor. pressure 30 (when this is planned).

The operating state of the valve 31 is controlled by the control module 25 as a function of a first comparison between the first pressure PI measured by the first sensor 29 and a first pressure limit as PMI.U _ΠI and a second comparison between the second pressure PO measured by the second sensor 30 and a second pressure limit that P o.um- The PMi.Lim and / or the P o.um are chosen smaller than the external pressure applied Pex _t .

The first PMI, L..T _. and second PM _O, _L. _ _T. limit pressures, as well as Pεx _t are stored in registers of a memory, preferably rewritable, so that the limit values can be adapted to the conditions of use for infusion or infusion-transfusion. The release of the external pressure comes after separation.

Of course, when a single pressure sensor (29 or 30) is provided, the control of the operating state of the valve 31 by the control module 25 is carried out according to a single comparison between the pressure measured (PI or PO) and the associated limit pressure (P i.um or PMo.um) -

Preferably, the flow rate of the mobile phase which feeds the chamber 1 and the external pressure applied to the layer 2 are also controlled by the control module 25 at the level of the feed micro-pumps.

Thus, by playing on the external pressurization means (flow of the external pressurization fluid or force applied to the stationary phase 2) and jointly on the valve 31 (and therefore on the collection flow of the mobile phase) and on the flow d 'mobile phase supply, it is possible to control with very good precision the linearity of the total humidity front which characterizes the movement of the mobile phase in the stationary phase 2, under the action of the external pressurization means.

In fact, and as indicated above, when the valve 31 is placed in a "closed" state (zero flow) in which it prohibits the evacuation of the mobile phase from chamber 1, the air "trapped" in the phase stationary, before the arrival of the mobile phase, is gradually compressed in front of the alpha front. The pressure on this alpha front will therefore gradually increase while its migration speed will gradually decrease. The width of the partially wet zone (or alpha zone), located between the alpha front and the total humidity front, will therefore gradually decrease, so that the total humidity front will gradually tend towards quasi-linearity.

This state of quasi-linearity corresponds to limiting pressures (PMI _. U _ΠI ,

PMo.Lim) which can be easily determined.

The control module 25 therefore only has to carry out its comparison (s) to monitor whether the pressure or pressures measured are respectively higher or lower than the associated limit pressures. When the measured pressures PI and PO becomes greater than or equal to limit pressure PMI, _L. _ _TI and PMO, U _.TI. who are smaller than Pεx _t , the control module 25 acts on the valve 31, and possibly on at least one of the supply micro-pumps, so that the mobile phase can, or not, leave the chamber 1 When the valve 31 has a variable flow rate (as opposed to binary operation of the "all or nothing" type), the authorized collection (or evacuation) flow rate is determined as a function of the difference between the measured pressure and the pressure associated limit, during the period during which the partially wet zone leaves stationary phase 2 and chamber 1.

On the other hand, in all or nothing type operation, which is currently preferred for reasons of simplicity of the regulation, the valve 31 is switched from the "closed" state to the "open" state, or the reverse depending on the result of comparisons.

The "closed" state corresponds to a flow of a zero value, while the "open" state corresponds to a flow of a maximum value.

Two modes of operation can be envisaged. The first mode corresponds to an operation of the “offline” or “infusion” type device, in which the processing in the device consists only in the separation of the constituents of the sample. The analysis (determination and / or quantification) of these constituents is carried out in an external analyzer after separation of the constituents then extraction of the stationary phase 2. Any type of analysis known to those skilled in the art can be envisaged.

In this first mode, where the sample can be placed before or after introduction of the stationary phase 2 into the chamber 1, it is preferably started from a stationary "dry" phase, that is to say before the phase stationary is powered in mobile phase. The control module 25 orders the complete closure of the valve 31 and then the mobile phase supply. The valve 31 is kept closed throughout the duration of separation, that is to say as long as the measured pressure PI of the upstream mobile phase remains below PMI, _L. _ _TI OR, as will be explained below with reference to FIG. 4b, to P _M i.umo-

Then, when the limit pressure P I.LJ _ΠI is reached or exceeded, the mobile phase supply is interrupted by action on the corresponding micro-pump, the valve 31 is open, which amounts to reducing the pressure of this mobile phase , for example at ambient pressure, and finally the external pressure is released. As indicated above, when the valve 31 operates in all or nothing mode, it is switched from the closed state to the open state. On the other hand, when the valve 31 is at variable flow, it is switched from the closed state to one of its open states at non-zero flow (strictly greater than zero and less than or equal to the maximum flow). This state is chosen by the control module according to a criterion depending on the separation conditions. It is based on the difference in viscosity between the air and the mobile phase.

The second mode corresponds to an operation of the “online” or “infusion-transfusion” type device. The processing in the device consists in the separation of the constituents of the sample coupled to an online analysis of its constituents and / or to an external analysis. The separate constituents are therefore identified and / or quantified on the stationary phase 2 and / or outside of this chamber by analysis of the mobile phase leaving this chamber, which comprises the separate constituents.

In this second mode, it is possible to introduce the sample on the stationary phase 2 before infusion, that is to say before the introduction of the mobile phase.

But it could be otherwise; an infusion step then preceding the introduction of the sample. The volume used for infusion is known to the control module, as long as the latter knows the type of stationary phase used.

The constituents of the sample are separated under the joint action of a permanent supply in mobile phase and a management of the state of the valve 31. In fact, just as in the infusion mode, the command 25 first orders complete closure of the valve 31 and then the mobile phase supply. In an embodiment comprising only one upstream pressure sensor, the valve 31 is kept closed as long as the measured pressure PI of the mobile phase upstream of the chamber remains below the limit pressure PMi.um or, as will be set out below with reference to FIG. 4b, at P i.umo-

However, when the limit PMI.U _ΠI (or P i.um o) is reached or exceeded, the increase in the mobile phase is maintained and the valve 31 is open, which amounts to reducing the pressure of this mobile phase. Of course, as indicated previously, when the valve 31 operates in all or nothing mode, it is switched from the closed state to the open state authorizing a maximum discharge rate for the mobile phase (out of the chamber). On the other hand, when the valve 31 is of variable flow, it is switched from the closed state to one of its open states authorizing a non-zero flow (strictly greater than zero and less than or equal to the maximum flow) for discharging the mobile phase (outside the room). This state is chosen by the control module according to a criterion depending on the separation conditions. When the separation is complete, the constituents are analyzed on the stationary phase and or outside using the mobile phase leaving the chamber with the constituents separated from the sample.

As illustrated in FIGS. 1 and 3, a current or voltage supply module 33 can be provided for supplying electrodes housed in the chamber 1 for separation by electro-chromatography or electrophoresis. These electrodes are placed parallel or perpendicular to the flow, the electrophoresis being carried out either simultaneously or sequentially, relative to the separation by the mobile phase.

However, electro-chromatography can be carried out after a pre-wetting (or pre-wetting) infusion in the open state of the valve 31, using electrodes perpendicular to the flow. After the pre-wetting, the electro-chromatography and the chromatography can be carried out simultaneously or sequentially. The chromatographic and electrophoretic separation can be carried out simultaneously or sequentially on the pre-wetted stationary phase, using electrodes parallel or perpendicular to the flow. The electrophoresis is of course carried out in the wet (or wet) phase.

The stationary phase can also include several identical or different zones, each making it possible to carry out a particular treatment (separation and / or analysis). The external pressurization means used in the different zones may possibly be different, or else they may be identical but provide different pressures.

These different cases are illustrated in the diagrams of Figures 4a (infusion) and 4b (infusion-transfusion). More specifically, we can see, in the parts upper diagrams, the evolutions compared over time of the positions of the alpha front (solid line and referenced 1) and the total humidity front (dotted line and referenced 2), and in the lower parts, the evolution during of the time of the measured pressure PI upstream of the chamber inlet 13 (in solid line and referenced 3) and of the measured pressure PO downstream of the chamber outlet 18 (in solid line and referenced 7).

The rectangles designated by A, B, C and D illustrate, for four successive instants, the instantaneous profiles of the alpha (1) and total humidity (2) fronts.

In the upper parts of the diagrams, M1 denotes the place of introduction of the mobile phase (or second place chosen 7), while MO denotes the place of collection of the mobile phase (or third place chosen 16). Reference (6) designates the location of the sample between Ml and MO. The reference (4) indicates the place and the instant when the alpha front (1) disappears. The reference (5) indicates the place and the instant when the total humidity front (2) disappears. Otherwise :

- P IU _ΓTI designates the limiting pressure of the mobile phase, linked to the external pressure, upstream of the inlet 13 of the chamber;

- PO designates the pressure measured downstream of the outlet 18 of the chamber;

- Pεxt designates the external pressure applied to the stationary phase by the external pressurization means (Pεx _t is always strictly greater than P _M i.um, typically PMi.um is chosen to be substantially equal to approximately 80% of Pgxt, for example; done, at the start of programming the external pressure is chosen and the control module automatically deduces the limit pressure);

- PMi.um o designates in the infusion-transfusion mode a limit pressure of the mobile phase, also linked to the external pressure, but chosen lower than the limit pressure Pιyιι, um- It is in fact a pressure of security. In other words, this pressure is the actual limit pressure which is preferably used by the control module to make the comparison with the pressure PI measured upstream of the chamber. It therefore corresponds, at the moment when it is decided to open the valve 31, while maintaining the supply in mobile phase. When the device operates in this mode, it is clear that the control module directly calculates P _M i.umo from PBΛ.

Reference is now made to FIGS. 5a to 5d to describe different variants of the devices illustrated in FIGS. 1 and 3. In these variants, all the elements substantially identical to those of FIGS. 1 and 3 bear identical references. Of course, these variants are only a few examples of implementation among many others.

FIG. 5a illustrates a first variant in which a unit 35 is provided which makes it possible to supply the stationary phase both in mobile phase and in sample. This unit 35 is therefore placed on the fluid supply tube 10, between the upstream pressure sensor 29 and the inlet 13 of the chamber 1, and it is connected to a tube 36 for injecting the sample which penetrates into the chamber by an inlet provided for this purpose and opens opposite the first location 6. A single tube 10 could be provided for injecting both the mobile phase and the sample.

FIG. 5b illustrates a second variant in which a unit 37 is provided, making it possible to supply the stationary phase at two different and independent second locations for performing a bidirectional type separation, or else two separations of samples placed on two different stationary phases. A tube 38 supplies the unit 37 in mobile phase, while two tubes 39 and 40 leave the unit 37 to each supply one of the two second places, after having entered the chamber by two inlets 41 and 42 and passed through the film 8 at two sealed openings 9 and 17 provided facing the two second places. In the bidirectional hypothesis, a substantially linear mobile phase line is produced between the two injection sites and the separation takes place substantially perpendicular to this line. This unit 37 also has the function of directing the (or the different) mobile phase (s) to appropriate zones of the stationary phase (s).

In this variant, two third independent places are provided for collecting each mobile phase which has served for the separation of the constituents of the sample (s) in each of the two parts of the chamber 1. This chamber therefore comprises two independent collection outlets 43 and 44 which supply two tubes 45 and 46 connected to the valve 31, which consequently has two inlets and one outlet.

FIG. 5c illustrates a third variant in which a unit 37 is provided allowing a parallel supply of the stationary phase in three second different and independent locations, possibly formed on the same stationary phase or on three different stationary phases. A tube 38 feeds the unit 37 in mobile phase, while three tubes 47, 48 and 49 leave the unit 37 to feed each one of the three second places, after having entered the chamber by three inlets 50, 51 and 52 and passed through the film 8 at three sealed openings 9 provided opposite the three second locations.

In this unidirectional type separation variant, three independent third places are provided for collecting each mobile phase having served for the separation of the constituents of the sample (s) in each of the three parts of the chamber 1. This chamber therefore comprises three independent collection outlets 53, 54 and 55 which supply three tubes 56, 57 and 58 connected to the valve 31, which in this variant is a triple valve making it possible to let out separately and independently each of the three mobile phases collected.

FIG. 5d illustrates a fourth variant in which a unit 59 is provided making it possible to supply the stationary phase both in mobile phase and in sample. The mobile phase supply can be carried out in parallel on three channels, as in FIG. 5c, just like the sample implantation which can be carried out in parallel on three channels. This unit 59 is therefore connected, on the one hand, to three mobile phase supply tubes 60, 61 and 62 which enter the chamber by three fluid inlets 66, 67 and 68 and pass through the film 8 at three watertight openings 9 provided opposite three second places, and on the other hand, three sample injection tubes 63, 64 and 65 which enter the chamber through three sample inlets 69, 70 and 71 and pass through the film 8 at three watertight openings provided opposite three first places.

In this variant of unidirectional type separation, three third independent places are provided for collecting each mobile phase having served to the separation of the constituents of the sample (s) in each of the three parts of the chamber 1. This chamber therefore comprises three independent collection outlets 72, 73 and 74 which supply three tubes 75, 76 and 77 connected to the valve 31, which in this variant is a triple valve allowing to let out separately and independently each of the three mobile phases collected.

The invention also relates to a method for processing a sample by forced flux separation (OPLC). This process includes the following steps.

In a first step, at least one stationary phase suitable for receiving is placed in a chamber, at least in a first chosen location, at least one sample to be treated. Of course, the chamber can be arranged to receive several stationary phases in parallel or in series, or else stacked on top of each other, as is well known to those skilled in the art.

This stationary phase, which is preferably a layer of the type presented in the description of the device according to the invention, is either directly placed in the chamber on a support provided for this purpose, or previously placed in a cassette which is then introduced in the bedroom. Similarly, the sample can be placed on the stationary phase intended to separate its constituents before the stationary phase is introduced into the chamber, or else implanted (or injected) once the stationary phase is introduced into the chamber.

In a second step, at least one second selected location of the stationary phase is supplied with the mobile phase, while applying an external pressure of selected intensity to an upper face of the stationary phase and by preventing the mobile phase from leaving the phase. stationary.

This external pressure is not necessarily uniform. One can in fact consider applying different external pressures on different zones of the same stationary phase, or on different stationary phases placed in the same chamber.

In a third step, the pressure of the mobile phase is measured upstream and / or downstream of the chamber.

In a fourth step, the upstream and / or downstream pressures are compared which has just been measured at an upstream and / or downstream limit pressure (s). Then, when the pressure (s) measured is (are) greater than or equal (s) to the associated limit pressure (s) and the pressure mobile phase limit, linked to the external pressure (Pεx _t ), which is greater than the limit pressure (s), the pressure of the mobile phase is reduced so that it remains lower or equal to the limit pressure. In an all or nothing type of operating mode, this pressure reduction is not adaptable (in fact the mobile phase is placed at ambient pressure). On the other hand, in a “variable” type operating mode, the value of the pressure can be chosen as a function of the result of the comparison.

As was previously indicated in the section detailing the device, the method can be applied to the infusion mode (or "offline") or to the infusion-transfusion mode (or "online").

Separation by conventional transfusion is also possible when the valve 31 is in its open state during the separation process.

In the infusion mode, during the fourth step of the process, the supply of the stationary phase in the mobile phase is definitively interrupted when the measured upstream pressure becomes greater than or equal to the pressure P i.Lim- En fact, as illustrated in figure 4b, it is possible to decide to use PMi.Lim o as limit pressure being used for comparison, which is strictly lower than PMi.um. Then, the mobile phase is placed at ambient pressure, preferably, which amounts to reducing the pressure of this mobile phase, and finally the external pressure is released.

In the infusion-transfusion mode, during the fourth step, the mobile phase is prohibited from leaving the room, then the mobile phase is supplied. The ban is maintained as long as the measured pressure of the mobile phase upstream of the chamber remains below the limit pressure. When the limit pressure PMI.UITI or PMi.um o (depending on the choice made at the start) is reached or exceeded, the mobile phase supply is maintained and the mobile phase is allowed to leave the chamber, which amounts to reducing the pressure of this mobile phase. It can be envisaged that once the valve is open, a flow regulation is carried out leaving the mobile phase so that the measured pressure (s) of this mobile phase remains (are) substantially between the associated limit pressure (s) ( s) and a minimum pressure, throughout the duration of the treatment. In a preferred embodiment, during the second step, the external pressure is applied to the stationary phase by a fluid means, preferably hydraulic. But of course, other modes of application can be envisaged, in particular by mechanical or pneumatic means.

We can, during the first step, start by supplying the stationary phase with a mobile phase, before implanting the sample. This supply consists of filling the stationary phase with a selected volume. Evacuation of the mobile phase from the chamber is therefore prohibited during this feeding phase. Advantageously, the volume of mobile phase which is admitted into the chamber corresponds approximately to the total volume of the stationary phase when the pressure measured is substantially equal to the limit pressure.

More generally, everything that has been said in the part describing the device also applies to the process.

The invention is not limited to the embodiments of devices and methods described above, only by way of example, but it encompasses all the variants that a person skilled in the art may envisage within the framework of the claims below .

Thus, devices have been described in which the flow regulation means advantageously comprise a control module coupled to a valve as well as to means for measuring the pressure of the mobile phase upstream and / or downstream of the chamber. . However, the invention also relates to devices in which the flow regulation means only comprise a valve for regulating the flow rate of the mobile phase in the chamber, this valve being controlled either manually or by a programmable control module.

Furthermore, devices have been described in which the chamber treated only one or more stationary phases placed next to each other on a same support. However, the chamber can be adapted to receive several stationary phases stacked one on the other, with or without support, and used in series or in parallel, with or without interlayer.

On the other hand, an embodiment has been described in which a liquid mobile phase was introduced to drive the constituents of the sample. However, the invention also applies when a solvent is first introduced and then a gas such as air is used to displace (expel) the solvent mixed with the constituents of the sample. The air then serves as a mobile phase. This technique is known as "flash" chromatography. It follows that in all of the above and what follows, the mobile phase must be taken in a broad definition, namely "driving fluid", whether gaseous or liquid.

Claims

claims

1. Device for processing a sample by separation under forced flow, of the type comprising: - a chamber (1) arranged to accommodate at least one stationary phase (2) suitable for receiving in at least one first chosen location (6) at the at least one sample to be treated, external pressurization means (8) suitable for applying an external pressure of selected intensity to an upper face of the stationary phase, means of distribution (10) of a mobile phase into at least a second chosen location (7) of the stationary phase (2), at least a first inlet (13) for supplying said distribution means (10) in the mobile phase, and a first outlet (18) for discharging the mobile phase out of the chamber (1), and

- Means arranged to supply said first input (13) in the mobile phase, characterized in that it comprises regulation means (31) suitable for controlling the pressure of the mobile phase upstream and / or downstream of the stationary phase (2), so that this pressure remains less than or equal to a limit pressure, linked to the chosen external pressure.

2. Device according to claim 1, characterized in that said regulation means comprise a valve (31) placed downstream of said first outlet (18) and adapted to vary the flow rate of the mobile phase at the outlet of the chamber (1 ) between a null value and a maximum value.

3. Device according to claim 2, characterized in that said regulating means further comprises means (29; 30) suitable for measuring the pressure of the mobile phase upstream of said first inlet (13) and / or downstream of said first outlet (18) and a module (25) arranged to control said valve (31) according to a comparison between the measured pressure and said limit pressure, said valve being placed in an open state when the measured pressure is higher or equal to said limit pressure.

4. Device according to claim 3, characterized in that said module control (25) is arranged to control said supply means and said external pressurization means (8), together with said valve (31), according to said comparison.

5. Device according to claim 4, characterized in that said control module (25) is able to order said supply means to interrupt the supply of the chamber (1) in mobile phase, then to said valve (31 ) to place themselves in an open state and to said external pressurization means to release the external pressure, as soon as the measured pressure becomes greater than or equal to the limit pressure.

6. Device according to claim 4, characterized in that said control module (25) is arranged, after said valve opening, to order said supply means (23) to continue to supply the first input in mobile phase (13 ) of said chamber, and to order the valve (31) to be placed in an open state authorizing said mobile phase to leave the chamber at a selected flow rate.

7. Device according to claim 6, characterized in that said chamber (1) houses means for analyzing the constituents of the sample.

8. Device according to one of claims 1 to 7, characterized in that said chamber (1) is arranged to receive an extractable cassette comprising said stationary phase.

9. Device according to one of claims 1 to 8, characterized in that said external pressurization means comprise a flexible film (8) housed opposite the upper face (5) of the stationary phase (2) and means d application suitable for pressing one against the other said film (8) and said stationary phase according to an external pressure of selected intensity, said film (8) comprising a sealed opening (9) for supplying the stationary phase (2) by the distribution means (10).

10. Device according to claim 9, characterized in that said application means comprise a pump coupled to a reservoir (27) for the fluid intended to generate the external pressure and suitable for supplying an upstream part (15) of a circuit opening below the stationary phase (2), and in that said chamber (1) comprises a second sealed outlet (21) arranged to collect the external pressurizing fluid to supply a downstream part (20 ) of the circuit, connected to said tank (27).

11. Device according to claim 10, characterized in that said external pressurization fluid pump (27) and said mobile phase supply means are placed in a fluid supply unit (23) controlled by said control module (25).

12. Device according to one of claims 1 to 11, characterized in that said stationary phase (2) is provided with said sample to be treated before being introduced into said chamber (1).

13. Device according to one of claims 1 to 11, characterized in that said chamber (1) has a second inlet for the transport of the sample at the first location (6) of said stationary phase (2).

14. Device according to one of claims 1 to 13, characterized in that said first (6) and second (7) locations are at least partially combined.

15. Device according to claim 14, characterized in that said supply means are arranged to supply said first input in stationary and mobile phases.

16. Device according to one of claims 13 to 15, characterized in that the control module (25) is arranged to order, before routing the sample, on the one hand, to said valve (31) of place themselves in a state such that it prohibits the evacuation of the mobile phase from the chamber and, on the other hand, the supply means to supply the stationary phase with a chosen volume of mobile phase.

17. Device according to claim 16, characterized in that said volume is chosen so that it is substantially equal to a volume necessary for the pressure measured to be substantially equal to the limit pressure.

18. Device according to one of claims 3 to 17, characterized in that said measuring means (29; 30) comprise a first sensor (29) suitable for measuring the pressure of the mobile phase upstream of said first inlet (13) and a second sensor (30) suitable for measuring the pressure of the mobile phase downstream of said first outlet (18), and in that said control module (25) is arranged to control said supply means and said valve (31) as a function of a first comparison between the pressure measured upstream of the chamber and a first limiting pressure and of a second comparison between the pressure measured in downstream of the chamber and a second limit pressure.

19. Method for processing a sample by separation under forced flow, characterized in that it comprises the following steps:

a) placing in a chamber at least one stationary phase suitable for receiving at least one first chosen location at least one sample to be treated,

b) supplying in mobile phase at least a second chosen location of the stationary phase, while applying an external pressure of selected intensity on an upper face of this stationary phase and by preventing said mobile phase from leaving the stationary phase,

c) measuring the pressure of the mobile phase upstream and / or downstream of the chamber,

d) compare this measured pressure with a limit pressure linked to the chosen external pressure, and, when the measured pressure becomes greater than or equal to said limit pressure, reduce the pressure of the mobile phase so that it remains less than or equal to this limit pressure.

20. The method of claim 19, characterized in that in step d), when the measured pressure becomes greater than or equal to the limit pressure, the supply of the mobile phase is permanently interrupted, then the mobile phase introduced is placed in the stationary phase at ambient pressure, and the external pressure is released.

21. The method as claimed in claim 19, characterized in that in step d), when the measured pressure becomes greater than or equal to the limit pressure, the mobile phase is allowed to exit the chamber at a chosen rate, not zero, while continuing to feed said stationary phase in mobile phase.

22. Method according to claim 19, characterized in that in step d) the mobile phase supply is carried out by pressurization and / or by an electric field.

23. Method according to one of claims 19 to 22, characterized in that in step b) the external pressure is applied to the stationary phase by hydraulic means.

24. Method according to one of claims 19 to 23, characterized in that in step a) the stationary phase is previously provided with sample (s) to be treated.

25. Method according to one of claims 19 to 23, characterized in that in step a) the stationary phase is provided with sample to be treated after a substep in which the stationary phase is supplied with the mobile phase, while prohibiting its evacuation from the chamber, until said stationary phase has received a selected mobile phase volume.

26. The method of claim 25, characterized in that said volume is chosen so that it corresponds to a measured pressure substantially equal to said limit pressure.