DE202014101518U1 - Device for field flow fractionation - Google Patents

Abstract

Device for field flow fractionation, comprising one or more storage containers (1), a pump (2), a first flow rate distribution device (3), a first flow rate control device (5, 6, 7), an injector (8), a separation channel (10) with a first connection (9) at a first end (11) and a second connection (13) at a second end (12), a second flow volume distribution device (14), a first back pressure element (15), one or more detectors (19) ), a second flow control device (22) and one or more waste containers (23), wherein the first flow distribution device (3) is suitable, the volume flow generated by the pump in a first flow path (4), which connects the pump (2) with the first Connection (9) of the separation channel (10), and a second flow path (16), which connects the pump (2) to the second connection (13) of the separation channel (10), to divide the first flow rate control device (5, 6, 7) and the injector (8) are arranged in the first flow path (4), the second flow volume distribution device (14) is arranged within the second flow path (16) and is suitable for controlling the volume flow of the second flow path (16 ) between the second connection (13) of the separation channel (10) and the detector (s) (19), the first back pressure element (15) is arranged in a third flow path (18) which the second flow volume distribution device (14) and connects the detector (s) (19), the second flow control device (22) is arranged in a fourth flow path (21) which is connected to the separation channel (10) via a further connection (20) and the waste container ( 23) is / are arranged behind the detector (s) (19) and the second flow rate control device (22), characterized in that in the second flow path (16) between the first flow rate distribution device (3) and the second Flow rate distribution device (14) a second back pressure element (17) is arranged.

Description

Technical area

The invention relates to a device for Feldflussfraktionierung.

State of the art

Field Flow Fractionation (FFF) is a quasi-chromatographic analysis method for the separation of macromolecular and colloidal samples. In contrast to chromatography, sample separation during field-flow fractionation does not take place in a packed column but in an open separation channel which does not contain a stationary phase.

Due to the low channel height, a laminar flow with a parabolic flow profile is formed within the channel. The separation of the samples is performed by a force field applied within the channel perpendicular to this flow. Under the influence of the applied force field and the counteracting diffusion of the sample particles, the separation of the individual sample components takes place. First, an equilibrium state arises, in which the individual sample constituents are present in separate layers with a specific layer thickness within the separation channel. Smaller particles with a larger diffusion coefficient get higher, d. H. further away from the channel wall, located layers and thus in areas of greater flow velocities. These elute first. Larger particles with a lower diffusion coefficient move in lower, d. H. closer to the channel wall, located layers and thus in areas with a lower flow velocity and elute thereby later.

Depending on the type of generation of force field used, d. H. In the physical nature of this field, various field flux fractionation techniques have been developed. These include flux field-flow fractionation, sedimentation field-flow fractionation such as. As the centrifugal Feldflussfraktionierung, and the thermal Feldflussfraktionierung. Of these, flow field flux fractionation is most prevalent with the sub-techniques of symmetric flow field flow fractionation, asymmetric flow field flow fractionation (AF4), and hollow fiber flow field flow fractionation (HF5).

The flows or volumetric flows required in flow field-fraction fractionation apparatuses are produced by conventional HPLC pumps. In this case, systems with at least two pumps or even just a pump, such. In WO 2012/038518 described, used. Here come in the in WO 2012/038518 described systems with only one pump additionally suitable switching means within the individual flow paths used.

For high-resolution AF4 and HF5 separations, a "focus", ie. H. the concentration required the sample to form a narrow band in the separation channel. For this purpose, during the focusing carrier liquid, d. H. Running agent, pumped through both the inlet and through the outlet of the separation channel into this, wherein the focusing of the sample takes place in the region of the colliding solvent flows within the channel. After completion of the focusing, the sample is then eluted from the separation channel and can be detected.

If the focus flow, d. H. the flow for focusing, to be generated by means of a single pump, the pump flow must be split, d. H. be split. Conventional flow distribution devices always work with the distribution of a given volume flow between the respective flow paths. The flow is z. B. divided between a first flow path and a second flow path. However, depending on the desired separation method, it may be necessary to keep the flow constant over a first flow path while simultaneously changing the flow rate in a second flow path so that the total volume of flow changes during a measurement.

To realize this, suitable mass flow controllers can be used. These usually have a measuring unit for measuring the flow, d. H. the volume flow, and a control valve that is opened or closed depending on a reference / actual value comparison until the desired volume flow is applied.

The main problem of the mass flow controllers is the limited dynamic range for the flow rates and the pressure range in which the mass flow controllers can be used. Usually, the control valves used require a pressure difference between inlet and outlet of at least 3-10 bar, so that they can work correctly. In addition, a corresponding valve nozzle must be preselected so that the desired flow rates can be set precisely for a given valve lift.

Therefore, the simple control valves used in the past have been exchanged for external needle valves (see, for example, the in WO 2012/038518 described devices) and only the measuring unit of the respective mass flow controller used. However, these needle valves require additional electronic components and a motor for the control, which is a corresponding Extra effort means. This additional effort causes higher costs. In addition, the use of needle valves increases the risk of failure because needle valves, once fully closed, will easily get caught in the valve seat. Furthermore, needle valves tend to be soiled that eventually the desired flow rates can not be accurately adjusted.

It is an object of the present invention to provide an improved flow field-flow fractionation apparatus characterized by a simplified structure that does not require the use of needle valves for flow control.

Presentation of the invention

This object is achieved by the devices of the present invention. The devices according to the invention are based on a novel flow scheme with only one pump, which allows the use of commercial mass flow controllers with simple control valves instead of needle valves. In particular, the new flow scheme solves the problems previously described for the use of mass flow controllers with respect to the dynamic flow rate range and the required working pressure. This enables the realization of a programmable focus flux with simultaneous reduction of components, which reduces the costs and the risk of failure accordingly.

Further, in a preferred embodiment, the devices of the present invention do not require switching valves within the individual flow paths, which also results in simplification of construction and cost reduction.

The devices according to the invention are devices for Feldflussfraktionierung.

• – die erste Durchflussmengenaufteilungseinrichtung geeignet ist, den von der Pumpe erzeugten Volumenstrom in einen ersten Flusspfad, der die Pumpe mit dem ersten Anschluss des Trennkanals verbindet, und einen zweiten Flusspfad, der die Pumpe mit dem zweiten Anschluss des Trennkanals verbindet, aufzuteilen,
• – die erste Durchflussmengensteuerungseinrichtung und der Injektor in dem ersten Flusspfad angeordnet sind,
• – die zweite Durchflussmengenaufteilungseinrichtung innerhalb des zweiten Flusspfads angeordnet ist und geeignet ist, den Volumenstrom des zweiten Flusspfads zwischen dem zweiten Anschluss des Trennkanals und dem/den Detektor(en) aufzuteilen,
• – das erste Rückdruckelement in einem dritten Flusspfad angeordnet ist, der die zweite Durchflussmengenaufteilungseinrichtung und den/die Detektor(en) verbindet,
• – die zweite Durchflussmengensteuerungseinrichtung in einem vierten Flusspfad angeordnet ist, der über einen weiteren Anschluss mit dem Trennkanal verbunden ist und
• – der/die Abfallbehälter hinter dem/den Detektor(en) und der zweiten Durchflussmengensteuerungseinrichtung angeordnet ist/sind,

und sind dadurch gekennzeichnet, dass im zweiten Flusspfad zwischen der ersten Durchflussmengenaufteilungseinrichtung und der zweiten Durchflussmengenaufteilungseinrichtung ein zweites Rückdruckelement angeordnet ist.In a first embodiment, the field flow fractionation apparatuses of the present invention comprise one or more reservoirs, a pump, a first flow rate divider, a first flow rate controller, an injector, a separation channel having a first port at a first end, and a second port at a second end, a second one Flow rate distribution device, a first back pressure element, one or more detector (s), a second flow rate control device and one or more waste container, wherein

• The first flow rate divider is adapted to divide the volume flow generated by the pump into a first flow path connecting the pump to the first port of the separation channel and a second flow path connecting the pump to the second port of the separation channel;
• The first flow rate control device and the injector are arranged in the first flow path,
• The second flow rate distribution device is arranged within the second flow path and is suitable for dividing the volume flow of the second flow path between the second connection of the separation channel and the detector (s),
• The first back pressure element is arranged in a third flow path connecting the second flow rate distribution device and the detector (s),
• The second flow rate control device is arranged in a fourth flow path, which is connected via a further connection to the separation channel and
• The waste container (s) is / are arranged behind the detector (s) and the second flow rate control device,

and are characterized in that a second back pressure element is arranged in the second flow path between the first flow rate distribution device and the second flow rate distribution device.

• – die erste Durchflussmengenaufteilungseinrichtung geeignet ist, den von der Pumpe erzeugten Volumenstrom in einen ersten Flusspfad, der die Pumpe mit dem ersten Anschluss des Trennkanals verbindet, und einen zweiten Flusspfad, der die Pumpe mit dem dritten Anschluss des Trennkanals verbindet, aufzuteilen,
• – die erste Durchflussmengensteuerungseinrichtung und der Injektor in dem ersten Flusspfad angeordnet sind,
• – das erste Rückdruckelement in einem dritten Flusspfad angeordnet ist, der den zweiten Anschluss des Trennkanals mit dem/den Detektor(en) verbindet,
• – die zweite Durchflussmengensteuerungseinrichtung in einem vierten Flusspfad angeordnet ist, der über einen weiteren Anschluss mit dem Trennkanal verbunden ist, und
• – der/die Abfallbehälter hinter dem/den Detektor(en) und der zweiten Durchflussmengensteuerungseinrichtung angeordnet ist/sind,

und sind dadurch gekennzeichnet, dass im zweiten Flusspfad zwischen der ersten Durchflussmengenaufteilungseinrichtung und dem dritten Anschluss des Trennkanals ein zweites Rückdruckelement angeordnet ist.In an alternative embodiment, the field flow fractionation apparatus of the invention, one or more reservoirs, includes a pump, a first flow rate divider, a first flow rate controller, an injector, an Asymmetric Flow Field Flow Fractionation (AF4) divider having a first port at a first end, a second port at a second end and a third port between the first end and the second end of the separation channel, a first back pressure element, one or more detectors, a second flow rate control device, and one or more waste containers

• The first flow rate divider is adapted to divide the volume flow generated by the pump into a first flow path connecting the pump to the first port of the separation channel and a second flow path connecting the pump to the third port of the separation channel;
• The first flow rate control device and the injector are arranged in the first flow path,
• The first back pressure element is arranged in a third flow path which connects the second connection of the separation channel to the detector (s),
• - The second flow rate control means is arranged in a fourth flow path, which is connected via a further connection to the separation channel, and
• The waste container (s) is / are arranged behind the detector (s) and the second flow rate control device,

and are characterized in that in the second flow path between the first flow rate distribution device and the third port of the separation channel, a second back pressure element is arranged.

• (i) Injektion einer Probe unter Verwendung eines Laufmittels in den Trennkanal,
• (ii) Fokussierung der Probe mit Hilfe des Laufmittels in dem Trennkanal und
• (iii) Elution der Probe mit dem Laufmittel aus dem Trennkanal unter dem Einfluss eines Trennfeldes und die Detektion der aufgetrennten Probe mit einem oder mehreren Detektor(en).

The devices according to the invention are devices for analyzing a sample by means of field-flow fractionation. It will go through the following steps:

• (i) injecting a sample into the separation channel using an eluant;
• (ii) focusing the sample by means of the eluent in the separation channel and
• (iii) eluting the sample with the eluent from the separation channel under the influence of a separation field and detecting the separated sample with one or more detector (s).

In the context of the present invention, the term "injection" means the introduction of the sample by means of the injector of the devices according to the invention into the mobile phase and the flushing of the sample together with the mobile phase into the separating channel.

In the context of the present invention, the term "focusing" means concentrating the sample into a narrow band in the separation channel. During focussing, in the devices according to the invention according to the first embodiment described above, through the first port and the second port of the separation channel and in the inventive devices according to the alternative embodiment described above by the first port and the third port of the separation channel in the Separating channel pumped into it. The focusing of the sample takes place in the region of the colliding solvent streams within the separation channel, usually at the beginning of the separation channel.

In a preferred embodiment of the devices according to the invention, the flow rate in the first flow path during the introduction of the sample and its focusing is 0.01 ml / min to 0.5 ml / min.

In the context of the present invention, the term "elution" means the rinsing of the sample with the aid of the eluent from the separation channel in the direction of / the detector / detectors, wherein the second flow path is not flowed through and the rinsing takes place under the influence of a separation field whose Strength can be varied depending on the type of sample to be analyzed.

During elution, in devices according to the invention with a mass flow controller as the first flow rate control device in the first flow path, the control valve of the mass flow controller is opened to a maximum. This will ensure that only the pump will adjust the flow rate in the first flow path. At the same time, the pressure resulting from the pressure drop across the first flow path is less than the back pressure required to open the second back pressure element in the second flow path so that the second back pressure element remains closed and no eluent flows through the second flow path during elution.

The separation field present during the elution and the detection is a liquid flow (cross flow), which is generated by means of the second flow rate control device connected to the separation channel perpendicular to the flow direction of the eluent in the separation channel. With the aid of the second flow rate control device, it is additionally possible to vary the transverse flow and thus the strength of the separation field.

In the context of the present invention, the term "detection" means the detection of the sample in the mobile phase by the detector (s) used and the generation of a corresponding measurement signal by the sample, if this is detected by the detector (s). en).

In the context of the present invention, the term "volume flow" means the volume of an eluent per unit time by the inventive devices or parts thereof, such. The first and / or second flow rate control means, the separation channel, the first and / or second back pressure element or the detector (s) flows. Instead of the term "volume flow", the term "flow rate" is also used below.

The liquid and volumetric flows required for the injection, the focusing and the elution and detection in steps (i), (ii) and (iii) in the devices according to the invention according to the first embodiment are determined by means of the pump and the first and second flow rate distribution devices, the first and second flow rate control means and the first and second back pressure member generates and controls.

The liquid and volumetric flows required for injection, focusing and elution and detection in steps (i), (ii) and (iii) in the devices according to the invention according to the alternative embodiment are, by means of the pump and the first flow rate distribution device, the first and second flow rate control means and the first and second back pressure elements are generated and controlled.

In the context of the present invention, the term "analysis" means the separation and detection of a sample by field-flow fractionation by means of the devices according to the invention.

In the context of the present invention, the term "sample" means any type of analyte which can be separated and detected with the aid of the devices according to the invention. These may be substances with a molecular weight of 500 Da to 16 MDa or with a size of 2 nm to 5 μm, which are dissolved or suspended in the solvent used in each case.

The term "sample" in particular also includes mixtures, wherein the molecular weight and / or the size of the individual constituents may be the same or different.

Samples for separation and detection by means of the devices according to the invention are e.g. As proteins from the fields of the pharmaceutical industry and research, nanoparticles and carbon nanotubes (carbon nanotubes) and natural and synthetic polymers, in particular silicates, pigments, colloids, peptides, virus cells, liposomes, antibodies, polysaccharides and other macromolecules.

The devices of the invention may include one or more reservoirs for one or more carrier fluids, d. H. for one or more eluents. Measurements are preferably carried out with the devices according to the invention with an eluent, so that the devices according to the invention comprise a reservoir in a preferred embodiment. Alternatively, the operation with more than one solvent is possible, so that a gradient elution can be performed. In a further embodiment, the devices according to the invention therefore comprise two or more storage containers.

As the eluent for use in the devices of the present invention, aqueous and non-aqueous organic solvents can be used. Examples include aqueous solutions containing 0.5-5 g / l NaCl and / or 0.1-5 g / l sodium dodecyl sulfate (SDS) and the organic solvents tetrahydrofuran (THF), toluene, acetone, methanol, ethanol, Chloroform, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) and mixtures thereof.

As the pump, any pump suitable for use in field-flow fractionation or liquid chromatography (LC), such as an HPLC pump, may be used in the devices of this invention. Preferably, an HPLC pump is used. The pump is provided to promote the eluent in a defined constant flow rate.

The devices of the invention comprise only one, d. H. no further, pump to promote the eluent.

The first and second flow rate distribution devices are, for. B. a T-connector or a manifold.

As a T-connector or manifold for use in the devices of the present invention, it is possible to use all commercially available T-connectors and manifolds which are suitable for incorporation into a field-flow fractionation system and / or an HPLC system. The material used may be any material that is inert to the particular solvent used and the sample to be analyzed. For example, T-connectors or manifolds made of metal or PEEK can be used.

The bore within the T-connector or manifold used as the first flow-rate divider and the T-connector or manifold used as the second flow-rate divider may be the same size and have an inside diameter of 100 microns up to max. 1000 μm and preferably 500 μm.

In a preferred embodiment, the bore within the T-connector or manifold used as the first flow-rate divider has a larger inside diameter than the bore within the T-connector or manifold used as the second flow-rate divider , The inner diameter of the bore within the T-connector or manifold, which is used as the first flow rate divider, in this preferred embodiment is 500 μm to max. 1000 μm and preferably 1000 μm. The bore within the T-connector or manifold, which is used as the second flow rate divider, in this preferred embodiment has an inside diameter of 125 μm to max. 250 μm and preferably 250 μm. This ensures the smallest possible dead volume. Holes within the T-connector or manifold used as a second flow-rate divider, with an inside diameter greater than 250 microns, can lead to peak broadening and hence degradation of the measurement result.

In a preferred embodiment, the first flow rate divider and the second flow rate divider are each a T-connector.

As the first flow rate control device of the devices according to the invention, it is possible to use all commercially available mass flow controllers which are suitable for installation in a field-flow fractionation system and / or an HPLC system.

Mass flow controllers consist essentially of three components, a measuring unit, a control unit, z. B. a control valve, and the corresponding electronics (control line). The measuring unit can determine the volume flow rate in a flow path based on various physical principles (Coriolis force, heat transport, pressure difference, etc.) and transmit this measured value to the electronics. Depending on the physical measuring principle, this measured value is calculated with appropriate material constants in order to determine a volume flow per unit of time. Depending on the physical measuring principle, further parameters such as density or temperature are determined in the measuring unit and transmitted to the electronics. This means that the devices can also work independently of preset material constants, depending on the physical measuring principle. The electronics have an interface so that a desired volume flow can be transmitted to the electronics. The electronics then perform a setpoint / actual value comparison of the data obtained with the measuring unit and accordingly controls the control unit to set the desired flow rate. In general, the control unit consists of a magnetically moving punch and a nozzle / bore. If the measured value / actual value exceeds the nominal value, the punch is moved slightly more in the direction of the bore by means of the electronics, thus reducing the opening width of the flow path in which the mass flow controller is integrated. Thus, it increases flow resistance of the mass flow controller and the flow rate through the control unit decreases. Since the setpoint and actual value are constantly monitored by the electronics, the plunger can be aligned so that the desired volume flow is set via the mass flow controller.

A suitable for use in the inventive devices mass flow controller comprises a measuring unit and a control valve, which are connected to each other via a control line. As described above, the opening and closing of the control valve takes place via the control line as a function of a setpoint / actual value comparison of the data obtained with the measuring unit.

In a preferred embodiment, the first flow rate control device is a mass flow controller, comprising a measuring unit, a control valve and a control line.

The control valve may be installed upstream or downstream of the measuring unit. Preferably, the control valve is installed downstream of the measuring unit.

In a further embodiment, the devices according to the invention comprise a shut-off valve. The shut-off valve is arranged in the first flow path, so that the control valve of the mass flow controller can be bridged in order to remove it completely from the first flow path as required. As a result, during the elution via the first flow path, extremely high flow rates can be achieved, as required in preparative systems.

In a preferred embodiment of the devices according to the invention, the flow rate in the first flow path of the devices according to the invention with a mass flow controller, comprising a measuring unit, a control valve and a control line, as the first flow rate control device during the introduction of the sample and their focusing 0.01 ml / min to 0, 5 ml / min. After the introduction of the sample and its focusing, d. H. During elution, the mass flow controller's control valve in the first flow path is no longer needed for control and therefore open maximum. The mass flow controller must therefore work correctly only in the range of 0.01 ml / min to 0.5 ml / min. Within this range, the control by means of conventional mass flow controller is easily and reliably possible.

The second flow rate control device is used to generate and control the cross flow and is either a syringe pump system or a mass flow controller, as it is also used for the first flow rate control device.

A syringe pump system suitable for use in the devices of the invention comprises two syringes operating in pendulum mode. This will prevent uncontrolled outflow of the crossflow from the separation channel

In a preferred embodiment, the second flow rate control means is a syringe pump system comprising two pendulum-type syringes or a mass flow controller comprising a measuring unit, a control valve and a control line.

In a particularly preferred embodiment, a syringe pump system comprising two pendulum-type syringes is used as the second flow rate control means.

An injector for use in the devices of the invention may be a manually operated injection valve or an autosampler upstream of the separation channel, i. H. upstream is installed.

In a preferred embodiment, the injector is an autosampler.

In a further preferred embodiment, the injector is arranged downstream of the first flow rate control device in the first flow path.

The separation channel of the devices according to the invention has a first port (inlet) at a first end and a second port (outlet) at a second end. The separation channel also has a further connection, via which the separation channel is connected to the second flow rate control device and which serves to dissipate the liquid flow arising during the application of the crossflow. The separation channel of the devices according to the invention is either a hollow fiber separation channel or a separation channel for asymmetric flow field flow fractionation (AF-4).

A hollow fiber separation channel for use in the devices of the invention comprises one or more hollow fibers and a housing.

The hollow fibers for use in the devices according to the invention are size exclusion membranes with a round cross-section. These have an inside diameter (ID) of 0.5 to 1 mm. In a preferred embodiment, the inner diameter of the hollow fibers used is 1 mm. The hollow fibers generally have a length of 100 to 370 mm, preferably from 300 to 320 mm and particularly preferably 305 mm. The cut-off of the hollow fibers is from 300 Da to 100 kDa, preferably from 300 Da to 30 kDa and particularly preferably 10 kDa. Regenerated cellulose, polyethersulfone (PES) or polyvinylidene fluoride (PVDF) is preferably used as material for the hollow fibers.

The hollow fibers for use in the separation channel of the devices according to the invention are contained in an encapsulated housing (hollow fiber housing). This has in each case a connection for the inlet and the outlet of the eluent and a further connection for the second flow rate control device.

A hollow fiber and the housing in which the hollow fiber is located form a hollow fiber separation channel.

In addition to one can also two, three, four, five, six or more, such as. B. up to ten, up to twenty, up to one hundred or up to five hundred, parallel connected the same hollow fibers or hollow fiber separation channels are used. These are combined to form a separation module.

The parallel connection is made either by introducing the desired number of hollow fibers in a single housing, so that a corresponding hollow fiber separation channel has more than one hollow fiber, or by the use of multiple hollow fiber separation channels, each containing only one hollow fiber.

The use of several parallel connected hollow fibers or hollow fiber separation channels allows the injection of larger amounts of sample or a higher sample loading of the device according to the invention.

Preferably, only one or two identical hollow fibers (n) connected in parallel or only one hollow fiber separation channel or two identical hollow fiber separation channels connected in parallel are used. Particular preference is given to using two identical hollow fibers or hollow fiber separation channels connected in parallel.

In a further particularly preferred embodiment, a hollow fiber separation channel with two parallel connected, contained in a housing same hollow fibers is used.

As the AF-4 separation channel for use in the devices according to the invention, it is possible to use conventional AF-4 separation channels comprising a support, a frit, a semipermeable membrane, a spacer and a cover. In a further embodiment, the frit and the semipermeable membrane may also be formed in one piece. Corresponding separation channels are for example in EP 1 879 025 A1 described.

In semipermeable membranes for use in an AF4 separation channel the Devices according to the invention are size exclusion membranes. The cut-off of the semipermeable membranes is 300 Da to 100 kDa, preferably 300 Da to 30 kDa and particularly preferably 10 kDa. Regenerated cellulose, polyethersulfone (PES) or polyvinylidene fluoride (PVDF) is preferably used as material for the semipermeable membranes.

An AF-4 separation channel for use in the devices of the present invention may have an additional (third) port between the first end and the second end of the separation channel besides the first port (inlet), the second port (outlet) and the further port for the second flow rate control device exhibit. During the focusing of the sample, eluent is pumped into the separation channel via this third connection. An AF-4 separation channel with a third connection is used in the previously described alternative embodiment of the devices according to the invention.

In a preferred embodiment, the separation channel is a hollow fiber separation channel.

In a further particularly preferred embodiment, the hollow fiber separation channel may comprise a hollow fiber separation channel or a plurality of hollow fiber separation channels, each comprising a hollow fiber housing and one or more hollow fiber (s).

In a further preferred embodiment, the separation channel is a separation channel for asymmetric flow field-flow fractionation (AF4).

In a further embodiment, the devices according to the invention may comprise both a hollow fiber separation channel and an AF-4 separation channel. The two separation channels are arranged side by side in this case, d. H. connected in parallel, so that, as needed, one or the other channel is flowed through during a measurement with eluent.

The first and second back pressure elements used in the devices of the present invention are either a back pressure capillary or a back pressure regulator.

In a particularly preferred embodiment, the first back pressure element is a back pressure capillary and the second back pressure element is a back pressure regulator.

The inner diameter and the length of a back pressure capillary for use in the devices according to the invention must be selected so that the resulting back pressure through the capillary is always greater than the pressure drop across the separation channel.

The dimensions of the back pressure capillary are also dependent on the particular separation method used and the corresponding selected flows during the individual phases of the fractionation and therefore variable. In particular, the dimensions of the back pressure capillary are dependent on the respective set and constant volume flow through the / the detector (s).

Typical or preferred values are a length of 100 to 500 mm and an inner diameter (ID) of 50 to 200 μm. Particularly preferred is an inner diameter of 125 microns. As the material for the Rückdruckkapillare any material can be used which is inert to the particular solvent used and the sample to be analyzed. Preferred materials are polyetheretherketone (PEEK), fluorinated ethylene-propylene copolymer (FEP), perfluoroalkoxy (PFA), high-purity perfluoroalkoxy (HPFA), and stainless steel (SS).

As a back pressure regulator for use in the devices of the invention, all commercially available back pressure regulator can be used, which are suitable for installation in a Feldflussfraktionierungssystem and / or a HPLC system. A suitable back pressure regulator has a plunger which is pressed by a spring against the flow direction into a sealing seat.

The volume flow generated by the pump creates a fluid pressure. This presses on the stamp. If the force acting on the punch is less than the counterforce preset by the spring, the punch remains in its seat and the back pressure regulator remains closed. If the fluid pressure increases correspondingly, the force acting on the piston increases until the spring force is overcome. As a result, the punch moves out of its seat and the liquid path is opened. Depending on the volume flow, the punch is pressed more or less far out of its seat, so that the opening width varies depending on the force generated by the volume flow and acting on the punch.

A back pressure regulator used as a second back pressure element in the second fluid path of the devices according to the invention is characterized in that it automatically closes or remains closed when the opening pressure is not reached. If, therefore, no eluent is to flow via the second flow path, the control valve of the mass flow controller in the first flow path is opened until the pressure drop across the first flow path resulting pressure is less than the pressure required for the opening of the back pressure regulator.

But with a back pressure capillary as the second back pressure element, this would not be possible. Instead, a switching valve would additionally have to block the second flow path, since a back pressure capillary, in contrast to a back pressure regulator, would always permit a flow, albeit a small one.

If solvent is to be passed via the second flow path, the control valve of the mass flow controller is closed until the resulting from the pressure drop across the first flow path pressure is greater than the pressure required for the opening of the back pressure regulator so that it opens. The mass flow controller then regulates only the pressure drop in the first flow path and the volume flows divide accordingly between the first and the second flow path.

Any detectors known in the field of field-flow fractionation or high-performance liquid chromatography (HPLC) can be used as the detector (s) in the devices according to the invention. Examples are UV detectors, refractive index (RI) detectors, (multi-angle) light scattering detectors, mass spectrometers, fluorescence detectors, ICP mass spectrometers, dynamic light scattering detectors (DLS) and small angle X-ray scattering (SAXS) detectors.

In a preferred embodiment, the detector (s) are a UV detector, a refractive index (RI) detector and / or a (multi-angle) light scattering detector.

In the first embodiment of the devices according to the invention, the first flow rate divider is suitable for connecting the volume flow generated by the pump to a first flow path connecting the pump to the first port of the separation channel and a second flow path connecting the pump to the second port of the separation channel to split up. The first flow rate control device and the injector are arranged in the first flow path. The second flow rate divider is disposed within the second flow path and is adapted to divide the volume flow of the second flow path between the second port of the separation channel and the detector (s). The first back pressure element is disposed in a third flow path connecting the second flow rate distribution device and the detector (s). The second flow rate control device is arranged in a fourth flow path, which is connected via a further connection to the separation channel. The waste bin (s) is located behind the detector (s) and the second flow rate controller. In addition, a second back pressure element is arranged in the second flow path between the first flow rate distribution device and the second flow rate distribution device.

In the alternative embodiment of the devices according to the invention, the first flow rate divider is adapted to connect the volume flow generated by the pump into a first flow path connecting the pump to the first port of the AF4 separation channel and a second flow path connecting the pump to the third port of the AF4 separation channel AF4 separation channel connects, split. The first flow rate control device and the injector are arranged in the first flow path. The first back pressure element is disposed in a third flow path connecting the second port of the separation channel to the detector (s). The second flow rate control device is arranged in a fourth flow path, which is connected via a further connection to the AF4 separation channel. The waste bin (s) is located behind the detector (s) and the second flow rate controller. In addition, a second back pressure element is arranged in the second flow path between the first flow rate distribution device and the third port of the AF4 separation channel.

The described flow diagram of the devices according to the invention is achieved by the installation of the second back pressure element in the second flow path. This makes it possible to use commercially available mass flow controllers instead of needle valves for flow rate control in the first flow path. In particular, this solves the problems known from the prior art with respect to the dynamic flow rate range and the required working pressure of mass flow controllers. As a result, this enables the realization of a programmable focus flux with simultaneous reduction of components, which reduces the costs and the risk of failure.

In the individual flow paths in addition to the other components of the devices according to the invention arranged switching means such. As switching valves, are not required for the realization of the flow scheme of the devices according to the invention. This also contributes to a simplification of the structure and to the cost reduction. In a preferred embodiment, the devices according to the invention therefore comprise no switching means in the first, second, third and fourth flow paths, and in particular no switching valves.

In a first particularly preferred embodiment, the devices according to the invention comprise one or more storage containers, one A pump, a first flow rate distribution device, a mass flow controller, comprising a measuring unit, a control valve and a control line, as a first flow rate control device, an autosampler as an injector, a hollow fiber or AF4 separation channel having a first terminal at a first end and a second terminal at a second end, a second flow rate divider, a back pressure capillary as a first back pressure element, a back pressure regulator as a second back pressure element, a UV detector, a refractive index (RI) detector and / or a (multi-angle) light scatter detector as the detector (s), a syringe pump system two reciprocating syringes as a second flow rate control device and one or more waste containers.

The inventive devices of this particularly preferred embodiment may additionally comprise a shut-off valve, by means of which the control valve of the mass flow controller can be bridged.

The devices according to the invention according to the described particularly preferred embodiment are characterized by the following flow scheme:
The first flow rate divider is adapted to connect the volume flow generated by the pump into a first flow path connecting the pump to the first port of the hollow fiber or AF4 separation channel and a second flow path connecting the pump to the second port of the hollow fiber or AF4 Separating channel connects, split. The mass flow controller and the autosampler are located in the first flow path with the autosampler located downstream of the mass flow controller. The second flow rate divider is disposed within the second flow path and is adapted to divide the volume flow of the second flow path between the second port of the hollow fiber or AF4 separation channel and the detector (s). The back pressure capillary is disposed in a third flow path that connects the second flow rate divider and the UV detector, refractive index (RI) detector, and / or (multi-angle) light scattering detector used as the detector (s). The syringe pump system is arranged in a fourth flow path which is connected via a further connection to the hollow fiber or AF4 separation channel. The waste bin (s) are located behind the detector (s) and the syringe pump system. The back pressure regulator is arranged in the second flow path between the first flow rate distribution device and the second flow rate distribution device. An optional shut-off valve is arranged in the first flow path, so that the control valve of the mass flow controller can be bridged in order to remove it completely from the first flow path as required.

In an alternative particularly preferred embodiment, the devices of the invention comprise one or more reservoirs, a pump, a first flow rate divider, a mass flow controller comprising a measuring unit, a control valve and a control line, as a first flow rate controller, an autosampler as an injector, an AF4 separating channel first port at a first end, a second port at a second end and a third port between the first end and the second end of the separation channel, a back pressure capillary as a first back pressure element, a back pressure regulator as a second back pressure element, a UV detector, a refractive index ( RI detector and / or a (multi-angle) light scattering detector as a detector (s), a syringe pump system with two oscillating spraying as a second flow rate control device and one or more waste containers.

The devices according to the invention of this alternative particularly preferred embodiment may additionally comprise a shut-off valve, by means of which the control valve of the mass flow controller can be bridged.

The devices according to the invention according to the described alternative particularly preferred embodiment are characterized by the following flow scheme:
The first flow rate divider is adapted to divide the volume flow generated by the pump into a first flow path connecting the pump to the first port of the AF4 separation channel and a second flow path connecting the pump to the third port of the AF4 separation channel. The mass flow controller and the autosampler are located in the first flow path with the autosampler located downstream of the mass flow controller. The back pressure capillary is located in a third flow path connecting the second port of the AF4 separation channel and the UV detector, refractive index (RI) detector and / or (multi-angle) light scattering detector used as the detector (s). The syringe pump system is arranged in a fourth flow path, which is connected via a further connection to the AF4 separation channel. The waste bin (s) are located behind the detector (s) and the syringe pump system. The back pressure regulator is arranged in the second flow path between the first flow rate distribution device and the third port of the AF4 separation channel. An optional shut-off valve is so in arranged first flow path, that so that the control valve of the mass flow controller can be bridged to completely remove this if necessary from the first flow path.

The devices according to the invention are devices for analyzing a sample by means of field-flow fractionation.

• – in Schritt (i) eine Probe in ein Laufmittel eingebracht,
• – in Schritt (ii) Laufmittel, in das die Probe eingebracht wurde, mit einem konstanten Volumenstrom über den ersten Anschluss (Einlass) des Trennkanals in diesen hineingespült und ebenfalls mit einem konstanten Volumenstrom zusätzliches Laufmittel durch die zweite Durchflussmengenaufteilungseinrichtung über den zweiten Anschluss (Auslass) des Trennkanals in diesen hineingepumpt, wobei das Laufmittel mit Hilfe des ersten Rückdruckelements und der zweiten Durchflussmengenaufteilungseinrichtung in einen ersten Teil, der in Richtung des Auslasses des Trennkanals strömt, und einen zweiten Teil, der in Richtung des/der Detektors/Detektoren strömt, aufgeteilt wird und
• – in Schritt (iii) zunächst der Volumenstrom über die zweite Durchflussmengenaufteilungseinrichtung in den Auslass des Trennkanals bis auf null reduziert und zeitgleich der Volumenstrom in den Einlass des Trennkanals um den entsprechenden Betrag erhöht und die Probe anschließend unter dem Einfluss eines Trennfeldes über die zweite Durchflussmengenaufteilungseinrichtung und das erste Rückdruckelement aus dem Trennkanal eluiert und in einem oder mehreren Detektor(en) detektiert, wobei der Volumenstrom und die Flussrichtung des Laufmittels durch den/die Detektor(en) gleich bleiben und wobei die Stärke des Trennfeldes mit Hilfe der zweiten Durchflussmengensteuerungseinrichtung, die über einen weiteren Anschluss mit dem Trennkanal verbunden ist, kontrolliert wird und gleichzeitig der Volumenstrom so variiert wird, dass bei einer Veränderung der Stärke des Trennfeldes der Volumenstrom durch den/die Detektor(en) gleich bleibt.

In the steps taken thereby, in the devices according to the invention according to the described particularly preferred embodiment:

• In step (i) a sample is introduced into a mobile phase,
• - in step (ii) eluent, in which the sample was introduced, with a constant volume flow through the first port (inlet) of the separation channel rinsed therein and also with a constant volume flow additional eluent through the second flow rate distribution device via the second port (outlet) the separation channel is pumped into the latter, wherein the eluent is divided by means of the first back pressure element and the second flow rate divider into a first part which flows in the direction of the outlet of the separation channel and a second part which flows in the direction of the detector / detectors and
• - In step (iii), first the volume flow through the second flow rate divider into the outlet of the separation channel is reduced to zero and at the same time the volume flow in the inlet of the separation channel increased by the corresponding amount and the sample then under the influence of a separation field on the second flow rate distribution device and the first back pressure element elutes from the separation channel and detected in one or more detector (s), wherein the volume flow and the flow direction of the eluent by the / the detector (s) remain the same and wherein the strength of the separation field by means of the second flow rate control means, the a further connection is connected to the separation channel, is controlled and at the same time the volume flow is varied so that when changing the strength of the separation field, the volume flow through the / the detector (s) remains the same.

• – in Schritt (i) eine Probe in ein Laufmittel eingebracht,
• – in Schritt (ii) Laufmittel, in das die Probe eingebracht wurde, mit einem konstanten Volumenstrom über den ersten Anschluss (Einlass) des AF4-Trennkanals in diesen hineingespült und ebenfalls mit einem konstanten Volumenstrom zusätzliches Laufmittel über den dritten Anschluss des AF4-Trennkanals in diesen hineingepumpt, wobei das Laufmittel innerhalb des AF4-Trennkanals mit Hilfe des ersten Rückdruckelements in einen ersten Teil, der in Richtung des Einlasses des AF4-Trennkanals strömt, und einen zweiten Teil, der aus dem zweiten Anschluss (Auslass) des AF4-Trennkanals in Richtung des/der Detektors/Detektoren strömt, aufgeteilt wird und
• – in Schritt (iii) zunächst der Volumenstrom durch den dritten Anschluss des AF4-Trennkanals bis auf null reduziert und zeitgleich der Volumenstrom in den Einlass des AF4-Trennkanals um den entsprechenden Betrag erhöht und die Probe anschließend unter dem Einfluss eines Trennfeldes über das erste Rückdruckelement aus dem AF4-Trennkanal eluiert und in dem/den Detektor(en) detektiert, wobei der Volumenstrom und die Flussrichtung des Laufmittels durch den/die Detektor(en) gleich bleiben und wobei die Stärke des Trennfeldes mit Hilfe der zweiten Durchflussmengensteuerungseinrichtung, die über einen weiteren Anschluss mit dem AF4-Trennkanal verbunden ist, kontrolliert wird und gleichzeitig der Volumenstrom so variiert wird, dass bei einer Veränderung der Stärke des Trennfeldes der Volumenstrom durch den/die Detektor(en) gleich bleibt.

In the devices according to the invention according to the described alternative particularly preferred embodiment:

• In step (i) a sample is introduced into a mobile phase,
• - In step (ii) Laufmittel, in which the sample was introduced, with a constant flow through the first port (inlet) of the AF4 separation channel rinsed therein and also with a constant volume flow additional eluent through the third port of the AF4 separation channel in this pumped in, wherein the eluent within the AF4 separation channel by means of the first back pressure element in a first part, which flows in the direction of the inlet of the AF4 separation channel, and a second part, from the second port (outlet) of the AF4 separation channel in Direction of the detector / detectors flows, is divided and
• - In step (iii), first the volume flow through the third port of the AF4 separation channel is reduced to zero and at the same time the volume flow in the inlet of the AF4 separation channel increased by the corresponding amount and the sample then under the influence of a separation field on the first back pressure element eluted from the AF4 separation channel and detected in the detector (s), wherein the volumetric flow and the flow direction of the eluent by the / the detector (s) remain the same and wherein the strength of the separation field by means of the second flow rate control device via a is connected to the AF4 separation channel is controlled and at the same time the volume flow is varied so that when changing the strength of the separation field, the volume flow through the / the detector (s) remains the same.

In a particularly preferred embodiment of the devices according to the invention, in step (i) and step (ii), d. H. during the introduction of the sample and its focusing, the flow rate in the first flow path of the devices according to the invention with a mass flow controller, comprising a measuring unit, a control valve and a control line, as the first flow rate control device 0.01 ml / min to 0.5 ml / min. After the introduction of the sample and its focusing, d. H. during the elution in step (iii), the control valve of the mass flow controller in the first flow path of the devices according to the invention with a mass flow controller as the first flow rate control device is no longer needed to control the flow rates and therefore opened maximum.

In the context of the present invention, the terms "and" or "remain the same" in relation to a volume flow mean that this volume flow may not vary by more than ± 2%, preferably not more than ± 1.2% , For example, a flow rate through the detector (s) of 1 ml / min should not fluctuate more than 12 μl / min.

Brief description of the illustrations

1 shows the schematic structure of a device according to the invention according to the first preferred embodiment.

2 shows the schematic structure of a device according to the invention according to the alternative preferred embodiment.

3 shows the schematic structure of a device according to the invention according to the first preferred embodiment, which additionally comprises a shut-off valve.

Ways to carry out the invention

Preferred embodiments of the devices according to the invention are explained below with reference to the accompanying drawings and the reference numerals used therein.

The 1 shows the schematic structure of a device for Feldflussfraktionierung according to the invention. After a storage container ( 1 ) and a pump ( 2 ) of the pump ( 2 ) via a T-connector ( 3 ) into a first flow path ( 4 ) and a second flow path ( 16 ) divided up. The first river path ( 4 ) leads first to a mass flow controller, comprising a measuring unit ( 5 ), a control valve ( 6 ) and a control line ( 7 ). Via the control line ( 7 ) the control valve ( 6 ) according to a setpoint / actual value comparison with the measuring unit ( 5 ) controlled data. The first flow path then leads via an injector ( 8th ) to a first connection (inlet) ( 9 ) at a first end ( 11 ) of the separation channel ( 10 ). The separation channel may be either a hollow fiber separation channel or an AF4 separation channel.

The second flow path ( 16 ) leads to a second T-connector ( 14 ). This divides the volume flow between a second connection (outlet) ( 13 ) at a second end ( 12 ) of the separation channel ( 10 ) and a third flow path ( 18 ), which via a first back pressure element ( 15 ) to the detector (s) ( 19 ) leads.

The first back pressure element ( 15 ) is preferably a back pressure capillary, which generates a corresponding back pressure on the system depending on the respective applied flow rate. The back pressure is required to separate the separation channel ( 10 ) and thus the second flow rate control device ( 22 ) via a fourth flow path ( 21 ) and another connection ( 20 ) with the separation channel ( 10 ) is pressurized so that it can work. The second flow rate control device ( 22 ) controls the flow through the size exclusion membrane of the separation channel ( 10 ), ie the separation field / crossflow, and thus affects the separation efficiency of the field flow fractionation device.

In the second flow path ( 16 ) is a second back pressure element ( 17 ) integrated. This second back pressure element ( 17 ) is preferably a back pressure regulator having a punch which is pressed by a spring against the flow direction in a sealing seat.

The eluent is removed from the second flow rate control device ( 22 ) and the detector (s) ( 19 ) z. B. in a waste container ( 23 ) derived from the device.

A sample separation by means of in 1 shown devices of the invention consists of three steps.

In the first step, the sample is introduced into the system via the injector ( 8th ) and the transport of the sample into the separation channel ( 10 ).

In the second step, the sample is focused at the beginning of the separation channel ( 10 ). During this step, eluant at a flow rate in the range of 0.01 ml / min to 0.5 ml / min along the first flow path (FIG. 4 ) through the injector ( 8th ) into the separation channel ( 10 ). At the same time a further correspondingly larger volume flow through the second flow path ( 16 ) via the second back pressure element ( 17 ). Due to the use of the first back pressure element ( 15 ) generated part of the eluent flows through the second T-connector ( 14 ) in the opposite direction through the outlet ( 13 ) into the separation channel ( 10 ), while another part of the eluent in the direction of the third flow path ( 18 ) flows. This will cause the sample at the beginning of the separation channel ( 10 ) focused. Meanwhile, the second flow rate control device ( 22 ) and is controlled so that the volume flow through the second flow rate control device ( 22 ) is slightly smaller than the volume flow from the pump ( 2 ) in the system. Thus, in the third flow path ( 18 ) a controlled volume flow in the direction of the detector / detectors ( 19 ). By means of the first back pressure element ( 15 ) results in the above-described back pressure in the separation channel ( 10 ) responsible for the work of the second flow rate control device ( 22 ) is required.

The third step is to use the injector ( 8th ) into the separation channel ( 10 ) introduced sample after focusing from the separation channel ( 10 ) elutes. For this purpose, the volume flow through the second flow path ( 16 ) being stopped. At the same time, the volume flow through the first flow path ( 4 ) by the amount of the previous volume flow through the second flow path ( 16 ) to increase a constant volume flow in the third flow path ( 18 ) and thus by the detector (s) ( 19 ) to ensure. During the elution then the whole of the pump ( 2 ) conveyed volume flow over the first flow path ( 4 ) into the separation channel ( 10 ) and the third flow path ( 18 ) and by the detector (s) ( 19 ). Within the separation channel ( 10 ) is at the same time a separation field (cross flow), by the second flow rate control device ( 22 ) is produced. In doing so, that of the pump ( 2 ) volume flow as a function of the strength of the cross-flow controlled so that the volume flow through the third flow path ( 18 ) and thus by the detector (s) ( 19 ) remains constant.

The flows described for the individual steps are regulated by appropriate software.

As described, mass flow controllers are limited in the dynamic flow rate range. In the novel flow scheme described with only one pump but this does not matter. During the introduction of the sample and its focusing, the flow rate in the first flow path is ( 4 ) with the mass flow controller 0.01 ml / min to 0.5 ml / min. After the introduction of the sample and its focusing, ie during the elution, the control valve ( 6 ) of the mass flow controller in the first flow path ( 4 ) no longer needed to control the flow rates and therefore simply open the maximum. The mass flow controller must therefore work correctly only in the range of 0.01 ml / min to 0.5 ml / min. Within this range, the control by means of conventional mass flow controller is easily and reliably possible.

The prerequisite for this is provided by the second back pressure element ( 17 ) in the second flow path ( 16 ) created. The second back pressure element ( 17 ) in the second fluid path ( 16 ) is adjusted so that when the control valve is active ( 6 ) of the mass flow controller in the first flow path ( 4 ) and at the same time the back pressure through the entire first flow path ( 4 ) is higher than the back pressure through the second flow path ( 16 ). This creates a critical condition for the use of a conventional mass flow controller. Because of this, the pressure at the inlet of the mass flow controller is significantly higher than the pressure in the separation channel ( 10 ), which is approximately at the pressure at the outlet of the control valve ( 6 ) corresponds. As a result, the pressure difference at the mass flow controller corresponds approximately to the pressure difference at the second back pressure element (FIG. 17 ). In principle, the pressure at the inlet of the mass flow controller should be 3-10 bar and preferably 6 bar higher than at the outlet of the mass flow controller. As a result, the mass flow controller is in the working range required for reliable control and is able to maintain the desired flow rate in the range of 0.01 to 0.5 ml / min.

If, after the sample has been introduced and focused, the control valve ( 6 ) of the mass flow controller open maximum, the back pressure through the first flow path ( 4 ) and falls below the threshold from which the second back pressure element ( 17 ) the second flow path ( 16 ) opens. Thus, the punch in the second back pressure element ( 17 ) and blocks the second flow path ( 16 ). This will get the whole of the pump ( 2 ) conveyed volume flow over the first flow path ( 4 ) into the separation channel ( 10 ). A regulation of the flow through the mass flow controller is now no longer necessary because the eluent only over the first flow path ( 4 ) enters the system and the flow rate through the pump ( 2 ) is determined.

The 2 shows the schematic structure of a device for Feldflussfraktionierung according to the alternative embodiment of the invention. After a storage container ( 1 ) and a pump ( 2 ) of the pump ( 2 ) via a T-connector ( 3 ) into a first flow path ( 4 ) and a second flow path ( 16 ) divided up. The first river path ( 4 ) leads first to a mass flow controller, comprising a measuring unit ( 5 ), a control valve ( 6 ) and a control line ( 7 ). Via the control line ( 7 ) the control valve ( 6 ) according to a setpoint / actual value comparison with the measuring unit ( 5 ) controlled data. The first flow path then leads via an injector ( 8th ) to a first connection (inlet) ( 9 ) at a first end ( 11 ) of the separation channel ( 10 ). The separation channel is an AF4 separation channel.

The second flow path ( 16 ) leads to a third connection ( 24 ) between the first end ( 11 ) and a second end ( 12 ) of the separation channel ( 10 ).

A third flow path ( 18 ) leads from a second port (outlet) ( 13 ) at the second end ( 12 ) of the separation channel ( 10 ) via a first back pressure element ( 15 ) to the detector (s) ( 19 ).

In the second flow path ( 16 ) is a second back pressure element ( 17 ) integrated. This second back pressure element ( 17 ) is preferably a back pressure regulator having a punch which is pressed by a spring against the flow direction in a sealing seat.

Regarding the operation and the effect of the first back pressure element ( 15 ) and in the second flow path ( 16 ) integrated second back pressure element ( 17 ) corresponds to the in 2 shown device of in 1 shown device. The corresponding explanations to 1 So meet for the 2 to.

The eluent is removed from the second flow rate control device ( 22 ) and the detector (s) ( 19 ) z. B. in a waste container ( 23 ) derived from the device.

A sample separation by means of in 2 The device according to the invention shown consists of three steps.

In the first step, the sample is introduced into the system via the injector ( 8th ) and the transport of the sample into the separation channel ( 10 ).

In the second step, the sample is focused at the beginning of the separation channel ( 10 ). During this step, eluant at a flow rate in the range of 0.01 ml / min to 0.5 ml / min along the first flow path (FIG. 4 ) through the injector ( 8th ) into the separation channel ( 10 ). At the same time, a further correspondingly larger volume flow through the second flow path ( 16 ) via the second back pressure element ( 17 ) and the eluent flows through the third port ( 24 ) into the separation channel ( 10 ). Due to the use of the first back pressure element ( 15 ), the solvent stream is thereby divided so that a portion of the eluent through the outlet ( 13 ) from the separation channel ( 10 ) in the direction of the third flow path ( 18 ) flows and another part of the eluent in the opposite direction through the separation channel ( 10 ) flows. This will cause the sample at the beginning of the separation channel ( 10 ) focused. Meanwhile, the second flow rate control device ( 22 ) and is controlled so that the volume flow through the second flow rate control device ( 22 ) is slightly smaller than the volume flow from the pump ( 2 ) in the system. Thus, in the third flow path ( 18 ) a controlled volume flow in the direction of the detector / detectors ( 19 ). Due to the first back pressure element ( 15 ) results in the above-described back pressure in the separation channel ( 10 ) responsible for the work of the second flow rate control device ( 22 ) is required.

The third step is to use the injector ( 8th ) into the separation channel ( 10 ) introduced sample after focusing from the separation channel ( 10 ) elutes. For this purpose, the volume flow through the second flow path ( 16 ) being stopped. At the same time, the volume flow through the first flow path ( 4 ) by the amount of the previous volume flow through the second flow path ( 16 ) to increase a constant volume flow in the third flow path ( 18 ) and thus by the detector (s) ( 19 ) to ensure. During the elution then the whole of the pump ( 2 ) conveyed volume flow over the first flow path ( 4 ) into the separation channel ( 10 ) and the third flow path ( 18 ) and by the detector (s) ( 19 ). Within the separation channel ( 10 ) is at the same time a separation field (cross flow), by the second flow rate control device ( 22 ) is produced. In doing so, that of the pump ( 2 ) volume flow as a function of the strength of the cross-flow controlled so that the volume flow through the third flow path ( 18 ) and thus by the detector (s) ( 19 ) remains constant.

The flows described for the individual steps are regulated by appropriate software.

Regarding the operation and the effect of the mass flow controller and its interaction with the second back pressure element ( 17 ) corresponds to the in 2 shown device of in 1 shown device. The corresponding explanations to 1 So meet for the 2 to.

3 shows the schematic structure of another device according to the invention. This is based on the in 1 shown device and additionally includes a shut-off valve ( 25 ).

The shut-off valve ( 25 ) is in the first flow path ( 4 ) arranged. When the shut-off valve is fully open ( 25 ) the control valve ( 6 ) bypasses the mass flow controller and in this way completely from the first flow path ( 4 ) away. As a result, extremely high flow rates, as required for preparative systems, can be achieved.

LIST OF REFERENCE NUMBERS

1

reservoir

2

pump

3

First flow rate distribution device

4

First river path

5

measuring unit

6

control valve

7

control line

8th

injector

9

First connection (inlet)

10

separation channel

11

First end

12

Second end

13

Second connection (outlet)

14

Second flow rate divider

15

First back pressure element

16

Second river path

17

Second back pressure element

18

Third river path

19

detector

20

Another connection

21

Fourth river path

22

Second flow rate control device

23

waste container

24

Third connection

25

shut-off valve

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2012/038518 [0005, 0005, 0010]
• EP 1879025 A1 [0065]

Claims (16)

Device for field-flow fractionation, comprising one or more storage containers ( 1 ), a pump ( 2 ), a first flow rate distribution device ( 3 ), a first flow rate control device ( 5 . 6 . 7 ), an injector ( 8th ), a separation channel ( 10 ) with a first connection ( 9 ) at a first end ( 11 ) and a second connection ( 13 ) at a second end ( 12 ), a second flow rate distribution device ( 14 ), a first back pressure element ( 15 ), one or more detectors (s) ( 19 ), a second flow rate control device ( 22 ) and one or more waste containers ( 23 ), wherein the first flow rate distribution device ( 3 ) is adapted, the volume flow generated by the pump in a first flow path ( 4 ), the pump ( 2 ) with the first connection ( 9 ) of the separation channel ( 10 ) and a second flow path ( 16 ), the pump ( 2 ) with the second connection ( 13 ) of the separation channel ( 10 ), splits the first flow rate control device ( 5 . 6 . 7 ) and the injector ( 8th ) in the first flow path ( 4 ), the second flow rate distribution device ( 14 ) within the second flow path ( 16 ) and is suitable for controlling the volume flow of the second flow path ( 16 ) between the second port ( 13 ) of the separation channel ( 10 ) and the detector (s) ( 19 ), the first back pressure element ( 15 ) in a third flow path ( 18 ) is arranged, the second flow rate distribution device ( 14 ) and the detector (s) ( 19 ), the second flow rate control device ( 22 ) in a fourth flow path ( 21 ), which is connected via a further connection ( 20 ) with the separation channel ( 10 ) and the waste bin (s) ( 23 ) behind the detector (s) ( 19 ) and the second flow rate control device ( 22 ) is / are arranged, characterized in that in the second flow path ( 16 ) between the first flow rate distribution device ( 3 ) and the second flow rate distribution device ( 14 ) a second back pressure element ( 17 ) is arranged. Device for field-flow fractionation, comprising one or more storage containers ( 1 ), a pump ( 2 ), a first flow rate distribution device ( 3 ), a first flow rate control device ( 5 . 6 . 7 ), an injector ( 8th ), a separation channel ( 10 ) for Asymmetric Flow Field Flow Fractionation (AF4) with a first port ( 9 ) at a first end ( 11 ), a second port ( 13 ) at a second end ( 12 ) and a third port ( 24 ) between the first end ( 11 ) and the second end ( 12 ) of the separation channel ( 10 ), a first back pressure element ( 15 ), one or more detectors (s) ( 19 ), a second flow rate control device ( 22 ) and one or more waste containers ( 23 ), wherein the first flow rate distribution device ( 3 ) is adapted, the volume flow generated by the pump in a first flow path ( 4 ), the pump ( 2 ) with the first connection ( 9 ) of the separation channel ( 10 ) and a second flow path ( 16 ), the pump ( 2 ) with the third connection ( 24 ) of the separation channel ( 10 ), splits the first flow rate control device ( 5 . 6 . 7 ) and the injector ( 8th ) in the first flow path ( 4 ), the first back pressure element ( 15 ) in a third flow path ( 18 ) is arranged, the second connection ( 13 ) of the separation channel ( 10 ) with the detector (s) ( 19 ), the second flow rate control device ( 22 ) in a fourth flow path ( 21 ), which is connected via a further connection ( 20 ) with the separation channel ( 10 ), and the waste bin (s) ( 23 ) behind the detector (s) ( 19 ) and the second flow rate control device ( 22 ) is / are arranged, characterized in that in the second flow path ( 16 ) between the first flow rate distribution device ( 3 ) and the third port ( 24 ) of the separation channel ( 10 ) a second back pressure element ( 17 ) is arranged. Apparatus according to claim 1 or claim 2, wherein in the first flow rate control means ( 5 . 6 . 7 ) around a mass flow controller, comprising a measuring unit ( 5 ), a control valve ( 6 ) and a control line ( 7 ), acts. Apparatus according to claim 3, wherein the flow rate in the first flow path ( 4 ) during introduction of the sample and its focusing is 0.01 ml / min to 0.5 ml / min. Device according to one of the preceding claims, wherein the device in the first, second, third and fourth flow path ( 4 . 16 . 18 . 21 ) includes no switching means. Device according to one of claims 1 and 3-5, wherein the separation channel is a hollow fiber separation channel or a separation channel for asymmetric flow field-flow fractionation (AF4). The device of claim 6, wherein the hollow fiber separation channel may comprise a hollow fiber separation channel or a plurality of hollow fiber separation channels, each comprising a hollow fiber housing and one or more hollow fiber (s). Device according to one of the preceding claims, wherein the injector ( 8th ) in the first flow path ( 4 ) downstream behind the first Flow rate control device ( 5 . 6 . 7 ) is arranged. Device according to one of the preceding claims, wherein in the second flow rate control device ( 22 ) is a syringe pump system comprising two pendulum-type syringes. Device according to one of the preceding claims, wherein the injector ( 8th ) is a manually operated injection valve or an autosampler. Device according to one of claims 1 and 3-10, wherein it is in the first flow rate distribution device ( 3 ) and the second flow rate distribution device ( 14 ) is in each case a T-connector. Device according to one of the preceding claims, wherein the first back pressure element ( 15 ) a back pressure capillary and the second back pressure element ( 17 ) is a back pressure regulator. Device according to one of the preceding claims, wherein the detector (s) ( 19 ) is a UV detector, a refractive index (RI) detector and / or a (multi-angle) light scattering detector. Apparatus according to claim 1, wherein the apparatus comprises a mass flow controller comprising a measuring unit ( 5 ), a control valve ( 6 ) and a control line ( 7 ), as the first flow rate control device ( 5 . 6 . 7 ), an autosampler as an injector ( 8th ), a hollow fiber or an AF4 separation channel as a separation channel ( 10 ), a back pressure capillary as the first back pressure element ( 15 ), a back pressure regulator as a second back pressure element ( 17 ), a UV detector, a refractive index (RI) detector and / or a (multi-angle) light scattering detector as detector (s) ( 19 ) and a syringe pump system with two pendulum-type syringes as a second flow rate control device ( 22 ). Apparatus according to claim 2, wherein the apparatus comprises a mass flow controller comprising a measuring unit ( 5 ), a control valve ( 6 ) and a control line ( 7 ), as the first flow rate control device ( 5 . 6 . 7 ), an autosampler as an injector ( 8th ), an AF4 separation channel as a separation channel ( 10 ), a back pressure capillary as the first back pressure element ( 15 ), a back pressure regulator as a second back pressure element ( 17 ), a UV detector, a refractive index (RI) detector and / or a (multi-angle) light scattering detector as detector (s) ( 19 ) and a syringe pump system with two pendulum-type syringes as a second flow rate control device ( 22 ). Device according to one of claims 3-4, 14 and 15, wherein the device additionally comprises a shut-off valve ( 25 ) for bridging the control valve ( 6 ) of the mass flow controller.

Images