Dead volume-free fraction collection apparatus

Abstract

An apparatus for collecting liquid fractions from a separation/reaction apparatus (1). A capillary (2) guides an extracted liquid fraction to a branching unit (3), a collection arrangement (4) carries a plurality of target vessels (5) receiving the liquid fraction from the capillary and a fluid line (6) is flow-connected to a fluid pump (7) and opens into the branching unit. The capillary and the fluid line each have outlet openings in the direct vicinity of one another at their end facing a target vessel such that liquid emerging from the outlet opening (2′) of the capillary transitions into the outlet opening (6′) of the fluid line. This precludes back mixing with earlier/later liquid fractions, precludes uncontrolled dripping of sample substance at the transfer point, speeds displacement of the target vessels in the collection arrangement, facilitates automation of the fraction processing procedure, and results in a more compact fraction collection apparatus.

Claims

1. A fraction collection apparatus for collecting sequential liquid fractions from a separation or reaction apparatus, comprising: a branching unit, a capillary arranged and configured to guide the liquid fractions from the separation or reaction apparatus to the branching unit, a collection arrangement carrying a plurality of target vessels configured and arranged to receive the liquid fractions from the capillary, and a fluid line flow-connected to a fluid pump, wherein the fluid line opens into the branching unit, wherein the capillary and the fluid line each have a respective outlet opening facing the target vessels and arranged to receive the liquid fractions, wherein the capillary outlet opening and the fluid line outlet opening are disposed in a direct vicinity of one another such that liquid emerging from the capillary outlet opening transitions into the fluid line outlet opening to form a common section with an end section, wherein the end section of the common section faces the target vessels and opens into a cannula, and wherein the fluid pump is configured to switch or is operable to be switched from pumping to aspirating the liquid fractions.

2. The fraction collection apparatus as claimed in claim 1, wherein the fluid line and the capillary extend in parallel from a flow point of view along a common section.

3. The fraction collection apparatus as claimed in claim 2, wherein the fluid line and the capillary extend concentrically from the flow point of view along the common section.

4. The fraction collection apparatus as claimed in claim 2, wherein the fluid line extends into the branching unit.

5. The fraction collection apparatus as claimed in claim 1, wherein the branching unit comprises a distributor piece with at least three inlet openings, a first segment of the capillary coming from the separation or reaction apparatus opening into the first inlet opening, a second segment leading to the collection arrangement and consisting of the fluid line and the capillary opening into the second inlet opening and a section of the fluid line leading to the fluid pump opening into the third inlet opening.

6. The fraction collection apparatus as claimed in claim 1, wherein the capillary sequentially guides the plurality of liquid fractions from the separation or reaction apparatus, wherein the collection arrangement carries the plurality of target vessels arranged in an XY or an XYZ arrangement and configured to receive the plurality of liquid fractions individually and sequentially, further comprising a control apparatus configured to cooperatively control the capillary and the collection arrangement to deposit the plurality of liquid fractions respectively in the plurality of target vessels.

7. A fraction collection apparatus for collecting sequential liquid fractions from a separation or reaction apparatus, comprising: a branching unit, a capillary arranged and configured to guide the liquid fractions from the separation or reaction apparatus to the branching unit, a collection arrangement carrying a plurality of target vessels configured and arranged to receive the liquid fractions from the capillary, and a fluid line flow-connected to a fluid pump, wherein the fluid line opens into the branching unit, wherein the capillary and the fluid line each have a respective outlet opening facing the target vessels and arranged to receive the liquid fractions, wherein the capillary outlet opening and the fluid line outlet opening are disposed in a direct vicinity of one another such that liquid emerging from the capillary outlet opening transitions into the fluid line outlet opening to form a common section with an end section, wherein the end section of the common section faces the target vessels and opens into a cannula a waste collection vessel for collecting discarded ones of the plurality of liquid fractions guided out of the branching unit via the fluid line for disposal purposes, and a first switching valve disposed in the fluid line between the fluid pump and the waste collection vessel, wherein an inlet opening of the first switching valve is connected to the fluid pump and wherein outlet openings of the fluid pump are configured to be selectively connected to the waste collection vessel or to a system liquid container via the outlet openings of the first switching valve.

8. The fraction collection apparatus as claimed in claim 7, wherein the first switching valve is a 3/2-way valve.

9. The fraction collection apparatus as claimed in claim 7, wherein the system liquid container is configured for rinsing and/or for preparing contained liquid samples.

10. The fraction collection apparatus as claimed in claim 7, further comprising a further switching valve disposed in the fluid line between the fluid pump and the branching unit, wherein an inlet opening of the further switching valve is connected to the branching unit and wherein outlet openings of the branching unit are configured to be selectively connected to the fluid pump or to a gas container via the outlet openings of the further switching valve.

11. The fraction collection apparatus as claimed in claim 10, wherein the further switching valve is a 3/2-way valve, and wherein the gas container contains dry inert gas.

12. The fraction collection apparatus as claimed in claim 10, further comprising lines and/or cavities, wherein the gas container is configured to blow clear, purge and/or dry the lines and/or cavities via the outlet openings of the further switching valve.

13. The fraction collection apparatus as claimed in claim 1, further comprising a delivery pump disposed at the separation or reaction apparatus and configured to convey the liquid in the capillary at a capillary flow rate v2.

14. A fraction collection apparatus for collecting sequential liquid fractions from a separation or reaction apparatus, comprising: a branching unit, a capillary arranged and configured to guide the liquid fractions from the separation or reaction apparatus to the branching unit, a collection arrangement carrying a plurality of target vessels configured and arranged to receive the liquid fractions from the capillary, and a fluid line flow-connected to a fluid pump, and a delivery pump disposed at the separation or reaction apparatus and configured to convey the liquid in the capillary at a capillary flow rate v2, wherein the fluid line opens into the branching unit, wherein the capillary and the fluid line each have a respective outlet opening facing the target vessels and arranged to receive the liquid fractions, wherein the capillary outlet opening and the fluid line outlet opening are disposed in a direct vicinity of one another such that liquid emerging from the capillary outlet opening transitions into the fluid line outlet opening to form a common section with an end section, and wherein the end section of the common section faces the target vessels and opens into a cannula wherein the fluid pump is subject to closed-loop control such that a fluid line flow rate v1 in the fluid line is at least equal to the capillary flow rate v2 in the capillary.

15. The fraction collection apparatus as claimed in claim 14, further comprising a control apparatus configured to drive the fluid pump electronically, wherein a duration of on-and-off cycles and the fluid line flow rate v1 are subject to the closed-loop control.

16. The fraction collection apparatus as claimed in claim 15, further comprising a switch to set the fraction collection apparatus between an aspiration operation and a pressure operation.

17. The fraction collection apparatus as claimed in claim 1, wherein the separation or reaction apparatus comprises a liquid chromatography device with a separation column and a detector, and wherein the liquid fraction is an eluate fraction.

18. A system comprising a separation or reaction apparatus, and a fraction collection apparatus for collecting liquid fractions from the separation or reaction apparatus as claimed in claim 1.

19. The system as claimed in claim 18, wherein the separation or reaction apparatus is a liquid chromatography device with a separation column.

20. A method for operating a fraction collection apparatus as claimed in claim 1, comprising: a) supplying a first of the liquid fractions via the capillary at a known flow rate v2, b) positioning the cannula over and/or in a first of the plurality of target vessels with simultaneous aspiration with the fluid pump through the fluid line, c) stopping the fluid pump and collecting the first liquid fraction in the first target vessel over a period of time T1, d) starting the fluid pump and aspirating the liquid fraction into the fluid line for changing the positioning of the cannula into a subsequent target vessel over a further period of time T2, e) repeating said steps c) and d) for further liquid fractions and further target vessels until terminating the method.

21. The method as claimed in claim 20, wherein said step e) comprises: c1) in a pressure operation of the fluid pump, releasing the partial fraction aspirated in said step d) to the target vessel.

22. The method as claimed in claim 20, wherein the separation or reaction apparatus is a liquid chromatography device with a separation column and a downstream detector, wherein a volume V in the capillary between the detector and the end section of the capillary facing the one target vessel of the collection arrangement is known, and wherein said step e) further comprises stopping the fluid pump as per said step c) with a time offset by a period of time T3, wherein T3 is the time during which the eluate passes the volume V.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is depicted in the drawing and will be explained in more detail on the basis of exemplary embodiments.

(2) In the Figures:

(3) FIG. 1 shows a schematic illustration of the core of a first, simple embodiment of the fraction collection apparatus according to the invention, comprising a fluid pump in the fluid line and a common end section of the fluid line and capillary with communicating outlet openings;

(4) FIG. 2A shows a system comprising a fraction collection apparatus according to FIG. 1 and comprising a separation or reaction apparatus in a first operating state, with a stopped fluid pump for transferring the liquid fraction taken with the capillary to a target vessel of a collection arrangement;

(5) FIG. 2B shows the system according to FIG. 2A in a second operating state, with a running fluid pump for transferring the liquid situated in the capillary to a waste collection vessel via the fluid line;

(6) FIG. 3A shows a schematic illustration of an extended embodiment comprising a first switching valve between the fluid pump and the waste collection vessel and a container with system liquid in a third operating state with a running fluid pump for rinsing the fluid line;

(7) FIG. 3B shows an extended embodiment as in FIG. 3A, but additionally comprising a further switching valve between the fluid pump and the branching unit and comprising a gas container, in a fourth operating state with a stationary fluid pump and an opened gas container for blowing clear the fluid line;

(8) FIG. 4 shows a schematic illustration of a system comprising a fraction collection apparatus according to the prior art, as per citation [1];

(9) FIG. 5 shows a schematic illustration of (top) differently designed end sections, collectively embodied as cannulas, of capillary and fluid line and (bottom) various cross-sections A-A of the respective cannulas imaged thereabove;

(10) FIG. 6 shows a specific embodiment of the common end section of the fluid line and capillary with its communicating outlet openings, the fluid line being guided to the capillary from the side;

(11) FIG. 7 shows a typical schematic time curve during the detection of a liquid fraction taken with the capillary; and

(12) FIG. 8 shows the temporal separation of two liquid fractions that immediately follow one another in time.

DETAILED DESCRIPTION

(13) The present invention considers a specially modified fraction collection apparatus for collecting liquid fractions from a separation or reaction apparatus 1. By way of example, the separation or reaction apparatus 1 can comprise a liquid chromatography device with a separation column, or else, for instance, a reactor for chemical reactions that progress in controlled fashion.

(14) FIGS. 1 to 3B and 5 and 6 in the drawing each show details of preferred embodiments of the fraction collection apparatus according to the invention in a schematic view, while a generic system according to the prior art as per citation [1] is illustrated in FIG. 4.

(15) The fraction collection apparatus comprises a capillary 2, which guides a liquid fraction from the separation or reaction apparatus 1 to a branching unit 3, and a collection arrangement 4, which carries a plurality of target vessels 5 for receiving the liquid fraction from the capillary 2. A taken liquid fraction can be emptied into one of these target vessels 5 via the outlet opening 2′ of the capillary 2, for storage purposes and optional further processing. However, the taken fraction can also be guided to a waste collection vessel 8 via the branching unit 3 and a fluid line 6.

(16) In the fraction collector as per the prior art illustrated in FIG. 4, the separation or reaction apparatus 1 usually also contains a delivery pump 14 for conveying the liquid fraction from a chromatography device or reaction vessel into the capillary 2. Moreover, a detector 16 is usually also present, said detector indicating whether a liquid fraction currently contains a target molecule. Hence, further processing of this fraction, which is often automated as well, can then be driven in the fraction collection apparatus via a valve 17.

(17) This known fraction collection apparatus always contains a certain dead volume. In this case, this is the volume in the line between the 3/2-way valve 17 and the cannula outlet 2′ and the internal volume of the valve itself. This 3/2-way valve 17 allows the taken liquid fraction to be steered either into the collection arrangement 4, like in the operating state shown in FIG. 4, or into the waste collection vessel 8 for disposal purposes.

(18) The fraction collection apparatus according to the invention fundamentally differs from this prior art predominantly in that the fluid line 6 opening into the branching unit 3 is flow-connected to a fluid pump 7 and in that the capillary and the fluid line 6 have outlet openings 2′, 6′ at their end facing a target vessel 5, said outlet openings being disposed in the direct vicinity of one another such that liquid emerging from the outlet opening 2′ of the capillary 2 transitions into the outlet opening 6′ of the fluid line 6. This reliably avoids a detrimental dead volume, which would necessarily lead to undefined intermediate fractions between successive liquid fractions, in the arrangement.

(19) This geometric configuration of the outlet openings 2′, 6′, as proposed in the present invention, can easily be seen in the embodiment of FIG. 1, which has been kept relatively simple. Here, the fluid line 6 and the capillary 2 extend in parallel from a flow point of view along a common section and extend in spatially concentric fashion. On one side, the common section of capillary 2 and fluid line 6 extends into the branching unit 3 and, at its other end, said common section opens into a cannula 13 on the side facing the target vessel 5 of the collection arrangement 4. The embodiment of the invention illustrated here moreover has the peculiarity of the branching unit 3 comprising a distributor piece (a T-piece in this case) with three inlet openings 3a, 3b, 3c. A first segment of the capillary 2 comes from the separation or reaction apparatus 1 opening into the first inlet opening 3a; a second segment leads to the collection arrangement 4 and consists of a part of respectively the fluid line 6 and the capillary 2 opening into the second inlet opening 3b; and a section of the fluid line 6 leads to the fluid pump 7 opening into the third inlet opening 3c. Finally, a control apparatus 15 is also present, and is configured and arranged to drive the fluid pump 7 electronically, and to subject the duration of the on and off cycles and the flow rate v1 to closed-loop control, with a switch from aspiration operation to pressure operation.

(20) FIGS. 2A to 3B illustrate systems with in each case a particular embodiment of the fraction collection apparatus according to the invention and a conventional separation or reaction apparatus 1 in different operating states.

(21) If the fluid pump 7 is at a standstill—as shown in FIG. 2A—the liquid fraction taken by the capillary 2 drips into the target vessel 5, provided therefor, at the transfer point for further processing.

(22) Once dispensing of the fraction has been completed, the fluid pump 7—as shown in FIG. 2B—aspirates at an approximately 1% higher flow rate than the delivery pump 14 in the separation or reaction apparatus 1. As a result, the drop of liquid fraction arising at the needle tip of the cannula 13 at the end of the capillary 2 is aspirated into the outlet opening 6′ of the fluid line 6 and transferred by the latter into the waste collection vessel 8. Dispensing the fraction into the collection arrangement 4 therefore stops immediately and without a time delay, and so uncontrolled dripping of parts of the liquid onto the collection arrangement 4 is reliably avoided.

(23) Then, the needle tip of the cannula 13 is moved safely to the next vessel position, which is advantageously positioned in directly adjacent fashion; as a rule, this requires less than 1 s. If the fluid pump 7 is stopped again, then the next fractioning begins ad hoc without further time delay and without back mixing with a preceding fraction.

(24) The fraction drawn during the position change is output again into the next target vessel 5 in the new position (added to the current fraction) where necessary, most easily by switching the fluid pump 7 to dispense, and hence 100% loss-free fractions are obtained.

(25) As a result of this simple structure, any available XYZ fraction collector can be converted into a dead volume-free fraction collector by merely replacing the cannula 13 and adapting the employed control software. The general fraction collector function may possibly be slightly restricted as a result of the internal volume conditions of the dual cannula now used, but it continues to be present.

(26) FIG. 3A shows an embodiment in which a first switching valve 9, in this case in the form of a 3/2-way valve, is disposed in the fluid line 6 between the fluid pump 7 and the waste collection vessel 8, an inlet opening of said first switching valve being connected to the fluid pump 7. By way of outlet openings of said first switching valve, the fluid pump 7 can be selectively connected to the waste collection vessel 8 or to a container 10 with system liquid, in particular for priming and/or for preparing liquid samples. In this way, it is possible to select between waste for aspirated fractions and the rinsing of the system, and actions for “normal” sample preparation.

(27) FIG. 3B illustrates a more specific embodiment: a further switching valve 11 is disposed in the fluid line 6 between the fluid pump 7 and the branching unit 3, an inlet opening of said further switching valve being connected to the branching unit 3 and the branching unit 3 being able to be selectively connected to the fluid pump 7 or to a gas container 12 with gas, preferably dry inert gas, in particular for blowing clear, purging and/or drying lines and liquid fractions of the liquid collection apparatus collected in target vessels, via the outlet openings of said further switching valve.

(28) This facilitates active evaporation and increased concentration of the liquid fraction. In this way, a definable, selectable volume of the fraction is obtained. This embodiment of the invention is advantageous for NMR measurements, in particular, since the chromatographic step can be carried out using a protonated solvent, which is then evaporated. The residue can subsequently be taken up again with a defined amount of a deuterated solvent. Deuterated solvents are substantially more expensive than protonated solvents and are therefore used very economically and only if absolutely necessary.

(29) The gas is additionally heated, increasing the evaporation rate, if at least parts of the cannula 13 are heated, for example using an electric resistor. A triple channel cannula with an additional separate gas line is an additional optional embodiment, in which chromatography, fraction collector and gas line extend separately and parallel to a common tip. As a result, the further switching valve 11 between the fluid pump 7 and the branching unit 3 is no longer required.

(30) FIG. 5 shows differently designed embodiments of the respective common end section of the fluid line 6 and capillary 2 with their communicating outlet openings, in the form of cannulas 13 that taper to a tip in needle-shaped fashion. Illustrated therebelow are different cross sections A-A transverse to the longitudinal axis of the respective cannula 13 imaged thereabove. The bottom row shows embodiments of the cannulas with subdivisions in the line itself, while the row second from the bottom in each case shows the line guidance with parallel tubes or pipes.

(31) If a multiple cannula is used instead of a simple dual cannula, which likewise has a needle with an angled tip and venting at the end, the fraction collection can also be undertaken in vessels sealed by a septum. Cannulas made of concentrically disposed feed and discharge lines are currently shown. However, a parallel arrangement of fluid line 6 and capillary 2 in the cannula 13, inter alia, is also possible, with the openings 2′, 6′ then opening next one another into the needle tip. The fraction collector function for preparations is not influenced thereby; i.e., the aspiration by the fluid pump 7 works equally well in both variants without drips falling from the cannula tip. Naturally, the dimension of the cannulas has to be considered because drop-free aspiration would no longer work reliably in the case of an interior cannula cross section that is too large or in the case of an elution agent with a very low surface tension. Preferably, the outer capillary has an internal diameter of 0.7 mm to 2.5 mm and the inner capillary, which contains the sample to be fractionated, has an external diameter of 0.3 mm to 2 mm. In the case of a dual channel cannula, the preferred internal channel dimension lies between 0.5 mm and 2 mm, wherein the channel provided for aspiration may ideally have a slightly larger dimension than the channel guiding the liquid fraction. The aspirating channel should at least have dimensions that are suitable for aspiration at the envisaged flow rate (e.g., >0.5 mm ID ∅ in the case of flow rates <1 ml/min).

(32) In the case of a four-channel cannula, as shown furthest on the right in FIG. 5, the fourth channel is provided for a deuterated solvent, with which an evaporated sample, for example, is solubilized again with a defined volume. This is because experience has shown that pumps for protonated and deuterated solutions must be strictly separated since a 99.999% solvent exchange can only be carried out with very large amounts of solvent. This rinsing consumption should be avoided in any case for the relatively expensive deuterated solvents for reasons of costs and time.

(33) FIG. 6 shows—very schematically—a specific embodiment of the common end section of the fluid line 6 and capillary 2 with their communicating outlet openings 2′, 6′, in which the fluid line 6 is guided to the capillary 2 from the side. Although this geometry differs fundamentally from the parallel, in particular concentric configurations presented above—for instance illustrated in FIG. 5—it equally meets the basic demand according to the invention, according to which the outlet openings 2′, 6′ are disposed in the direct vicinity of one another such that liquid emerging from the outlet opening 2′ of the capillary 2 transitions into the outlet opening 6′ of the fluid line 6.

(34) FIG. 7 illustrates a typical schematic time curve when detecting a liquid fraction taken with the capillary 2.

(35) It is possible to identify the relationships of the following physical variables:

(36) FL=Flow rate (μl/s)

(37) L.sub.1=Detection level for recording a liquid fraction (mV) in a defined time window

(38) T.sub.3=Time delay from the detector 16 to the outlet opening 2′ of the capillary 2volume/flow rate=μl/(μl/s)

(39) T.sub.FR=Time for recording a liquid fraction (s)T.sub.FR×FL=V.sub.Fr

(40) S.sub.1=Start of the fraction

(41) S.sub.2=end of the fraction

(42) V=Internal volume (μl) from the detector 16 to the outlet opening 2′

(43) V.sub.Fr=Volume of the liquid fraction (μl).

(44) The upper line in FIG. 7 represents the time curve in the detector and the lower line is the time-offset arrival of the detected eluate at the outlet opening 2′. The time offset is easily determinable automatically by virtue of the known internal volume V being divided by the flow rate FL, which is set by the control apparatus.

(45) If the sought-after fraction passes the detector 16 during the period of time T.sub.FR, the fraction can be captured precisely at the time offset T.sub.3, as illustrated in the drawing by the black area.

(46) Finally, FIG. 8 illustrates the (temporal and hence also spatial) separation of two liquid fractions that immediately follow one another in time. Here, the following variables are of importance:

(47) L.sub.1=Detection level for recording a liquid fraction (mV) in a defined time window

(48) S.sub.1=Start of fraction 1

(49) S.sub.1′=End of fraction 1

(50) S.sub.2=Start of fraction 2

(51) S.sub.2′=End of fraction 2

(52) T.sub.FR1=Time for recording a liquid fraction 1

(53) T.sub.FR2=Time for recording a liquid fraction 2

(54) T.sub.PC=time for a mechanical change in position to a new target vessel 5

(55) V.sub.FR1=Volume of the liquid fraction 1

(56) V.sub.FR2=Volume of the liquid fraction 2

(57) V.sub.asp=Volume of the aspirated liquid fraction during the change in position (V.sub.asp=V.sub.disp)

(58) V.sub.disp=Volume of the aspirated liquid fraction that is output following the change in position.

(59) The figure illustrates the goal of sharply separating the two fractions 1 and 2, which directly follow one another. To this end, it is necessary to work without a dead volume and to know the precise volume V. Then, fraction 1 is captured in a first target vessel 1 only during the period of time T.sub.FR1. Once the detector 16 identifies a drop in the peak, which is directly correlated to the concentration of the target molecule in fraction 1, the fluid pump 7 is activated such that no mixed fraction drops into the first target fraction. While the fluid pump 7 aspirates the eluate during the period of time T.sub.PC, the fraction collector changes the target vessel 5 in order to collect fraction 2. Here, the control apparatus 15 can deactivate the pump 7 once the detector 16 detects a significant increase in the concentration in fraction 2. This type of procedure using the fraction collection apparatus according to the invention allows fractions that lie close together to be collected in a sharply separated manner. The time offset from the detector 16 to the outlet opening 2′ is captured as mentioned in FIG. 7.

LIST OF REFERENCE SIGNS

(60) 1 Separation or reaction apparatus 2 Capillary 2′ Outlet opening of the capillary 3 Branching unit 3a First inlet opening 3b Second inlet opening 3c Third inlet opening 4 Collection arrangement 5, 5′ Target vessel(s) 6 Fluid line 6′ Outlet opening of the fluid line 7 Fluid pump 8 Waste collection vessel 9 First switching valve 10 Container with system liquid 11 Further switching valve 12 Gas container 13 Cannula 14 Delivery pump 15 Control apparatus 16 Detector 17 Valve A-A Horizontal section through the cannulas

LIST OF CITATIONS

(61) Publications considered for the assessment of the patentability:

(62) [0] US 2005/0158215 A1 [1] Handbuch, “Foxy® R1 and Foxy® R2, Fraction Collectors”, Teledyne Isco, Revision H, 21 Jun. 2016 [2] US 2002/0011276 A1 [3] US 2002/0088946 A1 [4] U.S. Pat. No. 6,704,880 A [5] US 2018/0136174 A1