SYSTEM AND METHOD FOR ANALYSING THE COMPOSITION OF A QUENCHED FLOW REACTION LIQUID

Abstract

The present invention relates to a system for analysing the composition of a quenched flow reaction liquid comprising a quenched flow reactor, and a high performance liquid chromatography (HPLC) apparatus; wherein the quenched flow reactor is in fluid communication with the HPLC apparatus.

Claims

1. A system for analyzing the composition of a quenched flow reaction liquid comprising a quenched flow reactor, and a high performance liquid chromatography (HPLC) apparatus; wherein the quenched flow reactor is in fluid communication with the HPLC apparatus.

2. The system according to claim 1, wherein the quenched flow reactor comprises: a first reagent release mechanism, a second reagent release mechanism, a reaction area, a quenching reagent release mechanism, and a quenching area.

3. The system according to claim 2, wherein the first reagent release mechanism is automated and/or wherein the second reagent release mechanism is automated and/or wherein the quenching reagent release mechanism is automated; and/or wherein the first reagent release mechanism is a syringe and/or wherein the second reagent release mechanism is a syringe and/or the quenching reagent release mechanism is a syringe.

4. The system according to claim 1, wherein the HPLC apparatus comprises a HPLC injection valve and a column, preferably wherein the column is a digestion column.

5. The system according to claim 4, wherein the HPLC injection valve comprises a HPLC injection valve loop for holding a proportion of the quenched flow reaction liquid prior to injecting the proportion of the quenched flow reaction liquid into the column.

6. The system according to claim 1, wherein the quenched flow reactor and the HPLC apparatus are connected by a bypass valve.

7. The system according to claim 6, wherein in a first position, the bypass valve directs a first proportion of the quenched flow reaction liquid to a non-HPLC apparatus location and wherein in a second position, the bypass valve directs a second proportion of the quenched flow reaction liquid into the HPLC injection valve.

8. The system according to claim 2, wherein the reaction area comprises a tube and/or wherein the quenching area comprises a mixer.

9. The system according to claim 8, wherein the length of the tube can be varied.

10. The system according to claim 8, wherein the length of the tube can be selected from at least 2 predetermined lengths.

11. The system according to claim 8, wherein the length of the tube or each tube is about 1 cm to about 30 cm.

12. The system according to claim 8, wherein the tube comprises a bore and the diameter of the bore is about 0.2 mm to about 2 mm.

13. The system according to claim 8, wherein the reaction area comprises a pathway extension valve, wherein adjusting the pathway extension valve varies the length of the tube.

14. The system according to claim 1, further comprising an analysis apparatus in fluid communication with the HPLC apparatus.

15. The system according to claim 2, wherein the first reagent release mechanism comprises a prereaction system.

16. A method for analyzing the composition of a quenched flow reaction liquid comprising: (a) providing a first reagent, (b) providing a second reagent, (c) mixing the first reagent and the second reagent in a reaction area, (d) allowing a reaction to take place between the first reagent and the second reagent in the reaction area for a predetermined reaction time, (e) quenching the reaction in a quenching area to form a quenched flow reaction liquid, (f) directly transferring a proportion of the quenched flow reaction liquid into a HPLC apparatus, and (g) analyzing the quenched flow reaction liquid by HPLC to form an HPLC analyte.

17. The method according to claim 16, wherein step (f) further comprises transferring the quenched flow reaction liquid from the quenched flow reactor through a bypass valve, adjusting the bypass valve to a first position to transfer a first proportion of the quenched flow reaction liquid to a non-HPLC apparatus location and adjusting the bypass valve to a second position to transfer a second proportion of the quenched flow reaction liquid into the HPLC apparatus.

18. The method according to claim 17, wherein the second proportion of the quenched flow reaction liquid is transferred into a HPLC injector valve of the HPLC apparatus, preferably into a HPLC injection valve loop.

19. The method according to claim 16, wherein the first reagent comprises a macromolecule.

20. The method according to claim 16, wherein the second reagent comprises a label, or induces a measurable change in the first reagent.

21. The method according to claim 20, wherein the second reagent comprises deuterium oxide.

22. The method according to claim 16, wherein in step (a) the first reagent is provided at a first rate, and wherein in step (b) the second reagent is provided at a second rate.

23. The method according to claim 16, wherein in step (e) the reaction is quenched by cooling, heating or adding a quenching reagent.

24. The method according to claim 16, wherein the reaction time is from about 5 ms to about 24 hours.

25. The method according to claim 16, further comprising (h) directly transferring the HPLC analyte into an analysis apparatus, and (i) analyzing the HPLC analyte.

26. The method according to claim 16, wherein step (a) further comprises providing a prereaction system, wherein the prereaction system comprises: providing a first precursor, providing a second precursor, mixing the first precursor and the second precursor in a prereaction area, and allowing a prereaction to take place between the first precursor and the second precursor in the prereaction area for a predetermined prereaction time, to form the first reagent.

27. The method according to claim 26, wherein the first precursor comprises a macromolecule and/or the second precursor comprises a ligand.

28. A method for analyzing the composition of a quenched flow reaction liquid comprising: (a) providing a first reagent, (b) providing a second reagent, (c) mixing the first reagent and the second reagent in a reaction area, (d) allowing a reaction to take place between the first reagent and the second reagent in the reaction area for a predetermined reaction time, (e) quenching the reaction in a quenching area to form a quenched flow reaction liquid, (f) directly transferring a proportion of the quenched flow reaction liquid into a HPLC apparatus, and (g) analyzing the quenched flow reaction liquid by HPLC to form an HPLC analyte, wherein the method is performed in a system according to claim 1.

Description

FIGURES

[0149] Example embodiments of the present invention will now be described with reference to the accompanying figures, in which

[0150] FIG. 1 shows an analysis system of the invention.

[0151] FIG. 2 shows a pathway extension valve in a first configuration.

[0152] FIG. 3 shows a pathway extension valve in a second configuration.

[0153] FIG. 4 shows a pathway extension valve in a third configuration.

[0154] FIG. 5 shows a pathway extension valve in a fourth configuration.

[0155] FIG. 6a shows a rotor of a pathway extension valve.

[0156] FIG. 6b shows a cross-sectional view of a rotor of a pathway extension valve.

[0157] FIG. 7a shows a stator of a pathway extension valve.

[0158] FIG. 7b shows a cross-sectional view of a stator of a pathway extension valve.

[0159] FIG. 8a shows a pathway extension valve.

[0160] FIG. 8b shows a cross-sectional view of a pathway extension valve.

[0161] FIG. 9 shows a prereaction system.

[0162] The present invention relates to a system and method for analysing the composition of a quenched flow reaction liquid. FIG. 1 shows a quenched flow reactor 5 in fluid communication with a HPLC apparatus 8. The quenched flow reactor comprises a first reagent release mechanism 1, a second reagent release mechanism 2 and a quenching reagent release mechanism 3. All are shown as a syringe, but other release mechanisms are envisaged. The valve 10 is used to fill the syringe from a reservoir and to allow external delivery of reagents from a separate flow release mechanism.

[0163] In use, the first reagent and the second reagent are released and mixed in a reaction area 11 which includes the pathway extension valve 4. Preferably the reaction area 11 comprises a mixer, such as a t-format mixer or a berger ball mixer. The pathway extension valve 4 comprises three loops 9 which may or may not form part of the fluid pathway, depending on the position of the valve. It will be appreciated that other lengths and numbers of option loops are included in the present invention. The liquid then flows to the quenching area 12, where it mixes with the quenching reagent released from the quenching reagent release mechanism 3 to form a quenched flow reaction liquid. The quenching area 12 preferably comprises a mixer, such as a t-format mixer or a berger ball mixer. The quenched flow reaction liquid is then transferred, preferably piped into a bypass valve 6. A first proportion of the quenched flow reaction liquid is then transferred, preferably piped out of the system to waste 7 or to a container 7. This allows the liquid to flow through the quenched flow reactor at a fast rate, such as about 0.2 to about 30 ml/s, preferably about 0.5 to about 20 ml/s while the first reagent and the second reagent are mixing and the reaction is taking place. It will be appreciate that the fast flow rates are required to mix the first reagent and second reagent effectively, and that the flow rates may be reduced, or even stopped to give the desired reaction time, prior to pushing the reaction liquid into the quenching area.

[0164] A second proportion of the quenched flow reaction liquid is directed into the HPLC injection valve 13, and in particular through the HPLC injection valve loop 14. The HPLC apparatus 8 comprises a HPLC pump 17 which pumps solvent to the HPLC injection valve 13 through the solvent line 16. The HPLC injection valve loop 14 has two positions. In a first position, the HPLC injection valve loop 14 is connected to waste 15 or to a container 15. This allows the HPLC injection valve loop 14 to be loaded with the desired first part of the second proportion of the quenched flow liquid and some of the quenched flow reaction liquid to be removed from the system. Once the desired first part of the second proportion of the quenched flow reaction liquid is loaded into the HPLC injection valve loop 14, the HPLC injection valve 13 is moved to a second position, in line with the solvent line 16 of the HPLC apparatus to load the selected quenched flow reaction liquid onto the column. The HPLC apparatus 8 may comprise a digestion column, such as a pepsin column. Further, the HPLC analyte resulting from the HPLC analysis may be further piped into an analysis apparatus, preferably a mass spectrometer (not shown).

[0165] FIG. 2 shows a pathway extension valve 4 in a first position, whereby the fluid pathway through the pathway extension valve is from the inlet 23, directly to the outlet 24. The additional passageway extensions 25, 26 and 27 do not form part of the passageway at the first position.

[0166] FIG. 3 shows a pathway extension valve 4 in a second position, whereby the fluid pathway through the pathway extension valve is from the inlet 23, through a first extension passageway 25 and then to the outlet 24. The additional passageway extensions 26 and 27 do not form part of the passageway at the second position.

[0167] FIG. 4 shows a pathway extension valve 4 in a third position, whereby the fluid pathway through the pathway extension valve is from the inlet 23, through a first passageway extension 25, through a second passageway extension 26 and then to the outlet 24. The additional passageway extension 27 does not form part of the passageway at the third position.

[0168] FIG. 5 shows a pathway extension valve 4 in a fourth position, whereby the fluid pathway through the pathway extension valve is from the inlet 23, through a first passageway extension 25, through a second passageway extension 26, through a third passageway extension 27 and then to the outlet 24. There are no unused passageway extensions in the fourth position.

[0169] It will be appreciated that each passageway extension is shown as a loop. Each loop may be the same length, or a different length to the other loops present. Further, the length of the passageway through the pathway extension valve can be selected from at least 2 predetermined lengths, preferably from 2 to about 10 predetermined lengths, preferably from about 3 to about 8 predetermined lengths, most preferably from 4 to 6 predetermined lengths. Further, the pathway extension valve may be arranged such that the liquid can flow through a first passageway extension, or a second passageway extension, or a third passageway extension, or a fourth passageway extension, or a fifth passageway extension, or a sixth passageway extension, or a seven passageway extension, or an eight passageway extension, or a ninth passageway extension or a tenth passageway extension, or any combination thereof where each length may be different. It will be appreciated that there may be any number of different passageway extensions in the pathway extension valve, such as at least one, preferably about 2 to about 10, preferably about 3 to about 8, preferably about 4 to about 6.

[0170] FIGS. 6a-8b show an example of the pathway extension valve. It will be appreciated that other arrangements are possible, such as a plug type valve with passageway extensions along the radius of the plug and the passageway path diagonally or right angled drilled through the middle of the plug to a common port.

[0171] FIG. 6a shows a rotor 31 of a pathway extension valve. The rotor 31 comprises a rotor sealing surface 34. The rotor sealing surface 34 comprises three passageway extensions 36 which may optionally be included in the passageway through the pathway extension valve. The sealing surface further comprises part of the passageway 33.

[0172] FIG. 6b shows a cross-section view of the rotor 31. One of the passageway extensions 36 and part of the passageway 33 are each shown as an indent in the sealing surface 34.

[0173] FIG. 7a shows a stator 32 of the pathway extension valve. The stator comprises a stator sealing surface 40. The stator sealing surface 40 comprises a plurality of threaded fluid tube sealing ports 38 shown arranged around the outer portion of the stator sealing surface 40. The stator sealing surface 40 has one common threaded fluid tube sealing port 39 shown in the centre of the sealing surface. The common threaded fluid tube sealing port 39 is either the inlet or the outlet. One of the plurality of threaded fluid tube sealing ports 38 is the other of the inlet or the outlet. The arrangement will depend on how the tubing is connected. The stator has a stator sealing surface 40. Adjusting the valve, such as by turning the valve will determine which of the passageway extensions are included in the passageway through the pathway extension valve.

[0174] FIG. 7b shows a cross-sectional view of the stator 32. The stator sealing surface 40 has threaded fluid tube sealing ports 38 through the surface. The common threaded fluid tube sealing port 39 is shown in the centre of the stator sealing surface 40.

[0175] FIG. 8a shows the rotor 31 in engagement with the stator 32. The threaded fluid tube sealing ports 38 and the common threaded fluid tube sealing port 39 are each engaged with a passageway extension 36 or a part of the passageway 33. The rotor sealing surface engages with the stator sealing surface 40.

[0176] FIG. 8b shows a cross-sectional view of the rotor 31 in engagement with the stator 32. Part of the passageway 33 lines up with the common threaded fluid tube sealing port 39. Each of the threaded fluid tube sealing ports 38 engage with a passageway extension 36. The rotor sealing surface 34 and the stator sealing surface 40 are in engagement. Adjusting the valve by turning will move the position of the passageway extensions 36 to move them to form part of the passageway or remove them from the passageway, thus allowing the length of the passageway through the pathway extension valve to be adjusted, and thus the length of the reaction area fluid pathway to be changed.

[0177] It will be appreciated that the pathway extension valve may have a different number, type and arrangement of sealing ports and tubing.

[0178] FIG. 9 shows a prereaction system 41 that can be optionally incorporated into the quenched flow reactor 5 of FIG. 1. The prereaction system 41 comprises a first precursor release mechanism 42, a second precursor release mechanism 43. All are shown as a syringe, but other release mechanisms are envisaged. The valve 10 is used to fill the syringe from a reservoir and to allow external delivery of reagents from a separate flow release mechanism. In use, the first precursor and the second precursor are released and mixed in a prereaction area 44, to form the first reagent. The prereaction area 44, may preferably comprise a pathway extension valve as described herein (not shown). The first reagent flows to the reaction area 11, where it mixes with the second reagent released from the second reagent release mechanism 2 (not shown). The system and method for analysing the composition of a quenched flow reaction liquid then continues to proceed as described above with reference to FIG. 1.