METHOD FOR OPTIMIZING A LIQUID CHROMATOGRAPHY SYSTEM AND SYSTEM FOR LIQUID CHROMATOGRAPHY

Abstract

The present invention relates to a method performed in a liquid chromatography system, the method comprising: in a first configuration (I), wherein a first separation column is fluidly connected to a separation pump and a detector, supplying a flow from the first separation column towards the detector by means of the separation pump in the first configuration (I); and switching the liquid chromatography system from the first configuration (I) to a second configuration (II), wherein the first separation column is fluidly connected to a second pump and to the detector, and supplying a flow from the first separation column towards the detector by means of the second pump in the second configuration (II). The present invention also relates to a corresponding system, use, computer program product, computer-readable medium and data carrier signal.

Claims

1. A method performed in a liquid chromatography system, the method comprising: in a first configuration (I), wherein a first separation column is fluidly connected to a separation pump and a detector, supplying a flow from the first separation column towards the detector by means of the separation pump in the first configuration (I), and switching the liquid chromatography system from the first configuration (I) to a second configuration (II), wherein the first separation column is fluidly connected to a second pump and to the detector, and supplying a flow from the first separation column towards the detector by means of the second pump in the second configuration (II).

2. The method according to claim 1, wherein in the first configuration (I) a second separation column is fluidly connected to the second pump and to a waste, wherein the method further comprises supplying a flow from the second separation column towards the waste by means of the second pump in the first configuration (I), wherein in the second configuration (II), the second separation column is fluidly connected to the separation pump and to the waste, wherein the method further comprises supplying a flow from the second separation column towards the waste by means of the separation pump in the second configuration (II).

3. The method according to claim 1, wherein the liquid chromatography system is switched from the first configuration (I) to the second configuration (II) at a first switching time (T.sub.II), wherein the method comprises switching the liquid chromatography system from the second configuration (II) to a third configuration (III), wherein the first separation column is fluidly connected to the second pump and to a waste, wherein the method further comprises supplying a flow from the first separation column towards the waste by means of the second pump in the third configuration (III), wherein the liquid chromatography system is switched from the second configuration (II) to the third configuration at a second switching time (T.sub.III), and wherein the second switching time (T.sub.III) is later than the first switching time (T.sub.II)

4. The method according claim 3, wherein the method comprises switching the liquid chromatography system from the third configuration (III) to a fourth configuration (IV), wherein the first separation column is fluidly connected to the separation pump and to a waste, wherein the method further comprises supplying a flow from the first separation column towards the waste by means of the separation pump in the fourth configuration (IV), and wherein the liquid chromatography system is switched from the third configuration (III) to the fourth configuration (IV) at a third switching time (T.sub.IV), wherein the third switching time (T.sub.IV) is later than the second switching time (T.sub.III).

5. The method according to claim 4, wherein in the fourth configuration (IV), the second separation column is fluidly connected to the second pump and to the detector, and wherein the method further comprises supplying a flow from the second separation column towards the detector by means of the second pump in the fourth configuration (IV).

6. The method according to claim 4, wherein the liquid chromatography system is switched from the fourth configuration (IV) back to the first configuration (I) at a fourth switching time (T.sub.I), thereby forming a cyclic process and thereby ending a previous cycle and thereby starting a subsequent cycle, wherein the fourth switching time (T.sub.I) is later than the third switching time (T.sub.IV), wherein the subsequent cycle follows the same temporal sequence such that times T.sub.II, T.sub.III, T.sub.IV, T.sub.I of the subsequent cycle correspond, respectively, to the times T.sub.II, T.sub.III, T.sub.IV, T.sub.I of the previous cycle.

7. The method according to claim 6, wherein a time difference t.sub.delay between the second switching time (T.sub.III) and the first switching time (T.sub.II) is based on a volume V.sub.5 of the second separation column, on a volume V.sub.con of fluidic connections connected to the second separation column, and on a flow rate F of the separation pump, wherein a time difference t.sub.delay between the fourth switching time (T.sub.I) and the third switching time (T.sub.IV) is based on a volume V.sub.8 of the first separation column, on a volume V.sub.con of fluidic connections connected to the first separation column, and on a flow rate F of the separation pump.

8. The method according to claim 6, wherein the liquid chromatography system comprises a pre-column switching valve, wherein the liquid chromatography system comprises a post-column switching valve, wherein the method comprises switching, via the pre-column switching valve, the liquid chromatography system from the first configuration (I) to the second configuration (II) at the first switching time T.sub.II, wherein the method comprises switching, via the pre-column switching valve, the liquid chromatography system from the third configuration (III) to the fourth configuration (IV) at the third switching time T.sub.IV, wherein the method comprises switching, via the post-column switching valve, the liquid chromatography system from the second configuration (II) to the third configuration (III) at the second switching time T.sub.III, wherein the method comprises switching, via the post-column switching valve, the liquid chromatography system from the fourth configuration (IV) to the first configuration (I) at the fourth switching time T.sub.I.

9. The method according to claim 7, wherein the method comprises utilizing an optimization procedure to optimize the time difference t.sub.delay and/or the time difference t.sub.delay, wherein the method comprises using a user interface, at least in part, in the steps of the optimization procedure.

10. A system for liquid chromatography, the system comprising: a first separation column, a separation pump, and a detector, wherein the first separation column is configured to be fluidly connected to the separation pump and the detector in a first configuration (I), wherein the system is configured to supply a flow from the first separation column towards the detector by means of the separation pump in the first configuration (I), and wherein the system is configured to switch the system from the first configuration (I) to a second configuration (II), wherein the first separation column is configured to be fluidly connected to the second pump and to the detector in the second configuration (II), and wherein the system is configured to supply a flow from the first separation column towards the detector by means of the second pump in the second configuration (II).

11. The system according to claim 10, wherein the system further comprises: a second separation column, and a waste, wherein the second separation column is configured to be fluidly connected to the second pump and to the waste in the first configuration (I), wherein the system is configured to supply a flow from the second separation column towards the waste by means of the second pump in the first configuration (I).

12. The system according to claim 10, wherein the system is configured to switch the system from the first configuration (I) to the second configuration (II) at a first switching time (T.sub.II), wherein the system is configured to switch the system from the second configuration (II) to a third configuration (III), wherein the first separation column configured to be fluidly connected to the second pump and to a waste, and wherein the system is configured to supply a flow from the first separation column towards the waste by means of the second pump in the third configuration (III), wherein the system is configured to switch the system from the second configuration (II) to the third configuration at a second switching time (T.sub.III), wherein the second switching time (T.sub.III) is later than the first switching time (T.sub.II).

13. The system according to claim 12, wherein the system is configured to switch the system from the third configuration (III) to a fourth configuration (IV), wherein the first separation column is fluidly connected to the separation pump and to a waste, and wherein the system is configured to supply a flow from the first separation column towards the waste by means of the separation pump in the fourth configuration (IV).

14. The system according to claim 13, wherein in the fourth configuration (IV), the second separation column is fluidly connected to the second pump and to the detector, and wherein the system is configured to supply a flow from the second separation column towards the detector by means of the second pump in the fourth configuration (IV).

15. The system according to claim 13, wherein system is configured to switch the system from the fourth configuration (IV) back to the first configuration (I) at a fourth switching time (T.sub.I), thereby forming a cyclic process and thereby ending a previous cycle and thereby starting a subsequent cycle, wherein the fourth switching time (T.sub.I) is later than the third switching time (T.sub.IV) and wherein a time difference t.sub.delay between the fourth switching time (T.sub.I) and the third switching time (T.sub.IV) is based on a volume V.sub.8 of the first separation column, on a volume V.sub.con of fluidic connections connected to the first separation column, and on a flow rate F of the separation pump.

16. The system according to claim 15, wherein a time difference t.sub.delay between the second switching time (T.sub.III) and the first switching time (T.sub.II) is based on a volume V.sub.5 of the second separation column, on a volume V.sub.con of fluidic connections connected to the second separation column, and on a flow rate F of the separation pump, wherein a time difference t.sub.delay between the fourth switching time (T.sub.I) and the third switching time (T.sub.IV) is based on a volume V.sub.8 of the first separation column, on a volume V.sub.con of fluidic connections connected to the first separation column, and on a flow rate F of the separation pump.

17. The system according to claim 10, wherein the liquid chromatography system comprises a pre-column switching valve, wherein the pre-column switching valve comprises a plurality of ports and a plurality of connecting elements for interchangeably connecting the ports, wherein one port of the pre-column switching valve is fluidly connected to the separation pump, one port of the pre-column switching valve is fluidly connected to the second pump, one port of the pre-column switching valve is fluidly connected to the first separation column, one port of the pre-column switching valve is fluidly connected to the second separation column.

18. The system according to claim 16, wherein the system is configured to utilize an optimization procedure to optimize the time difference t.sub.delay and/or the time difference t.sub.delay.

19. A computer-readable medium comprising instructions which, when executed by a processor, cause the processor to control a system for liquid chromatography to carry out a method, the method comprising: in a first configuration (I), wherein a first separation column is fluidly connected to a separation pump and a detector, supplying a flow from the first separation column towards the detector by means of the separation pump in the first configuration (I), and switching the liquid chromatography system from the first configuration (I) to a second configuration (II), wherein the first separation column is fluidly connected to a second pump and to the detector, and supplying a flow from the first separation column towards the detector by means of the second pump in the second configuration (II).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0927] FIG. 1 illustrates exemplary chromatograms of a liquid chromatography system for different values of the time difference between the start of gradient delivery and the start of detection in the liquid chromatography system.

[0928] FIG. 2 depicts, as an example, a preferred embodiment of a liquid chromatography system according to an embodiment of the present invention in a first configuration, which may be referred to as first steady state;

[0929] FIG. 3 depicts, as an example, a preferred embodiment of the system of FIG. 2 in a second configuration, which may be referred to as first intermediate state;

[0930] FIG. 4 depicts, as an example, a preferred embodiment of the system of FIG. 2 in a third configuration, which may be referred to as second steady state;

[0931] FIG. 5 depicts, as an example, a preferred embodiment of the system of FIG. 2 in a fourth configuration, which may be referred to as second intermediate state;

[0932] FIG. 6 depicts an exemplary visualization of a workflow of a tandem liquid chromatography system with an optimized acquisition time window based on four configurations of the tandem liquid chromatography system;

[0933] FIG. 7 illustrates, as an example, UV chromatograms of Cytochrome C showing the influence on UV chromatograms of Cytochrome C of the time difference between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system.

[0934] FIG. 8 depicts, as an example, preferred embodiments of a liquid chromatography system utilizing a double barrel electrospray source and adopting four configurations according to embodiments of the present invention.

DETAILED DESCRIPTION OF THE FIGURES

[0935] It is noted that not all the drawings carry all reference signs. Instead, in some of the drawings, some of the reference signs have been omitted for sake of brevity and simplicity of illustration.

[0936] Hereafter, exemplary embodiments of the present invention will be described in detail, referring to the accompanying figures.

[0937] While in the following preferred embodiments of the present invention will be described, the person skilled in the art will understand that the preferred embodiments are provided for illustrative purposes only and to render the disclosure of the present invention complete, and should by no means be construed to limit the scope of the present invention, which is defined by the claims.

[0938] From a very general viewpoint, embodiments of the present invention relate to executing a workflow in tandem a liquid chromatography (LC) system. Embodiments relate to executing a workflow in a tandem liquid chromatography system, characterized by the utilization of two pumps and two separation columns.

[0939] In order to perform LC, in general, a sample is subjected to flow, by means of the action of a pump, through a separation column and towards a detector. The pump that is utilized in a liquid chromatography system may deliver a gradient into the separation column. That it, the composition of the mobile phase over time may be changed using the separation pump. The mobile phase, moreover, may traverse the separation column in a given amount of time. Such amount of time may in turn depend on the volume of the separation column, as well as on the fluidic connection linking the separation column to the pump and the separation column to the detector. It may further depend on the flow rate of the pump. In other words, the gradient delivery at the separation pump at a given point in time may be different from the gradient delivery at the detector at the same given point. For example, right when the gradient starts being delivered by the pump, the gradient delivery at the pump is equal to the starting gradient delivery, while the gradient delivery at the detector will be equal to the starting gradient delivery at a later time.

[0940] In many prior art LC systems, the detector is configured to start the detection window as the pump starts delivering the gradient into the separation column. Such a workflow, however, hinders an optimized utilization of the detection window, since the detector is actively used beginning from when the pump starts delivering the gradient. In other words, the detector is actively used beginning from when the gradient delivery at the separation pump is the starting gradient and not when the gradient delivery at the detector is the starting gradient.

[0941] The present invention relates, at least in part, to a workflow, wherein the time difference between the start of gradient delivery and the start of detection is substantially different from zero, and may be optimized.

[0942] FIG. 1 illustrates exemplary chromatograms of a liquid chromatography system for different values of the time difference between the start of the gradient delivery and the start of the detection in the liquid chromatography system. Put differently, the effect the different values of said time difference is illustrated.

[0943] FIG. 1A) shows an exemplary periodic time variation of the gradient delivery at the pump 14. Generally, a pump may deliver a gradient in a liquid chromatography procedure. That is, the solvent composition may change over time. For example, different solvents A and B may be mixed at different ratios. FIG. 1A) (as FIG. 1 B)) depicts the volume % of solvent B in the solvent mixture over time. As depicted in FIG. 1A), the amount of solvent B increases monotonously with different rates, is then held constant and is then decreased again during run I (see item 15), but it will be understood that this is merely exemplary and that other solvent compositions over time may be used. The subsequent runs i+1 and i+2 have a corresponding solvent composition delivered over time. The start of the gradient delivery at the pump 14 may coincide, in this exemplary embodiment, with the start of the sample run 15. The gradient delivery at the pump 14 at the start of the sample run 15 may substantially coincide with the starting gradient delivery 16. Analogously, the gradient delivery at the pump 14 at the end of the sample run 15 may substantially coincide with the ending gradient delivery 17. The sample run 15 may be temporally followed by subsequent sample runs.

[0944] Generally, it should be understood that FIG. 1A) depicts the solvent composition at the pump, which may also be referred to as chromatographic pump or separation pump. Furthermore, it will be understood that the solvent composition at a detector downstream of the pump will be different to the solvent composition at the pump. In particular, there typically is a time lag or detail time t.sub.delay between the two. Consider, for example, that the pump delivers with a flow rate of 10 l/min and further consider that the fluidic path between the pump (e.g., connecting tubes, column, valves) have a total inner volume of 20 l. In this example, it would take any change of solvent composition at the pump 2 min to arrive at the detector.

[0945] This is visible in FIG. 1 B). FIG. 1 B) depicts the solvent composition over time at the detector. In simple terms, this solvent composition corresponds to the solvent composition at the detector, but there is a time delay t.sub.delay between the two. In other words, FIG. 1 B) shows an exemplary periodic time variation of the gradient delivery at the detector 18. It will be understood that the time difference t.sub.delay 19 between the time at which the detector starts detecting and the time at which the gradient delivery starts at the pump 14 is usually different from zero.

[0946] FIG. 1 C) depicts exemplary chromatograms when not accounting for the delay time t.sub.delay, i.e., in case that acquisition time windows 20 were chosen to coincide with the gradients runs (see 15) as delivered by the gradient pump. In this example, there are three acquisition time windows 20 a, 20 b, and 20 c, which coincide with the gradient delivery runs i, i+1, and i+2 at the pump. However, due to the time delay t.sub.delay, the acquisition time windows are not ideal. In particular, there is a portion at the beginning of the first acquisition time window 20 a, where the gradient as delivered be the pump has not yet reached the detector. Furthermore, there is a portion 21 at the beginning of the second acquisition time window 20 b, where the gradient (and thus also sample) from the run i still arrives at the detector. That is, this portion 21 actually corresponds to the run i, but is (in the example discussed with reference to FIG. 1 C)) part of the data acquisition time window 20 a. It will be understood that similar considerations also apply to the data acquisition time window 20 c in FIG. 1 C).

[0947] FIG. 1 D) depicts exemplary chromatograms when the data acquisition time windows 20 account for the delay time t.sub.delay. It will be appreciated that the data acquisition time windows 20 are shifted in the time domain with respect to the gradient runs 15 at the pump. Thus, the portion 21 is correctly assigned to the first run. Furthermore, with regard to FIGS. 1 B) and 1 D), it will be appreciated that the data acquisition time windows correspond to the gradient as delivered at the detector.

[0948] Generally, in embodiments of the present invention, data acquisition time windows at the detector may be shifted with respect to analytical runs (or more specifically gradient runs) as delivered by the pump. This shift accounts for the time delay t.sub.delay, i.e., the time it takes for the solvent to travel from the pump to the detector. It will be appreciated that better and more reproducible chromatograms are typically generated when taking the delay time into consideration by shifting the data acquisition time windows as described.

[0949] In tandem LC applications the workflows may be typically highly optimized for high throughput to obtain one chromatogram right after another. Therefore, the size/duration of the elution window may be consuming a significant portion of the duration of the gradient. Under these conditions, it may occur that the chromatogram may lack fractions of the compounds of interest. This is, for instance, illustrated in FIG. 1C. Additionally, it may occur that the resulting chromatogram is composed of compounds eluted partly from one separation column and partly from the other, which may not be intended as the chromatogram would not be strictly associated with a single sample run and even an individual sample. This is, for instance, illustrated in FIG. 1C. Hence, it may be advantageous to optimize t.sub.delay.

[0950] It will be understood that embodiments of the herein presented approach allow to optimize the position of the elution window in the temporal domain, i.e., the period when compounds of interest are eluted from the separation column to achieve high sample throughput and uncompromised chromatographic performance. This may be realized by adjusting the start of the detector data acquisition relative to the start of the gradient as is illustrated in FIG. 1.

[0951] FIG. 2 depicts, as an example, a preferred embodiment of a liquid chromatography system according to an embodiment of the present invention in a first configuration I.

[0952] Embodiments of the present invention may be directed to utilizing a tandem liquid chromatography system, wherein two separation columns and two pumps are used. The liquid chromatography system, as illustrated in FIG. 2, may comprise a first separation column 8, a second separation column 5, a separation pump 1, a reconditioning pump 12 and a detector 22. The first separation column and the second separation column may be hosted in a column compartment 2. The system may comprise an autosampler 3, which may comprise an injection valve 10. The liquid chromatography system may further comprise a pre-column switching vale 13 and a post-column switching valve 7. The pre-column switching vale 13 and the post-column switching valve 7 may be hosted in the column compartment 2. The liquid chromatography system may comprise a waste.

[0953] Each of the injection valve 10, the pre-column switching valve 13 and the post-column switching valve 7 may comprise a stator, a rotor and a rotatable drive, respectively. Each stator may comprise a multitude of ports to which different elements in the liquid chromatography system may be fluidly connected to. Each rotor may comprise connecting elements, for example grooves, that may fluidly connect different ports of the stator. The rotor may be rotated relative to the stator using the rotatable drive, allowing the connecting elements of the rotor to establish fluidic connections between different ports of the stator.

[0954] One port of the injection valve 10 may have a fluidic connection 9 to one port of the pre-column switching valve 13. Another port of the injection valve 10 may have a fluidic connection 11 to the reconditioning pump 12. The port of the injection valve 10 which may have a fluidic connection 9 to one port of the pre-column switching valve 13, and the port of the injection valve 10 which may have a fluidic connection 11 to the reconditioning pump 12 may be fluidly connected by means of the connecting elements of the injection valve 10 in the configuration of FIG. 2. Furthermore, one port or a plurality of further ports of the injection valve 10 may have a fluidic connection to one or a plurality of sample reservoirs containing one or a plurality of samples.

[0955] One port of the pre-column switching valve 13 may have a fluidic connection 4 to the separation pump 1; another port of the pre-column switching valve 13 may have a fluidic connection to the first separation column 8; another port of the pre-column switching valve 13 may have a fluidic connection to the second separation column 5.

[0956] One port of the post-column switching valve 7 may have a fluidic connection 6 to the detector 22; another port of the post-column switching valve 7 may have a fluidic connection to the first separation column 8; another port of the post-column switching valve 7 may have a fluidic connection to the second separation column 5; another port of the post-column switching valve 7 may have a fluidic connection to a waste.

[0957] The configuration assumed by the pre-column switching valve 13 and by the post-column switching valve 7 may determine the fluidic connection between different elements of the chromatography system, or, in other words, the configuration of the liquid chromatography system. In particular, the configuration of the connecting elements of the pre-column switching valve 13 and of the post-column switching valve 7 determines the configuration of the liquid chromatography system.

[0958] As an example, in FIG. 2, an embodiment the liquid chromatography system is depicted, according to the present invention, in a first configuration I.

[0959] The port of the pre-column switching valve 13, which may have a fluidic connection to a port of the injection valve 10, and the port of the pre-column switching valve 13, which may have a fluidic connection to the second separation column 5, may be fluidly connected by means of the connecting elements of the pre-column switching valve 13 in the first configuration I. The port of the pre-column switching valve 13, which may have a fluidic connection to the separation pump 1, and the port of the pre-column switching valve 13, which may have a fluidic connection to the first separation column 8, may be fluidly connected by means of the connecting elements of the pre-column switching valve 13 in the first configuration I.

[0960] The port of the post-column switching valve 7, which may be have a fluidic connection to the first separation column 8, and the port of the post-column switching valve 7, which may have a fluidic connection to the detector 22, may be fluidly connected by means of the connecting elements of the post-column switching valve 7 in the first configuration I. The port of the post-column switching valve 7, which may be have a fluidic connection to the second separation column 5, and the port of the post-column switching valve 7, which may have a fluidic connection to the waste, may be fluidly connected by means of the connecting elements of the post-column switching valve 7 in the first configuration I.

[0961] The present invention, according a preferred embodiment, is at least in part directed to supplying a flow from the first separation column 8 into the detector 22 by means of the separation pump 1, in the first configuration I, as depicted in the preferred embodiment of FIG. 2. The first configuration I may be referred to as first steady state. In the first configuration I, the separation pump creates a flow from the first separation column 8 into the detector 22.

[0962] The present invention, according a preferred embodiment, is also at least in part directed to supplying a flow from the second separation column 5 into the waste by means of the reconditioning pump 12 in the first configuration I.

[0963] The method may further comprise the injection of a sample into the liquid chromatography system via the injection valve 10 and the pushing of the sample into the second separation column 5 by means of the reconditioning pump 12 in the first configuration I.

[0964] FIG. 3 depicts, as an example, a preferred embodiment of the system of FIG. 2 in a second configuration II.

[0965] The port of the pre-column switching valve 13, which may be have a fluidic connection to a port of the injection valve 10, and the port of the pre-column switching valve 13, which may have a fluidic connection to the first separation column 8, may be fluidly connected by means of the connecting elements of the pre-column switching valve 13 in the second configuration II. The port of the pre-column switching valve 13, which may be have a fluidic connection to the separation pump 1, and the port of the pre-column switching valve 13, which may have a fluidic connection to the second separation column 5, may be fluidly connected by means of the connecting elements of the pre-column switching valve 13 in the second configuration I.

[0966] The port of the post-column switching valve 13, which may be have a fluidic connection to the first separation column 8, and the port of the post-column switching valve 13, which may have a fluidic connection to the detector 22, may be fluidly connected by means of the connecting elements of the post-column switching valve in the second configuration II. The port of the post-column switching valve 13, which may be have a fluidic connection to the second separation column 5, and the port of the post-column switching valve 13, which may have a fluidic connection to the waste, may be fluidly connected by means of the connecting elements of the post-column switching valve 13 in the second configuration II.

[0967] The present invention is at least in part directed to supplying a flow from the first separation column 8 into the detector 22 by means of the reconditioning pump 12 in the second configuration II, as depicted in the preferred embodiment of FIG. 3. The second configuration II may be referred to as first intermediate state, wherein the reconditioning pump 12 creates a flow from the first separation column 8 into the detector 22.

[0968] The present invention is also at least in part directed to supplying a flow from the second separation column 5 into the waste by means of the separation pump 1 in the second configuration I as depicted in preferred the embodiment of FIG. 3.

[0969] The present invention is also at least in part directed to injecting a sample into the liquid chromatography system via the injection valve 10 and the pushing of the sample into the first separation column 8 by means of the reconditioning pump 12 in the second configuration II.

[0970] FIG. 4 depicts, as an example, a preferred embodiment of the system of FIG. 2 in a third configuration.

[0971] The port of the pre-column switching valve 13, which may be have a fluidic connection to a port of the injection valve 10, and the port of the pre-column switching valve 13, which may have a fluidic connection to the first separation column 8, may be fluidly connected by means of the connecting elements of the pre-column switching valve 13 in the third configuration. The port of the pre-column switching valve 13, which may be have a fluidic connection to the separation pump 1, and the port of the pre-column switching valve 13, which may have a fluidic connection to the second separation column, may be fluidly connected by means of the connecting elements of the pre-column switching valve 13 in the third configuration III.

[0972] The port of the post-column switching valve 13, which may be have a fluidic connection to the first separation column 8, and the port of the post-column switching valve 13, which may have a fluidic connection to the waste, may be fluidly connected by means of the connecting elements of the post-column switching valve 13 in the third configuration III. The port of the post-column switching valve 13, which may be have a fluidic connection to the second separation column 5, and the port of the post-column switching valve 13, which may have a fluidic connection to the detector 22, may be fluidly connected by means of the connecting elements of the post-column switching valve 13 in the third configuration III.

[0973] The present invention is at least in part directed to supplying a flow from the first separation column 8 into the waste by means of the reconditioning pump 12 in the third configuration III, as depicted in the preferred embodiment of FIG. 3.

[0974] The present invention is also at least in part directed to supplying a flow from the second separation column 5 into the detector 22 by means of the separation pump 1 in the third configuration III, as depicted in the preferred embodiment of FIG. 3. The third configuration III may be referred to as second steady state, wherein the separation pump 1 creates a flow from the second separation column 5 into the detector 22.

[0975] The present invention is also at least in part directed to injecting a sample into the liquid chromatography system via the injection valve 10 and the pushing of the sample into the first separation column 8 by means of the reconditioning pump 12 in the third configuration III.

[0976] FIG. 5 depicts, as an example, a preferred embodiment of the system of FIG. 2 in a fourth configuration.

[0977] The port of the pre-column switching valve 13, which may be have a fluidic connection to a port of the injection valve 10, and the port of the pre-column switching valve, which may have a fluidic connection to the second separation column 5, may be fluidly connected by means of the connecting elements of the pre-column switching valve 13 in the fourth configuration IV. The port of the pre-column switching valve 13, which may be have a fluidic connection to the separation pump 1, and the port of the pre-column switching valve 13, which may have a fluidic connection to the first separation column 8, may be fluidly connected by means of the connecting elements of the pre-column switching valve 13 in fourth configuration IV.

[0978] The port of the post-column switching valve 13, which may be have a fluidic connection to the first separation column 8, and the port of the post-column switching valve 13, which may have a fluidic connection to the waste, may be fluidly connected by means of the connecting elements of the post-column switching valve 13 in the fourth configuration IV. The port of the post-column switching valve 13, which may be have a fluidic connection to the second separation column 5, and the port of the post-column switching valve 13, which may have a fluidic connection to the detector 22, may be fluidly connected by means of the connecting elements of the post-column switching valve 13 in the fourth configuration III.

[0979] The present invention is at least in part directed to supplying a flow from the first separation column 8 into the waste by means of the separation pump 1 in the fourth configuration IV, as depicted in the preferred embodiment of FIG. 4.

[0980] The present invention is also at least in part directed to supplying a flow from the second separation column 5 into the detector 22 by means of the reconditioning pump 12 in the fourth configuration IV, as depicted in the preferred embodiment of FIG. 4. The fourth configuration IV may be referred to as second intermediate state, wherein the reconditioning pump 12 creates a flow from the second separation column 5 into the detector 22.

[0981] The present invention is also at least in part directed to injecting of a sample into the liquid chromatography system via the injection valve 10 and the pushing of the sample into the second separation column 5 by means of the reconditioning pump 12 in the fourth configuration IV.

[0982] Furthermore, as depicted in FIG. 2, FIG. 3, FIG. 4 and FIG. 5, the system may also comprise a controller 42. The controller 42 can be operatively connected to other components, as depicted by dashed lines in FIG. 2, FIG. 3, FIG. 4 and FIG. 5. For instance, the controller 42 may be operatively connected to the reconditioning pump 12, to the injection valve 10, to the detector 22, to the post-column switching valve 7, to any component that may serve, at least in part, a similar purpose to the column switching valve 7, to the separation pump 1, and to the pre-column switching valve 13. The controller 420 can include a data processing unit and may be configured to control the system and carry out particular method steps. The controller can send or receive electronic signals for instructions. The controller can also be referred to as a microprocessor. The controller can be contained on an integrated-circuit chip. The controller can include a processor with memory and associated circuits. A microprocessor is a computer processor that incorporates the functions of a central processing unit on a single integrated circuit (IC), or sometimes up to a plurality of integrated circuits, such as 8 integrated circuits. The microprocessor may be a multipurpose, clock driven, register based, digital integrated circuit that accepts binary data as input, processes it according to instructions stored in its memory and provides results (also in binary form) as output. Microprocessors may contain both combinational logic and sequential digital logic. Microprocessors operate on numbers and symbols represented in the binary number system.

[0983] FIG. 6 depicts an exemplary visualization of a workflow of a tandem liquid chromatography system with optimized acquisition time window based on four configurations of the tandem liquid chromatography system.

[0984] The present invention is at least in part directed to using a tandem direct injection workflow of a liquid chromatography system with optimized elution time window and optimized detection time window, based on the four configurations of the preferred embodiments of FIG. 2 to FIG. 5.

[0985] In particular, the elution window and the window for detector data acquisition may be aligned such, that consistent, reproducible chromatograms are obtained at optimum throughput, without relevant portions of the chromatogram being lost. This approach may be particularly beneficial with regard to tandem LC applications.

[0986] More in particular, four moments in time may be significant: a first switching time T.sub.II, a second switching time T.sub.III, a third switching time T.sub.IV and a fourth switching time T.sub.I, wherein T.sub.I is later than T.sub.IV, which is later than T.sub.III, which is later than T.sub.II. The system may be switched from the first configuration I to the second configuration II at the first switching time T.sub.II. The system may be switched from the second configuration II to the third configuration III at the second switching time T.sub.III. The system may be switched from the third configuration III to the fourth configuration IV the third switching time T.sub.IV. The system may be switched from the fourth configuration IV back to the first configuration I at the fourth switching time T.sub.I. In other words, the invention relates to a process where the system is switched among four configurations. The process may be a cyclic process, i.e. a process that repeats itself.

[0987] The system, in particular, may be switched from a steady state of the first configuration I to an intermediate state of the second configuration II, and subsequently to a steady state of the third configuration III, and subsequently to an intermediate state of the fourth configuration IV. It will be understood that a steady state of the liquid chromatography system identifies a state where the separation pump 1 provides a flow, via the first 8 or the second separation column 4, into the detector. It will also be understood that a temporary state of the liquid chromatography identifies a state where the reconditioning pump 12 provides a flow, via the first 8 or the second separation column 4, into the detector.

[0988] The time difference between T.sub.III and T.sub.II may be identified with t.sub.delay. The time difference between T.sub.I and T.sub.IV may also be identified with t.sub.delay.

[0989] With regard to the preferred embodiment of FIG. 6, the liquid chromatography system is intended to be in the first configuration I right before T.sub.II. At T.sub.II the pre-column switching valve 13 can switch the system from the first configuration I to the second configuration II. At T.sub.II, a fluidic connection between the separation pump 1, the second separation column 5 and the waste can be achieved.

[0990] In other words, at T.sub.II, the pre-column switching valve 13 may be switched to direct the gradient flow, which is simultaneously started, to the freshly conditioned second separation column 5. Simultaneously, the reconditioning pump 12 may deliver the last fraction of the gradient, that may have been generated by the separation pump 1 in a preceding step in the cycle through the first separation column 5, towards the post-column switching valve 7 where it may be directed towards the detector 22. There the compounds which may have been eluted from this last fraction of the gradient can be detected. During this phase, the flow rate of the reconditioning pump 12 may preferably be identical to the gradient flow rate of the separation pump 1, to assure consistent flow of the gradient solvents to the detector 22.

[0991] The separation pump 1 can start providing a gradient at T.sub.II, thereby starting an elution window. However, the detector may not start a new acquisition window 38 yet. This is due to the fact that the gradient, started at T.sub.II, may need an amount of time to traverse the fluidic connections between the separation pump 1 and the second separation column 5, and further to traverse the second separation column 5.

[0992] At T.sub.II, furthermore, a fluidic connection between the reconditioning pump 12, the autosampler 3, the first separation column 8 and the detector 22 can be achieved.

[0993] The reconditioning pump 12 can provide a flow from the first separation column 8 into the detector 22. During this stage, which may be called Wait, the reconditioning pump 12 can push contents of the first separation column 8 into the detector 22. The flow rate of the reconditioning, during this stage, can be the same as the separation pump. The detector 22 may not start a new acquisition window 38 at T.sub.II, but rather continue detecting in a previous acquisition window, and, in particular, continue detecting the flow from the first separation column 8 provided my means of the reconditioning pump 12. The detector can also stop detecting after T.sub.II and before T.sub.III, thereby ending the previous acquisition window, and wait to start a new acquisition window until T.sub.III.

[0994] At T.sub.III the post-column switching valve 7 can switch the system from the second configuration II to the third configuration III.

[0995] At T.sub.III, a fluidic connection between the separation pump 1, the second separation column 5 and the detector 22 can be achieved.

[0996] The separation pump 1 may have started providing the gradient at T.sub.II. Therefore, at T.sub.III, the gradient provided by the separation pump may have had the time to traverse the fluidic connections between the separation pump 1 and the second separation column 5, and further the second separation column 5, and further the fluidic connection between the second separation column 5 and the detector 22. Therefore, at T.sub.III, the detector 22 may start detecting in an acquisition window 38.

[0997] At T.sub.III, furthermore, a fluidic connection between the reconditioning pump 12, the autosampler 3, the first separation column 8 and the waste can be achieved.

[0998] The reconditioning pump 12 can provide a flow from the first separation column 8 into the waste. During this stage, the reconditioning pump 12 conditions the first separation column 8. The flow rate of the reconditioning pump 12, during this stage, can be the larger than the separation pump 1. It will be understood that the autosampler 3 may start injecting a sample into the liquid chromatography system at T.sub.II and may stop injecting the sample into the liquid chromatography after T.sub.III and before T.sub.IV.

[0999] In other words, at T.sub.III, which may be at t.sub.delay after T.sub.I, the post-column switching valve 7 may switch and the flow from the second separation column 5, which may contain solvent at a gradient start condition (for example, 16), may be directed towards the detector 22. In the case of the reconditioning pump 12, at timepoint at T.sub.III, which may be at t.sub.delay after T.sub.I, the reconditioning pump 12 will have delivered the last fraction of the gradient representing the gradient end concentration (for example, 17) through the post-column switching valve 7 towards the detector 22. It may now start conditioning the first separation column 8. To this, the flow of the reconditioning pump 12 may be increased to speed up washing and equilibration of the first separation column 8. At the post-column switching valve 7 the flow may be directed towards a waste container.

[1000] It should be noted that in the case that a mass spectrometry detector with a double barrel electrospray source is used for detection, instead of a post-column switching valve 7, also a switching of the high voltage between the two electro-sprayers could be applied, analogously, to act as the switching of the post-column switching valve 7.

[1001] Embodiments regarding the use of a mass spectrometry detector with a double barrel electrospray source is used for detection, instead of a post-column switching valve 7, will be discussed with regard to FIG. 8.

[1002] At T.sub.IV the pre-column switching valve 13 can switch the system from the third configuration III to the fourth configuration IV.

[1003] At T.sub.IV, a fluidic connection between the separation pump 1, the first separation column 8 and the waste can be achieved.

[1004] The separation pump 1 can start providing the gradient at T.sub.IV, thereby starting an elution window. However, the detector may not start a new acquisition window yet. This is due to the fact that the gradient may need an amount of time to traverse the fluidic connections between the separation pump 1 and the first separation column 8, and further to traverse the first separation column 8.

[1005] At T.sub.IV, furthermore, a fluidic connection between the reconditioning pump 12, the autosampler 3, the second separation column 5 and the detector 22 can be achieved.

[1006] The reconditioning pump 12 can provide a flow from the second separation column 5 into the detector 22. During this stage, which may be called Wait, the reconditioning pump 12 can push the contents of second separation column 5 into the detector 22.

[1007] In other words, the last fraction of the gradient, provided by the separation pump 1 into the second separation column 5 starting from T.sub.II, can be eluted into the detector by the reconditioning pump 12 starting from T.sub.IV. The flow rate of the reconditioning pump 12, during this stage, can be the same as the separation pump 1.

[1008] The detector 22 may not start a new acquisition window at T.sub.IV, but rather continue detecting, and, in particular, continue detecting the flow from the second separation column 5 provided my means of the reconditioning pump 12. In other words, the detector 22 may continue detecting the last fraction of the gradient, provided by the separation pump 1 into the second separation column 5 the starting from T.sub.II. The detector can also stop detecting after T.sub.IV, thereby ending the acquisition window 38, and before T.sub.I and wait to start a new acquisition window until T.sub.I.

[1009] At T.sub.I the post-column switching valve 7 can switch the system from the fourth configuration IV back to the first configuration I.

[1010] At T.sub.I, a fluidic connection between the separation pump 1, the first separation column 8 and the detector 22 can be achieved.

[1011] The separation pump 1 can start providing the gradient at Tv. Therefore, at T.sub.I, the gradient provided by the separation pump 1 may have had the time to traverse the fluidic connections between the separation pump 1 and the first separation column 8, and further the first separation column 8, and further the fluidic connection between the first separation column 8 and the detector 22. Therefore, at T.sub.I, the detector 22 may start detecting in a new acquisition window.

[1012] At T.sub.I, furthermore, a fluidic connection between the reconditioning pump 12, the autosampler 3, the second separation column 5 and the waste can be achieved.

[1013] The reconditioning pump 12 can provide a flow from the second separation column 5 into the waste. During this stage, the reconditioning pump 12 conditions the second separation column 5. The flow rate of the reconditioning pump 12, during this stage, can be the larger than the separation pump 1. It will be understood that the autosampler 3 may start injecting a sample into the liquid chromatography system at T.sub.I.

[1014] It will be understood that the system, after T.sub.I, can repeat the process described in relation to the embodiment of FIG. 6 in a periodic manner, switching from the first configuration I to the second configuration II at a time T.sub.II, which is later than T.sub.I. In other words, a cyclic process may be realized by system, wherein the system is switched among four configurations.

[1015] The subsequent cycle follows the same temporal sequence such that times T.sub.II, T.sub.III, T.sub.IV, T.sub.I of the subsequent cycle correspond, respectively, to the times T.sub.II, T.sub.III, T.sub.IV, T, in terms of temporal as well as functional characteristics.

[1016] The present invention is furthermore at least in part directed to optimizing the time difference t.sub.delay between T.sub.III and T.sub.II, and between T.sub.I and T.sub.IV. The optimization of the time difference t.sub.delay can lead, for example in a tandem chromatography system, to consistent and reproducible chromatograms, where a single detection window can contain only and all of the chromatographic peaks of a single compound and/or group of compounds of interest. The optimization of the time difference t.sub.delay can further lead, for example in a tandem chromatography system, to an optimum throughput of the tandem chromatography system.

[1017] In other words, because of the optimized delay between T.sub.III and T.sub.II, and between T.sub.I and T.sub.IV, the detector widow of acquisition can be optimally aligned with respect to the time when the single compound and/or group of compounds of interest reach the detector.

[1018] Generally, embodiments of the present invention may be described as follows. FIG. 2 depicts a liquid chromatography system in a first configuration I. In this configuration, the separation pump 1 is connected to the first separation column 8 and further to the detector 22. Furthermore, the reconditioning pump 12 is fluidly connected to the second separation column 5 and to a waste (not depicted in FIG. 1). This is a normal or steady state, where the second separation column 5 may be reconditioned and the separation pump 1 may provide a gradient to the first separation column 8 and further to the detector 22.

[1019] It will be understood that FIG. 4 depicts another normal or steady state, where the separation pump 1 is connected to the second separation column 5 and to the detector 22, and the reconditioning pump 12 is connected to the first separation column 8 and to the waste.

[1020] Embodiments of the present invention are directed in that the system additionally also assumes the state or configuration II depicted in FIG. 3 and/or IV depicted in FIG. 5. In the configuration II in FIG. 3, the reconditioning pump 12, which may also be referred to as second pump 12, is connected to the first separation column 8 to the detector 22. That is, different to the configurations I and III depicted in FIGS. 2 and 4, the system also assumes a configuration, wherein the second pump 12 is connected to the detector.

[1021] The system is switched from the configuration I in FIG. 2 to the configuration II in FIG. 2 at a first switching time T.sub.II, the index denoting the configuration the system is switched to. At the first switching time T.sub.II, it is preferred that the solvent composition and the flow rate delivered by the second pump 12 is identical to the one delivered by the separation pump 1. It will be understood that the flow rate and solvent composition should be identical as delivered at the pre-column switching valve 13, as this is where the change of the fluidic connections becomes effective.

[1022] As discussed above, there generally is a delay time t.sub.delay between when a certain solvent composition is present at the separation pump 1 (as part of a gradient operation) and when this solvent composition arrives at the detector 22. In embodiments of the present invention, the system is operated in the configuration II of FIG. 3 for a duration corresponding to this delay time t.sub.delay. That is, for a duration t.sub.delay, fluid is delivered from the first separation column 8 to the detector 22 by means of the second pump 12.

[1023] This is also depicted in FIG. 6. According to this Figure, the pre-column switching valve 13 is switched at time T.sub.II. Subsequently, for the delay time t.sub.delay, the reconditioning pump 12 waits, i.e., is used to effect a flow from the first separation column 8 to the detector 22. During this time, there is data acquisition and a short wait time at the detector 22.

[1024] Once the last part of the gradient has reached the detector 22, the system is switched from the configuration II in FIG. 3 to the configuration III in FIG. 4. In this configuration, the first separation column 8 is connected to the reconditioning pump 12 and to the waste. Further, the second separation column 5 is connected to the separation pump 12 and to the detector 22. Relating to the separation column 5, reference is again made to the configuration II of FIG. 3. In this configuration II, the second separation column 5 is already connected to the separation pump 1. However, it is not yet connected to the detector 22, but to the waste. In this configuration, the separation pump 1 may start its gradient delivery (see FIG. 6 at the first switching time T.sub.II). Again, it will be understood that a solvent composition provided by the separation pump at a certain point of time will only reach downstream components later. In particular, this solvent composition at the start of the gradient delivery may take approximately the delay time t.sub.delay to reach the post-column switching valve. Only at this point of time, the solvent present at the post-column switching valve may be of interest for further detection. Thus, at the second switching time T.sub.III, which is the delay time t.sub.delay later than the first switching time T.sub.II, the system may be switched from the second configuration II of FIG. 3 to the third configuration III of FIG. 4. At this time, a new data acquisition time window may start (see 38 in FIG. 6), which corresponds to the gradient delivered by the separation pump 1 arriving at the detector 22.

[1025] Furthermore, it will be appreciated that in the configuration III of FIG. 4, the first separation column 8 is no longer connected to the detector 22, but to waste. In this configuration, the first separation column 8 may thus be reconditioned. This is depicted in FIG. 6, where, staring at the third switching time T.sub.III, the reconditioning pump 12 provides a column reconditioning. It should be understood that this column reconditioning may be performed with flow rates different from the flow rate delivered during the gradient delivery. In particular, it may be performed with higher flow rates. After the first separation column 8 has been reconditioned, it may be loaded with another sample. Further, the reconditioning pump 12 may perform an aligning step before the third switching time T.sub.IV.

[1026] In this regard, it will be understood that the fourth configuration IV depicted in FIG. 5 essentially corresponds to the second configuration II depicted in FIG. 3, but with reversed roles for the first separation column 8 and the second separation column 5. Again, the second separation column 5, in the configuration III assumed before configuration IV, is connected to the separation pump 1 and to the detector 22. In the configuration IV (see FIG. 5), the second separation column 5 is still connected to the detector 22, but no longer to the separation pump 1, but instead to the reconditioning pump 12. Similar to the configuration in FIG. 3, the reconditioning pump 12 effects the last sections of the gradient to flow from the second separation column 5 to the detector 22. Again, it may be beneficial if the operational parameters of the solvent provided a the third switching time T.sub.IV, i . . . e, when the system switches to configuration IV depicted in FIG. 5, are identical for the separation pump 1 and the second pump 12, which may also be referred to as reconditioning pump 12. Again, it will be understood that when switching to the fourth configuration IV in FIG. 5, the reconditioning pump 12 takes over the function of the separation pump 1 in configuration IV. It is thus beneficial for the operational parameters (in particular flow rate and solvent composition) to be identical at the switching time T.sub.IV. This is why prior to the switching time T.sub.IV (see FIG. 6), the reconditioning pump 12 and the separation pump 1 are aligned with one another, meaning that there operational parameters are identical at the switching time T.sub.IV. At T.sub.IV, the pre-column switching valve is switched, such that the configuration IV of FIG. 5 is assumed.

[1027] Again, this configuration IV essentially corresponds to the configuration II of FIG. 3, with the roles of the separation columns 5 and 8 reversed. It will thus be understood that at a fourth switching time T.sub.I, the system may again switch to configuration I depicted in FIG. 2 and that the overall operation be cyclical between the configurations I, II, III, and IV.

[1028] With regard to FIG. 6, the following considerations will be appreciated. Firstly, as described, the fluid is caused to arrive at the detector 22 both by the separation pump 1 and the reconditioning pump 12 (see boxes indicating with wait for the reconditioning pump 12 in FIG. 6 and configurations II and IV, where the reconditioning pump 12 is connected to the detector 22). Secondly, the acquisition time windows 38 are shifted with regard to the gradient delivery of the separation pump 1 (see FIG. 6) to account for time delay t.sub.delay between a certain solvent composition being delivered by the pump and arriving at the detector 22.

[1029] Overall, embodiments of the present technology thus allow for an increased usage time of the described system. In particular, the detector 22 may be supplied with solvents containing samples a higher percentage of the time as compared to other configurations. Further, by shifting the data acquisition time windows, signals may be more correctly assigned to a certain sample than is possible without such a shift.

[1030] FIG. 7 illustrates, as an example, UV chromatograms of Cytochrome C showing the influence on UV chromatograms of Cytochrome C of the time difference between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system.

[1031] More specifically, in FIG. 7, at the example of a UV Chromatogram of Cytochrome C, the elution window and thus the resulting chromatogram is step-wise shifted to the left by adjusting the start of the detector acquisition relative to the start of the gradient delivery.

[1032] The grey areas of the chromatograms of the embodiment of FIG. 7 indicate areas of the chromatograms that are not included in the detection window. The full UV chromatogram of Cytochrome C, can show fifteen peaks, with a first peak 23 and a last peak 37 in the sequence.

[1033] FIG. 7A depicts a UV chromatogram of Cytochrome C measured with a delay time of zero minutes between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system. In this case, the last peaks 35, 36, and 37 in the sequence of peaks of the full UV chromatogram of Cytochrome C may not be included in the detection window.

[1034] FIG. 7B depicts a UV chromatogram of Cytochrome C measured with a delay time of one minute between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system. In this case, the last peaks 36, and 37 (see FIG. 7 D) in the sequence of peaks of the full UV chromatogram of Cytochrome C may not be included in the detection window.

[1035] Overall, peaks at the far end (i.e. at high retention times) of the chromatogram may be missing, as illustrated in FIG. 7A and FIG. 7B.

[1036] FIG. 7C depicts a UV chromatogram of Cytochrome C measured with a delay time of three minutes between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system. In this case, all peaks in the sequence of peaks of the full UV chromatogram of Cytochrome C may be included in the detection window.

[1037] FIG. 7D depicts a UV chromatogram of Cytochrome C measured with a delay time of four minutes between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system. In this case, all peaks in the sequence of peaks of the full UV chromatogram of Cytochrome C may be included in the detection window.

[1038] FIG. 7E depicts a UV chromatogram of Cytochrome C measured with a delay time of five minutes between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system. In this case, all peaks in the sequence of peaks of the full UV chromatogram of Cytochrome C may be included in the detection window.

[1039] Overall, all peaks may be present in the chromatogram, as illustrated in FIG. 7C, FIG. 7D and FIG. 7E.

[1040] FIG. 7F depicts a UV chromatogram of Cytochrome C measured with a delay time of six minutes between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system. In this case, the first peaks 23, and 24 in the sequence of peaks of the full UV chromatogram of Cytochrome C may not be included in the detection window.

[1041] FIG. 7G depicts a UV chromatogram of Cytochrome C measured with a delay time of seven minutes between the start of detector acquisition and the start of gradient delivery in a liquid chromatography system. In this case, the first peaks 23, 24, 25, and 26 in the sequence of peaks of the full UV chromatogram of Cytochrome C may not be included in the detection window.

[1042] Overall, if the time difference between the start of detector acquisition and the start of gradient delivery is even further increased, the resulting chromatograms may be lacking the first part of the eluting compounds to an increasing degree, as illustrated FIG. 7F and FIG. 7G.

[1043] Overall, this shows that a suitable delay time t.sub.delay should ideally be chosen to include all the relevant peaks for a sample analysis. In the depicted example, delay times t.sub.delay of 2, 3 and 4 minutes (see FIGS. 7 C), D), E)) would be suitable to include all the peaks in the chromatogram, and it will be understood that suitable delay times may either be found by a user, or by a software driven approach.

[1044] It will be understood that embodiments of the herein presented approach may allow the period in which no compounds may be eluted from a separation column owing to a lack of eluting solvents during this time can be reduced (see, e.g., the shaded areas in FIG. 7). This may allow to reduce the cycle time and thus increase the throughput. Additionally, it may save storage space as no detector data may be recorded during the timeframe that does not contain any data that would be relevant for the analysis.

[1045] FIG. 8 depicts, as an example, preferred embodiments of a liquid chromatography system utilizing a double barrel electrospray source and adopting four configurations according to embodiments of the present invention.

[1046] The configuration I, II, III, IV of FIG. 8 may correspond, at least in part, to features of, e.g., the embodiments of any of the preceding figures.

[1047] The liquid chromatography system may comprise a double barrel electrospray source 39. The double barrel electrospray source 39 may be used, for instance, in place of the post-column switching valve 7 of the embodiments of FIG. 2, FIG. 3, FIG. 4, and/or FIG. 5. The liquid chromatography system may comprise a detector 22, which may be mass spectrometry detector.

[1048] A double barrel electrospray source 39 may allow to selectively introduce fluid from one of the separation columns 5, 8 into the detector 22, while for example connecting the other separation column 8, 5 to waste (not depicted).

[1049] In the embodiment depicted in FIG. 8, the first separation column 8 and the second separation column 5 are not contained in the column compartment 2. Instead, they may be contained in a column heater outside the column compartment 2.

[1050] Also in this embodiment, a detector 22 is provided, which may be a mass spectrometer (MS) 22. The first separation column 8 and the second separation column 5 may be arranged close to the detector 22. Each of the columns 5, 8 may comprise an outlet. The outlet of the first column 8 may be connected to a first emitter and the outlet of the second column 5 may be connected to a second emitter. The emitters are configured to spray directly into the MS 22. Generally, the system is configured to selectively apply a high voltage to one of the emitters. Thus, only the liquid arriving at the emitter where the high voltage is applied is sprayed into the detector chamber. This functionality is depicted in FIG. 8 by element 39. Selectively applying the high voltage thus effectively has the functionality of a valve, as only the liquid at the emitter where the high voltage is applied is sprayed towards the detector. In configurations I and II, the emitter connected to the first separation column 8 is supplied with high voltage, such that liquid in this branch is supplied to the MS 22, while in configurations III and IV, the emitter connected to the second separation column 5 is supplied with high voltage, such that liquid in this branch is supplied to the MS 22. Overall, this therefore defines two different barrels which may be connected to the MS 22 by applying the high voltage, which is why this system may also be referred to as a double barrel electrospray system.

[1051] As discussed, the columns 5, 8 may be located outside of the column compartment and generally close to the MS detector 22. Thus, at the outlet of the columns, there is directly the emitter where the electrospray is generated. This spray advantageously is in direct vicinity of an inlet of the MS 22 to allow for transfer of the generated charged species into the MS 22.

[1052] Generally, the liquid chromatography system depicted in FIG. 8 may be configured in such a way that a high voltage is applied to only a barrel at a time for electrospray ionization, i.e. to either the first barrel or to the second barrel at a time. Put differently, only one emitter at a time may spray into the detector 22. For example, the first barrel may be subject to a high voltage and the first emitter of the first barrel may therefore spray into the detector 22, while the contents of the second barrel may evaporate while traversing the second barrel or be directed to a waste, both of which is referred to as the barrel or the respective separation column being fluidly connected to waste. Vice versa, the second barrel may be subject to a high voltage and the second emitter of the second barrel may therefore spray into the detector 22, while the contents of the first barrel may evaporate while traversing the first barrel or be directed to a waste, i.e., the first barrel is fluidly connected to waste.

[1053] By alternatively switching the high voltage that the first barrel and of the second barrel are subject to, the double barrel electrospray source 39 may serve, at least in part, a similar purpose to the post column switching valve 7 depicted in FIGS. 2 to 5. The use of the double barrel electrospray source 39 may be preferably employed when, e.g., the flowrates determined by a pump for liquid chromatography are smaller than 1 L/min, as in, for instance, nano flow liquid chromatography-mass spectrometry applications. The use of the double barrel electrospray source 39 may have the benefit that the volume of the fluidic connection between at least one separation column and the detector in a liquid chromatography may be minimized. This may result in lower dispersion and lower gradient delay resulting in better chromatographic performance in terms of, but limited to, peak resolution and/or throughput.

[1054] In one embodiment of FIG. 8, the first barrel may be subject to a high voltage in the first configuration I and the first emitter of the first barrel may therefore be enabled to spray into the detector 22 in the first configuration I. In one embodiment of FIG. 8, the first barrel may be subject to a high voltage in the second configuration II and the first emitter of the first barrel may therefore be enabled to spray in to the detector 22 in the second configuration II. In one embodiment of FIG. 8, the second barrel may be subject to a high voltage in the third configuration III and the second emitter of the second barrel may therefore be enabled to spray in to the detector 22 in the third configuration III. In one embodiment of FIG. 8, the second barrel may be subject to a high voltage in the fourth configuration IV and the second emitter of the second barrel may therefore be enabled to spray in to the detector 22 in the fourth configuration IV.

[1055] Generally, it will thus be understood that the first configuration I depicted in FIG. 8 functionally corresponds to the configuration I depicted in FIG. 2. In this configuration, the separation pump 1 is connected to the first separation column 8, and the high voltage is supplied to the first barrel, such that the first emitter of the first barrel sprays into the detector 22 (which functionally corresponds to the first separation column 8 being connected to the detector).

[1056] Furthermore, the configuration II in FIG. 8 generally corresponds to the configuration II depicted in FIG. 3. In this configuration, the reconditioning pump 12 is fluidly connected to the first separation column 8, and the high voltage is supplied to first barrel, such that the first emitter of the first barrel sprays into the detector 22.

[1057] Similarly, it will be understood that the configurations III and IV depicted in FIG. 8 generally correspond to the configurations III and IV depicted in FIGS. 4 and 5, respectively.

[1058] The skilled person will thus understand that advantages as described above for the system comprising a post-column switching valve 7 may also be achieved when using the system of FIG. 8 with a double barrel electrospray source.

[1059] In this regards, it will also be understood that a controller 42 as depicted in FIGS. 2 to 5 is also typically present in the system of FIG. 8, but has been omitted in FIG. 8 for ease of illustration.

[1060] Whenever a relative term, such as about, substantially, essentially or approximately is used in this specification, such a term should also be construed to also include the exact term. That is, e.g., substantially straight should be construed to also include (exactly) straight.

[1061] Whenever steps were recited in the above or also in the appended claims, it should be noted that the order in which the steps are recited in this text may be accidental. That is, unless otherwise specified or unless clear to the skilled person, the order in which steps are recited may be accidental. That is, when the present document states, e.g., that a method comprises steps (A) and (B), this does not necessarily mean that step (A) precedes step (B), but it is also possible that step (A) is performed (at least partly) simultaneously with step (B) or that step (B) precedes step (A). Furthermore, when a step (X) is said to precede another step (Z), this does not imply that there is no step between steps (X) and (Z). That is, step (X) preceding step (Z) encompasses the situation that step (X) is performed directly before step (Z), but also the situation that (X) is performed before one or more steps (Y1), . . . , followed by step (Z). Corresponding considerations apply when terms like after or before are used.