METHODS FOR INJECTING SAMPLES IN LIQUID CHROMATOGRAPHY, PARTICULARLY IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Abstract

A sample injection method for liquid chromatography is performed with an injection valve having a waste port, two sample loop ports, and two high-pressure ports. One high-pressure port can be connected to a pump and the other high-pressure port can be connected to a chromatography column. A sample loop is connected to one of the sample loop ports on one end and to a pump volume of a sample conveying device on the other end. A section of the sample loop can be separated to facilitate receiving a sample fluid in the sample loop. A control unit controls the injection valve and the sample conveying device. The sample injector allows a sample to be loaded into the sample loop and then pressurized to an operating pressure prior to injecting the sample into the chromatography column. The sample loop may also be isolated from the operating pressure for facilitating depressurization of the loop.

Claims

1. A method of operating a liquid chromatography system, the method including: (a) isolating a sample loop of the liquid chromatography system from a high-pressure loop of the liquid chromatography system; (b) performing a pressure compensation operation including one of (i) when isolating the sample loop from the high-pressure loop leaves the sample loop at essentially an operating pressure of a liquid chromatography column, increasing a volume connected in the sample loop to reduce the pressure in the sample loop to essentially ambient pressure, or (ii) when the isolated sample loop is at essentially ambient pressure preparatory to injecting a sample liquid into the high-pressure loop, decreasing the volume connected in the sample loop to increase the pressure in the sample loop to essentially the operating pressure of the liquid chromatography column; and (c) when the pressure compensation operation comprises decreasing the volume connected in the sample loop to increase the pressure in the sample loop to essentially the operating pressure of the liquid chromatography column, further including connecting the sample loop to the high-pressure loop so that a pump pressure from a high-pressure pump of the liquid chromatography system is applied to the sample loop and a sample liquid in the sample loop is free to flow from the sample loop through a portion of the high-pressure loop to the chromatography column.

2. The method of claim 1 further including, after performing step (c) of claim 1 to introduce the sample liquid into the chromatography column: (a) isolating the sample loop from the high-pressure loop once the sample liquid is introduced into the chromatography column; and (b) increasing the volume connected in the sample loop to reduce the pressure in the sample loop to essentially ambient pressure.

3. The method of claim 1 wherein isolating the sample loop from the high-pressure loop includes placing an injection valve in a PRESSURE COMPENSATION position in which (i) first and second sample loop ports of the injection valve are closed so as to facilitate pressurization of the sample loop, and (ii) first and second high-pressure ports of the injection valve are connected so as to operatively connect the high-pressure pump to the chromatography column.

4. The method of claim 1 wherein placing the injection valve in the PRESSURE COMPENSATION position includes rotating a rotor of the injection valve with respect to a stator of the injection valve.

5. The method of claim 1 wherein the volume connected to the sample loop is defined by a cavity in which a movable element is slidably mounted, and wherein changing the volume connected to the sample loop includes sliding the movable element within the cavity.

6. The method of claim 5 further including measuring the pressure of a fluid in at least one of the sample loop or the volume connected to the sample loop with a pressure sensor.

7. The method of claim 5, wherein the movable element is connected to a drive device which is operable to move the movable element within the cavity, and further including measuring a force exerted upon the movable element by the drive device.

8. A method of injecting a sample in a liquid chromatography system, the method including: (a) isolating a sample loop of the liquid chromatography system from a high-pressure loop of the liquid chromatography system; (b) placing a sample liquid in the sample loop preparatory to injecting the sample liquid into the high-pressure loop; (c) with the sample liquid placed in the sample loop and with the sample loop remaining isolated from the high-pressure loop, performing a pressure compensation operation in which a volume connected in the sample loop is decreased to raise the pressure in the sample loop from ambient pressure to the operating pressure of a liquid chromatography column of the liquid chromatography system; and (d) connecting the sample loop to the high-pressure loop so that a pump pressure from a high-pressure pump is applied to the sample loop to cause the sample liquid in the sample loop to flow from the sample loop through a portion of the high-pressure loop to the chromatography column.

9. The method of claim 8 further including: (a) isolating the sample loop from the high-pressure loop of the liquid chromatography system after the sample liquid has flowed into the high-pressure loop; and (b) increasing the volume connected in the sample loop to reduce the pressure in the sample loop to ambient pressure.

10. The method of claim 8 wherein isolating the sample loop from the high-pressure loop includes placing an injection valve in a PRESSURE COMPENSATION position in which (i) first and second sample loop ports of the injection valve are closed so as to facilitate pressurization of the sample loop, and (ii) first and second high-pressure ports of the injection valve are connected so as to operatively connect the high-pressure pump to the chromatography column.

11. The method of claim 10 wherein placing the injection valve in the PRESSURE COMPENSATION position includes rotating a rotor of the injection valve with respect to a stator of the injection valve.

12. The method of claim 8 wherein the volume connected to the sample loop is defined by a cavity in which a movable element is slidably mounted, and wherein decreasing the volume connected to the sample loop includes sliding the movable element within the cavity.

13. The method of claim 12 further including measuring the pressure of a fluid in at least one of the sample loop or the volume connected to the sample loop with a pressure sensor.

14. The method of claim 12 wherein the movable element is connected to a drive device which is operable to move the movable element within the cavity, and further including measuring a force exerted upon the movable element by the drive device.

15. A method of preparing a liquid chromatography system for receiving a sample liquid, the method including: (a) isolating a sample loop of the liquid chromatography system from a high-pressure loop of the liquid chromatography system; and (b) with the sample loop isolated from the high-pressure loop and with the pressure in the sample loop remaining at an operating pressure of the liquid chromatography column, performing a pressure compensation operation in which a volume connected in the sample loop is increased to reduce the pressure in the sample loop to ambient pressure.

16. The method of claim 15 wherein isolating the sample loop from the high-pressure loop includes placing an injection valve in a PRESSURE COMPENSATION position in which (i) first and second sample loop ports of the injection valve are closed so as to facilitate pressurization of the sample loop, and (ii) first and second high-pressure ports of the injection valve are connected so as to operatively connect the high-pressure pump of the liquid chromatography system to the chromatography column.

17. The method of claim 16 wherein placing the injection valve in the PRESSURE COMPENSATION position includes rotating a rotor of the injection valve with respect to a stator of the injection valve.

18. The method of claim 15 wherein the volume connected to the sample loop is defined by a cavity in which a movable element is slidably mounted, and wherein increasing the volume connected to the sample loop includes sliding the movable element within the cavity.

19. The method of claim 18 further including measuring the pressure of a fluid in at least one of the sample loop or the volume connected to the sample loop with a pressure sensor.

20. The method of claim 18 wherein the movable element is connected to a drive device which is operable to move the movable element within the cavity, and further including measuring a force exerted upon the movable element by the drive device.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The invention is described in greater detail below with reference to the drawings. In these drawings:

[0025] FIG. 1 shows a schematic representation of an HPLC system with a sample injector according to the invention, to which a chromatography column is connected, wherein the injection valve is situated in the LOAD position and the process of taking in a sample volume can begin in the state shown;

[0026] FIG. 2 shows the HPLC system of FIG. 1, wherein the plunger of the syringe was moved into the end position (position C) in order to take in the sample volume;

[0027] FIG. 3 shows the HPLC system of FIG. 2, wherein the sample needle was moved into the injection port;

[0028] FIG. 4 shows the HPLC system of FIG. 3, wherein the injection valve was changed over from the LOAD position into the PRESSURE COMPENSATION position;

[0029] FIG. 5 shows the HPLC system of FIG. 4, wherein the plunger was moved into the position B in order to realize a pressure compensation (pressure increase) in the sample loop;

[0030] FIG. 6 shows the HPLC system of FIG. 5, wherein the injection valve was changed over from the PRESSURE COMPENSATION position into the INJECT position;

[0031] FIG. 7 shows the HPLC system of FIG. 6, wherein the injection valve was changed over from the INJECT position into the PRESSURE COMPENSATION position after the injection of the sample volume;

[0032] FIG. 8 shows the HPLC system of FIG. 7, wherein the plunger was moved into the end position (position C) in order to realize a pressure compensation (pressure reduction), and

[0033] FIG. 9 shows the HPLC system of FIG. 8, wherein the injection valve was changed over from the PRESSURE COMPENSATION position into the LOAD position.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0034] FIG. 1 shows a schematic representation of an HPLC system with a sample injector 10 that operates in accordance with the Split Loop Principle and features a sample conveying device 5, an injection valve 3 and a high-pressure pump 40. The sample injector 10 furthermore features a sample loop that includes a first connecting piece 51 and a second connecting piece 52, 44. These may be comprised of a pressure-resistant line with a small diameter, for example in the form of a capillary tube of glass or stainless steel. The connecting piece 51 is connected to a first sample loop port 16 of the injection valve 3 and to the sample conveying device or its pump volume V, respectively. The second connecting piece is comprised of an intake segment 44 and a feed segment 52 and is realized in a separable fashion. For this purpose, the feed segment 52 leads into an injection port 45 that is connected to a second sample loop port 13 of the injection valve 3 via the feed segment 52. The intake segment 44 that is connected to the pump volume V of the sample conveying device 5 with one end features on its other end a sample needle 42, by means of which the intake segment 44 can be connected to the injection port 45.

[0035] However, the sample needle 42 can also be moved to a sample container 43 and take in a defined sample volume into the intake segment 44 as described in greater detail below. Furthermore, the sample needle 41 can also be moved to a (not-shown) container for a flushing fluid in order to withdraw flushing fluid for a flushing process and to clean the sample loop 51, 52, 44, the pump volume V and, if applicable, also the ports and the grooves or channels of the injection valve. Due to the special topology of the Split Loop Principle shown, flushing of the sample loop 51, 52, 44 and of the sample conveying device 5 is normally not required because they are flushed during an injection process anyway, namely with eluent supplied by the pump 40. However, the outside of the sample needle 42 can also be cleaned by immersing the needle into a container with cleaning or flushing fluid.

[0036] In the embodiment shown, the sample conveying device 5 comprises a syringe 50, in which a plunger 53 is guided in a displaceable and pressure-tight fashion. The plunger 53 is driven by means of a drive 55 that is realized, for example, in the form of a stepping motor. The drive 55 is controlled by a control unit 60. The control unit 60 also controls the change-over processes of the injection valve 3 that features a not-shown controllable drive.

[0037] A waste port 12 of the injection valve is connected to a waste line 47, from which fluid can be discharged into a not-shown waste reservoir.

[0038] The high-pressure pump 40 is connected to a high-pressure port 15 of the injection valve. A chromatography column 41 is connected to the other high-pressure port 14. The high-pressure pump 40 may be integrated into and form part of the sample injector or be arranged in another unit or a separate pump unit.

[0039] The injection valve 3 includes a stator 1 and a rotor 2. The stator 1 features the two high-pressure ports 14, 15, the two sample loop ports 13, 16 and the waste port 12. The injection valve 3 is connected to the other functional elements of the HPLC system via these ports and the above-described connecting lines that may be realized in the form of capillary connections. The high-pressure screw connections required for this purpose are not illustrated in FIG. 1 in order to provide a better overview. For reasons of simplicity, the injection valve is illustrated in the interface between the stator 1 and the rotor 2, wherein the design of the face of the stator 1 and the design of the face of the rotor 2 are shown in order to better comprehend the function of the injection valve. Within the injection valve 3, the ports are realized in the form of bores that lead to the other side of the stator 1. The rotor 2 features a number of arc-shaped grooves 21, 23, 25 that are exactly aligned with the bores of the input and output ports.

[0040] The rotor 2 is pressed against the stator with a certain pressing force such that a common interface between the rotor 1 and the stator 2 is formed, at which both components are mutually sealed. In this case, the pressing force is chosen so high that the arrangement also remains sealed at the highest pressures to be expected.

[0041] In the first LOAD position of the valve 3 illustrated in FIG. 1, the grooves 21, 23, 25 are aligned relative to the ports 12-16 in such a way that the grooves 23 and 25 respectively connect the two high-pressure ports 14, 15 and the waste port 12 to the sample loop port 13. In this LOAD position, the high-pressure pump 40 therefore conveys fluid in the direction of the chromatography column 41. Furthermore, the sample loop port 16 is closed in a pressure-tight fashion.

[0042] In the state illustrated in FIG. 1, the sample needle 42 is moved into the sample container 43 such that a sample volume can be taken in. For this purpose, the plunger 53 is situated in the position A and can be moved into the position C by the control unit 60 in order to take in the sample volume. The desired defined sample volume is then withdrawn into the intake segment 44, wherein the volume of the sample is smaller than the volume of the intake segment 44 such that the sample fluid cannot mix with the fluid supplied by the high-pressure pump in the pump volume. FIG. 2 shows the state of the HPLC system after the intake process is completed.

[0043] In order to inject the sample volume situated in the intake segment 44, the sample needle 42 is moved into the injection port 45. This port seals the needle point in a high-pressure-resistant fashion. This state is illustrated in FIG. 3.

[0044] In the next step, the pressure in the sample loop is adjusted to the operating pressure of the chromatography column 41, i.e., to the pressure, with which the high-pressure pump 40 supplies fluid to the inlet of the chromatography column 41. For this purpose, the injection valve is initially changed over into a PRESSURE COMPENSATION position, in which the connecting piece 51 and the second connecting piece or the feed segment 52 of the sample loop are not connected to the other components connected to the injection valve 3 (FIG. 4).

[0045] In this PRESSURE COMPENSATION position, the plunger 53 of the high-pressure-resistant sample conveying device is moved into the position B (FIG. 5). In order to prevent an interruption of the flow through the chromatography column 41 while conveying the volume required for the compression of the sample loop content, the groove 25 in the rotor 2 of the valve is realized in a correspondingly elongated fashion such that the two high-pressure ports 14, 15 are still connected in the PRESSURE COMPENSATION position. The travel of the plunger 53 from position C into position B required for building up the pressure can be calculated from the compressibility of the fluid volume enclosed in the sample conveying device 5 and in the sample loop, the elasticity of the arrangement and the current pump pressure. Alternatively, a pressure compensation can be achieved with the aid of a control circuit for the pressure in the high-pressure-resistant sample conveying device. For this purpose, the pressure needs to be measured at a suitable location and the position of the plunger 53 in the sample conveying device 5 needs to be adjusted by the drive 55 in such a way that the pressure corresponds to the required target pressure (=column pressure). Pressure measurement may be realized with a pressure sensor such as sensor 56 or indirectly by means of a force measurement. Conceivable solutions are force measurements on the plunger 53 or in the drive 55. After pressure equality is achieved, the valve is changed over into the INJECT position in order to inject the sample volume into the column 41 (FIG. 6).

[0046] In the embodiment shown, the control unit 60 measures the force that the drive 55 needs to exert in order to achieve a corresponding compression in the sample loop. For this purpose, the drive 55 may feature an integrated sensor 57, the signal of which is fed to the control unit 60 (as indicated with a double arrow between the drive 55 and the control unit 60). Due to this measure, the control unit can determine the actual pressure in the pump volume and therefore in the sample loop (the pressure drop in the connecting pieces and in the valve is negligibly small) and adjust this pressure to the desired value.

[0047] After the entire sample volume has been conveyed from the intake segment 44 to the column 41 by means of the fluid conveyed by the pump 40, the valve can be once again changed over into the PRESSURE COMPENSATION position in order to decompress the sample loop (FIG. 7).

[0048] The plunger 53 is moved from the position illustrated in FIG. 7 into position C. This causes the pressure in the sample loop to be adjusted to the atmospheric pressure. This state of the HPLC system is illustrated in FIG. 8. During this decompression time in the PRESSURE COMPENSATION position of the injection valve 3, the column 41 is already connected to the pump 40 via the elongated groove 25 in order to prevent pressure drops. The travel of the plunger 53 from position B to position C can either be calculated analogous to the compression in FIG. 5 or determined by measuring and controlling the pressure. Alternatively, the pressure can also be determined indirectly by means of a force measurement on the plunger 53 or on the drive 55 of the plunger.

[0049] After the sample loop has been decompressed, the valve 3 is changed over into the LOAD position (FIG. 9). No damaging flows in the injection valve occur during this process.

[0050] The plunger 53 of the high-pressure-resistant sample conveying device 5 can now be moved back into the starting position A. The excess quantity of fluid is discharged via the waste connection 47. The unpressurized needle 42 can subsequently be moved from the needle seat of the injection port 45 to the corresponding sample bottle in order to take in the next sample.

[0051] The position C during the decompression may also differ from the starting position A prior to the compression. For example, if gradients (time-controlled mixing ratio of the eluent) are pumped through the column, the position C at the end of the decompression may differ because the compressibility of the loop content may have changed.

[0052] The control unit 60 can store predetermined positions A, B, C and/or differences in the distance between these positions as a function of parameters of the entire sample injector, particularly the compressibility of the eluent, elasticity properties of the sample loop and the sample conveying device, etc. The plunger can then be automatically moved into these positions (i.e., without a control) or these positions may serve as approximate values or initial values for a controlled movement.

[0053] In order to determine the positions A, B, C and the respective travel of the plunger, a change-over of the injection valve 3 may be carried out without compression or decompression, respectively. The pressure drop can then be determined by means of a pressure sensor and the required travel as well as the respective positions B or C can be determined based on this pressure drop. The thusly determined values can then be stored and used for other change-over processes, in which a compression or decompression takes place. A corresponding sensor may also be provided in the pump 40. Pumps of this type for HPLC always feature a pressure sensor for controlling the conveyed eluent anyway. The compressibility of the medium, particularly of the eluent, can also be determined by means of the pump 40. Such pumps are realized, for example, in the form of dual-plunger pumps, in which the change-over from one plunger to the other plunger is suitably controlled or regulated by means of a pressure sensor and a control unit in such a way that a highly constant flow rate is achieved. Since the compressibility of the medium also needs to be taken into account during this change-over process, the compressibility can be determined by suitably controlling the dual-plunger pump during the change-over from one plunger to the other plunger and fed to the control unit 60 as information. This connection between the pump 40 and the control unit 60 is merely illustrated with broken lines in FIG. 9.

[0054] In the automatic sample injector shown, it is therefore ensured that the pressure in the sample loop is adjusted to the current operating pressure of the chromatography column by means of decompression in the sufficiently (high) pressure-resistant sample conveying device when the injection valve is in a special intermediate position, namely the PRESSURE COMPENSATION position, before the intake segment is moved into the flow path toward the chromatography column, i.e., before the injection valve is changed over into the INJECT position.

[0055] In addition, the pressure in the sample loop is adjusted to the atmospheric pressure (decompression) in the same intermediate position of the injection valve, namely the PRESSURE COMPENSATION position, by taking in an exactly defined additional fluid quantity into the sample conveying device before the sample loop is separated in order to take in a sample volume from a sample container, i.e., before the injection valve is changed over into the LOAD position.

[0056] The compression and decompression volumes do not flow through the injection valve. Consequently, the service life of the (high-pressure) injection valve of the sample injector is only limited by the unavoidable abrasion between the rotor and the stator and, if applicable, the abrasive effect, for example, of dirt particles or sample material.

[0057] As used herein, whether in the above description or the following claims, the terms “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, that is, to mean including but not limited to. Any use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another, or the temporal order in which acts of a method are performed. Rather, unless specifically stated otherwise, such ordinal terms are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term).

[0058] The above described preferred embodiments are intended to illustrate the principles of the invention, but not to limit the scope of the invention. Various other embodiments and modifications to these preferred embodiments may be made by those skilled in the art without departing from the scope of the present invention.