Method for feeding a sample into an analysis branch of a liquid chromatography system

Abstract

The invention relates to a method for feeding a sample into an analysis branch of a liquid chromatography system, in particular a high-performance liquid chromatography system. A solvent or a solvent mixture from at least one solvent branch is supplied as volume flow {dot over (A)} into the analysis branch. At least one sample from at least one sample branch is fed as volume flow into the analysis branch within a predetermined time interval. The volume flow {dot over (A)} is reduced to an extent during the predetermined time interval, and a volume flow resulting from the sum of the volume flows {dot over (A)} and remains substantially constant in the analysis branch. The invention further relates to a sampler for carrying out a method of this kind.

Claims

1. A method for feeding a sample into an analysis branch of a liquid chromatography system, the method comprising: supplying a solvent or a solvent mixture from at least one solvent branch at a volume flow {dot over (A)} into the analysis branch, the at least one solvent branch is an input into a unidirectional valve, the analysis branch is an output out of the unidirectional valve; feeding at least one sample from at least one sample branch at a volume flow into the analysis branch within a predetermined time interval, the at least one sample branch is another input into the unidirectional valve reducing the volume flow {dot over (A)} during the predetermined time interval, in which the reducing of the volume flow {dot over (A)} causes an increase in the volume flow , and a control device controls the volume flow {dot over (A)} and the volume flow ; outputting a substantially constant volume flow in the analysis branch resulting from a sum of the volume flows {dot over (A)} and .

2. The method of claim 1, wherein the volume flow {dot over (A)} comes to a stop during the predetermined time interval at least after introducing the sample into the analysis branch where a solvent pump is stopped and a metering device pump is started, the solvent pump being configured to pump the solvent into the solvent branch, the metering device pump being configured to pump the sample into the sample branch.

3. The method of claim 2, wherein the volume flow {dot over (A)} comes to a stop during the predetermined time interval, at least after a first transition phase where the sample is introduced into the analysis branch, the solvent pump is stopped, and the metering device pump is started, and at least before a second transition phase where the sample has already been introduced into the analysis branch, the solvent pump is started, and the metering device pump is stopped.

4. The method of claim 1, wherein the volume flow corresponds to a maximum of the volume flow {dot over (A)} or .

5. The method of claim 3 further comprising: stopping the volume flow of the sample into the unidirectional valve before a first rear end of the sample, with respect to a direction of a flow of the sample, reaches the analysis branch, the rear end being a transition between an end of the sample and the solvent, whereby a second rear end arises on the sample introduced into the volume flow of the analysis branch.

6. The method of claim 1 further comprising: collecting the sample from a vial; and after collecting the sample from the vial, pressing an injection needle into a needle seat to introduce the sample to the sample branch, in which the needle seat is connected to the analysis branch via the unidirectional valve.

7. The method of claim 6 further comprising: after introducing the sample to the sample branch and prior to collecting a further sample, automatically cleaning the injection needle by flushing.

8. The method of claim 1, wherein the supplying of the solvent or the solvent mixture into the analysis branch is after or before the feeding of the at least one sample.

9. The method of claim 2, in which the supplying of the solvent or the solvent mixture to the at least one solvent branch causes a ball in the unidirectional valve to close the sample branch.

10. The method of claim 9, in which the solvent pump is stopped and causes the ball to open the sample branch allowing the sample to feed into the analysis branch.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is explained in more detail below on the basis of an illustrative embodiment depicted in the drawings.

(2) FIG. 1 shows a schematic view of a sampler according to the invention.

(3) FIG. 2 shows a schematic view of a detail from FIG. 1.

(4) FIG. 3 shows a diagram of the volume flows in a sampler according to FIG. 1.

DETAILED DESCRIPTION OF EMBODIMENTS

(5) The arrangement in FIG. 1 shows an automatic sampler, or autosampler, and its integration into a chromatography system. The autosampler comprises an injection needle 1 which, in a manner not shown in detail in the drawing, can be moved under the control of a motor in the x-y-z direction (x horizontal, z vertical in the plane of the drawing, and y perpendicular to the plane of the drawing), and also a sample loop connected thereto, and a metering device 10.

(6) By way of the injection needle 1, a sample from a vial 8 is drawn into a sample loop 2 by means of the metering device 10 (for example, as shown in the drawing, in the form of a piston pump). Thereafter, the injection needle 1 is pressed sealingly into a needle seat 6, to which a check valve 3 is attached. In normal operation, a solvent or mobile phase is aspirated (solvent branch 19) via a pump 20 (HPLC pump) and is guided via the check valve 3 to the column 9 (analysis branch 17). In this state, the port on the valve leading to the needle seat 6 is closed by a closure means, for example a ball 5, optionally pretensioned by a restoring element, for example a spring 4.

(7) If the sample is now to be fed in or introduced, the pressure that the metering device 10 has to apply must be greater than the prevailing system pressure, so that the check valve 3 opens. Moreover, the solvent pump 20 has to stop its flow during the sample injection time, so as to prevent a pressure increase in the connected solvent branch 19 and analysis branch 17.

(8) The sample is now pumped by the metering device 10 out of the sample branch 18 (content of the pressed-in needle 1 as far as the connected port of the check valve 3) in the direction of the column 9. After a desired amount of the sample located in the needle 1 has been fed in and the metering device 10 has stopped the supply, the check valve 3 closes automatically and the solvent pump 20 starts supplying again.

(9) In order to avoid increases or decreases in flow (volume flow in the analysis branch) and in pressure in this injection process, data communication takes place (for example by means of a control device 40) between autosampler (metering device 10, sample loop 2 and needle 1) and solvent pump 20, which data communication coordinates the flow responsibility between metering device 10 and solvent pump 20.

(10) In this way, a desired and definable amount of a sample is fed directly into the solvent stream to the separating column 9, without the sample being diluted with the solvent stream. Moreover, the low sample dispersion and the low longitudinal intermixing not only increase the precision of the analysis (in the schematically depicted detector 30) but also prolong the useful life of the column 9.

(11) As can be seen from FIG. 1, the sample loop 2 and injection needle can be flushed and cleaned via the metering device 10 by means of a flushing pump 14, which is connected to a flushing agent reservoir 16, after which the sample loop 2 and the inside of the needle 1 are filled by means of pump 15 which, like the pump 20, is also connected to a reservoir for the solvent 25 (fluid). Of course, it is also conceivable to use the solvent 25 also for cleaning purposes, in which case the additional pump 14 and flushing agent 16 can be dispensed with. The sample loop is filled with solvent all the way to the tip of the injection needle 1, in order to avoid gassing of a sample generally degassed in HPLC and to avoid undesired mixing of the sample. To hold the corresponding solvent column in the autosampler, it is possible, as shown schematically in the drawing, to provide a corresponding check valve 13, in order to prevent reverse flow and also forward ejection from the needle 1.

(12) Since the needle 1 is sterile before use and is contaminated during a sampling procedure in which a septum of a vial 8 is usually pierced, an aforementioned cleaning procedure according to the invention takes place after an injection of sample but before a renewed collection of sample, such that a sterile state is ensured once again after such cleaning.

(13) As is shown schematically in FIG. 1 by the four vials 8, it is possible according to the invention to collect samples successively from several different vials 8 (as is customary in HPLC) and then inject them. The vials 8 can be arranged in an autosampler, for example in the form of a tray or a (micro)titer plate (well plates).

(14) To permit flushing directly after the injection of a sample, the needle seat 6 has, on its upper face, an overflow container 7 which extends around the needle seat and is open toward the top, such that any emerging flushing liquid and/or solvent can collect in this overflow container and can flow off and be discarded as indicated by the bent arrow. In this way, a large number of samples can be collected and injected using the sampler according to the injection, wherein the flushing after the injection and before renewed collection of a sample prevents contamination of the subsequent sample and therefore of the next sample run.

(15) To control the aforementioned procedures and, in particular, to keep the volume flow in the analysis branch 17 constant, it is possible for the pump 20 and the metering device 10, and the drive motor 12 thereof, to be suitably controlled via the control device. By contrast, the corresponding control of the pumps 14 and/or 15 takes place exclusively, as has been explained above, for cleaning purposes and for keeping solvent in the sampler. To permit a particularly high level of precision of the control, it is possible, as is shown schematically in FIG. 1, to additionally provide a pressure sensor 21 in the solvent branch 19 and a pressure sensor 11 in the sample branch 18, said pressure sensors transmitting actual states to the control device, which actual states are evaluated there. Pressure sensors 21 and 11 of this kind can also be integrated in the pump 20 and the metering device 10.

(16) The enlarged detail of the needle seat 6 in four positions in FIG. 2 shows how, in a first position, the valve 3 in the blocked state closes the port to the needle seat 6 by means of ball 5, and the volume flow thus corresponds to the volume flow {dot over (A)}. In this valve position, the needle 1 can still be located outside the needle seat 6, without solvent emerging through the closed port of the check valve to the needle seat. Thereafter, the needle tip 1 is pressed sealingly into the needle seat 6 (as is shown in the second partial figure from the left in FIG. 2), such that the port of the valve 3 to the needle seat 6 is sealingly closed and, in this position, the port is also closed by the ball 5.

(17) The third partial figure in FIG. 2 shows how the volume flow {dot over (A)} has already been stopped, the ball 5 comes loose, upon pressure equality with the pressure present in the system or solvent branch 19 and the analysis branch 17 connected thereto, and frees the port to the needle seat, and the sample is injected into the analysis branch 17 through the volume flow . The view on the right in FIG. 2 shows once again how the sample injection has been stopped preferably after introduction of a predefined amount of volume flow , smaller than the amount of sample contained in the needle 1 and sample loop 2, and the volume flow is once again fed in from the volume flow {dot over (A)} of the solvent. Although, in a preferred embodiment of the method, not all of the sample amount contained in the sample loop 2 and needle 1 is fed into the volume flow , and part of the contained sample is thereby lost, this method can be advantageous since in this way intermixing effects at the rear separation surface can be avoided. Such intermixing effects otherwise occur, since the rear separation surface has been moved rearward in the sampling procedure and been moved forward again during the sample injection, until it is present as separation surface in the volume flow . However, through these movements, the separation surface is unclear because of intermixing effects, and this has a disadvantageous effect on the accuracy of the analysis in the detector 30.

(18) The diagram of volume flow per time in FIG. 3 shows how a volume flow is composed as resultant of the sum of volume flows {dot over (A)} and . In valve position closed (partial figures on the left, second from the left, and on the right in FIG. 2), the volume flow {dot over (A)} is constant and continues as constant volume flow . A volume flow does not exist at this time (before T1 and after T4).

(19) In the opened position, as shown in the third view from the left in FIG. 2, the volume flow {dot over (A)} has been stopped by stopping the pump 20, such that in the area between T2 and T3 the volume flow continues to the volume flow . In the transition phases T1-T2 and T3-T4 which, compared to the sample injection duration of for example 10 ms to 1 min, are short (for example 1 ms or less, but at least under a few ms), the volume flow rises (or falls) to the extent that the volume flow {dot over (A)} falls (or rises). Although the respective flanks are shown schematically in FIG. 3 as straight lines, the corresponding rise and fall can of course also take place according to suitable curves. In a preferred embodiment of the invention, however, suitable control of the pump 20 and of the metering device 10, even during opening of the valve 3 in the transition phase T1-T2 and during closure of the valve 3 in the transition phase T3-T4, ensures that at each time the sum of the volume flows reaches the same level as the preceding volume flow {dot over (A)} and the succeeding volume flow (transition closed to open) or the preceding volume flow and the succeeding volume flow {dot over (A)} (at the transition open to closed). By maintaining a constant volume flow not only before, during and after an injection of sample but also in the transitions (T1-T2 and T3-T4), a sample or a sample plug is fed in while maintaining clear separation surfaces, and, as has already been explained above, it is possible to prevent reverse intermixing, seen in the direction of flow, on account of movements in needle 1 and sample loop 2 by a separation of a predefined amount of sample from the entire contained sample.

(20) As is customary in HPLC, a sample run takes place at very high pressure, for example in excess of 500 bar or even in excess of 1000 bar, such that, with the required narrow cross sections in the analysis branch (10 m-max. 1 mm), it is possible to generate volume flows of several l-10 ml per minute, preferably under 100 ml per minute, in particular under 300 l per minute, but at the respectively desired level with a constancy of a deviation of under 25%, for example under 10% or under 5%, in particular 1%, in order to protect the separating column from disadvantageous and excessively high fluctuations in flow. A whole sample run can in this way last from a few minutes or so to one hour, until the substance and sample pass through the column 9 to the detector 30.

LIST OF REFERENCE SIGNS

(21) 1 injection needle 2 sample loop 3 check valve 4 spring 5 ball 6 needle seat 7 overflow container 8 vials 9 separating column 10 metering device (piston pump) 11 pressure sensor 12 drive for metering device 10 13 check valve 14 flushing pump 15 flushing pump 16 flushing agent 17 analysis branch 18 sample branch 19 solvent branch 20 HPLC pump 21 pressure sensor 25 solvent (fluid) 30 detector 40 control device {dot over (A)} volume flow for supplying solvent 25 from a solvent branch volume flow for feeding the sample from the sample branch into the analysis branch volume flow in the analysis branch to the separating column 9, which volume flow results from the sum of volume flows {dot over (A)} and