GB2487941A

GB2487941A - Fluid separation system for determining an injection time

Info

Publication number: GB2487941A
Application number: GB1102217.5A
Authority: GB
Inventors: Klaus Witt; Konstantin Choikhet
Original assignee: Agilent Technologies Inc
Current assignee: Agilent Technologies Inc
Priority date: 2011-02-09
Filing date: 2011-02-09
Publication date: 2012-08-15
Also published as: GB201102217D0

Abstract

A fluid separation system, for separating compounds of a sample fluid, comprises a mobile phase drive, preferably a pumping system 505, which drives a mobile phase through the fluid separation system; and a sample injector 510, for introducing the sample fluid into the mobile phase. At least one sensor 500 is provided, which is able to measure a physical property of at least one of the mobile phase and the sample fluid as a function of a reference variable, such as time, or volume of fluid. A separation unit, preferably a chromatography column 525, is also provided, for separating compounds of the sample fluid. The fluid separation system, or at least one of its units, is configured to determine an actual injection event from a course of the physical property measured by the at least one sensor, or from a value derived there from.

Description

DETERMINING AN ACTUAL INJECTION EVENT IN A FLUID

SEPARATION SYSTEM

BACKGROUND ART

[0001] The present invention relates to a fluid separation system for separating compounds of a sample fluid in a mobile phase, in particular in a high performance liquid chromatography application. The present invention further relates to a method for determining an actual injection time.

[0002] In high performance liquid chromatography (H PLC), a liquid has to be provided usually at a very controlled flow rate (e. g. in the range of microliters to milliliters per minute) and at high pressure (typically 20-1 00 MPa, 200-1000 bar, and beyond up to currently 200 MPa, 2000 bar) at which compressibility of the liquid becomes noticeable. For liquid separation in an HPLC system, a mobile phase comprising a sample fluid with compounds to be separated is driven through a stationary phase (such as a chromatographic column), thus separating different compounds of the sample fluid which may then be identified.

[0003] The mobile phase, for example a solvent, is pumped under high pressure typically through a column of packing medium (also referred to as packing material), and the sample (e.g. a chemical or biological mixture) to be analyzed is injected into the column. As the sample passes through the column with the liquid, the different compounds, each one having a different affinity for the packing medium, move through the column at different speeds. Those compounds having greater affinity for the packing medium move more slowly through the column than those having less affinity, and this speed differential results in the compounds being separated from one another as they pass through the column.

[0004] The mobile phase with the separated compounds exits the column and passes through a detector, which identifies the molecules, for example by spectrophotometric absorbance measurements. A two-dimensional plot of the detector measurements against elution time or volume, known as a chromatogram, may be made, and from the chromatogram the compounds may be identified. For each compound, the chromatogram displays a separate curve or "peak". Effective separation of the compounds by the column is advantageous because it provides for measurements yielding well defined peaks having sharp maxima inflection points and narrow base widths, allowing excellent resolution and reliable identification of the mixture constituents. Broad peaks, caused by poor column performance, so called "Internal Band Broadening" or poor system performance, so called "External Band Broadening" are undesirable as they may allow minor components of the mixture to be masked by major components and go unidentified.

[0005] An HPLC column typically comprises a stainless steel tube having a bore containing a packing medium comprising, for example, silane derivatized silica spheres having a diameter between 0.5 to 50 pm, or 1-10 pm or even 1-7 pm. The medium is packed under pressure in highly uniform layers which ensure a uniform flow of the transport liquid and the sample through the column to promote effective separation of the sample constituents. The packing medium is contained within the bore by porous plugs, known as "frits", positioned at opposite ends of the tube. The porous frits allow the transport liquid and the chemical sample to pass while retaining the packing medium within the bore. After being filled, the column may be coupled or connected to other elements (like a control unit, a pump, containers including samples to be analyzed) by e.g. using fitting elements. Such fitting elements may contain porous parts such as screens or frit elements.

[0006] During operation, a flow of the mobile phase traverses the column filled with the stationary phase, and due to the physical interaction between the mobile and the stationary phase a separation of different compounds or components may be achieved.

In case the mobile phase contains the sample fluid, the separation characteristics is usually adapted in order to separate compounds of such sample fluid. The term compound, as used herein, shall cover compounds which might comprise one or more different components. The stationary phase is subject to a mechanical force generated in particular by a hydraulic pump that pumps the mobile phase usually from an upstream connection of the column to a downstream connection of the column. As a result of flow, depending on the physical properties of the stationary phase and the mobile phase, a relatively high pressure occurs across the column.

[0007] U.S. patent 4,095,455 describes a pneumatic detector for a chromatographic analyzer. U.S. patent 5,897,781 relates to active pump phasing to enhance chromatographic reproducibility.

DISCLOSURE

[0008] It is an object of the invention to provide an improved fluid separation system that operates with improved accuracy, in particular for HPLC applications. The object is solved by the independent claim(s). Further embodiments are shown by the dependent claim(s).

[0009] A fluid separation system according to embodiments of the present invention is configured for separating compounds of a sample fluid. The fluid separation system comprises a mobile phase drive, preferably a pumping system, configured to drive a mobile phase through the fluid separation system, a sample injector configured to introduce the sample fluid into the mobile phase, at least one sensor configured to measure a physical property of at least one of the mobile phase and the sample fluid as a function of a reference variable, a separation unit, preferably a chromatographic column, configured for separating compounds of the sample fluid, wherein the fluid separation system or at least one of its units is configured to determine an actual injection event from a course of the physical property measured by the at least one sensor, or from a value derived there from.

[0010] According to embodiments of the present invention, in order to determine the actual injection event of a liquid sample with high accuracy, a sensor is provided, said sensor being configured to measure a physical property of the liquid in the separation flow path. By analyzing the course of the physical property detected by the sensor, the actual injection event, which is the event when the sample liquid is actually injected into the separation flow path, can be detected. This actual injection event may be taken as a reference point of the separation process. Then, the sample separation is performed, and the peaks of the detected signal are evaluated with regard to the actual injection event, which is taken as a reference point.

[0011] According to embodiments of the present invention, an actual injection event derived from actual measurements is taken as a reference point for the sample separation. In prior art solutions, an estimated or assumed target injection event has been taken as a reference point, and due to the inaccuracy of the estimated target injection event, there may have been a systematic or random error. By deriving the actual injection event from measurements of a physical property of the liquid, the actual injection event can be determined with high accuracy. This actual injection event may then be taken as a reference point for the sample separation. For example, peaks detected by a detection unit may be related to the actual injection event, which is taken as a reference point. For example, the actual injection time may be taken as a zero point of time scale of the chromatographic separation. In this respect, retention times of the various components of a sample may be determined with high accuracy.

Compared to an estimated target injection time, the accuracy of the actual injection time is much better, and therefore, the sample separation can be performed with improved accuracy.

[0012] According to a preferred embodiment of the invention, the reference variable is a variable indicating progress of a separation process.

[0013] According to a preferred embodiment of the invention, the reference variable is a variable indicating a progression of time.

[0014] According to a preferred embodiment, the reference variable is time.

[0015] According to a preferred embodiment, the reference variable is a delivered volume of fluid.

[0016] According to a preferred embodiment, the actual injection event corresponds to a certain value of the reference variable, said value of the reference variable being taken as a point of reference for a separation process.

[0017] According to a preferred embodiment, said value derived from the course of the physical property measured by the at least one sensor is: any specific feature, disruption or discontinuity in the course of the measured physical property, or a derivative of first or higher order that relates to a fluid property change caused by the actual injection event, or any mathematical function of one or more of: the measured physical property, fluid parameters, any specific feature, disruption or discontinuity in the course of the measured physical property, a derivative of first or higher order that relates to a fluid property change caused by the actual injection event, or any combination of one or more of: the measured physical property, fluid parameters, any specific feature, disruption or discontinuity in the course of the measured physical property, a derivative of first or higher order that relates to a fluid property change caused by the actual injection event, any mathematical functions derived from these properties.

[0018] According to a preferred embodiment, the at least one sensor comprises a pressure sensor configured for measuring fluid pressure as a function of the reference variable.

[0019] According to a preferred embodiment, the at least one sensor comprises a flow sensor configured for measuring flow of the fluid as a function of the reference variable.

[0020] According to a preferred embodiment, the at least one sensor comprises a sensor configured for measuring conductivity of the fluid.

[0021J According to a preferred embodiment, the at least one sensor comprises a sensor configured for measuring an index of refraction of the fluid.

[0022] According to a preferred embodiment, the at least one sensor comprises a sensor configured for determining a temperature of the fluid.

[0023] According to a preferred embodiment, the at least one sensor comprises a sensor located within the mobile phase drive.

[0024] According to a preferred embodiment, the at least one sensor comprises a sensor located downstream of the mobile phase drive and upstream of the sample injector.

[0025] According to a preferred embodiment, the at least one sensor comprises a sensor located within the sample injector.

[0026] According to a preferred embodiment, the at least one sensor comprises a sensor is located downstream of the sample injector and upstream of the separation unit.

[0027] According to a preferred embodiment, the sample injector comprises a multiport valve and a sample loop fluidically connected to ports of the multiport valve, wherein in a first position of the multiport valve, the sample loop is not included in a separation flow path, and wherein in a second position of the multiport valve, the sample loop is included in the separation flow path.

[0028] According to a preferred embodiment, the sample injector comprises a set of valves which are configured and operated to switch a flow stream such that a sample loop is fluidically connected into the flow stream going to the separation unit.

[0029] According to a preferred embodiment, the at least one sensor comprises a pressure sensor located upstream of the sample injector, the data processing unit being configured to detect a rise of pressure when the switching of the multiport valve starts, a pressure drop when the sample loop is introduced into the flow path, and a subsequent rise of the pressure.

[0030] According to a preferred embodiment, the at least one sensor comprises a pressure sensor located within the sample injector, the data processing unit being configured to detect a rise of the pressure in the sample loop when the sample fluid is introduced into the separation flow path.

[0031] According to a preferred embodiment, the at least one sensor comprises a pressure sensor located downstream of the sample injector, the data processing unit being configured to detect a decline of the pressure when the switching of the multiport valve starts and a subsequent rise of the pressure when the sample loop is introduced into the flow path.

[0032] According to a preferred embodiment, the at least one sensor comprises a flow sensor located upstream of the sample injector, the data processing unit being configured to detect a drop of fluid flow when the switching of the multiport valve starts, and a rise of the fluid flow when the sample loop is introduced into the flow path.

[0033] According to a preferred embodiment, the at least one sensor comprises a flow sensor located within the sample injector, the data processing unit being configured to detect a rise of fluid flow when the sample fluid is introduced into the separation flow path.

[00341 According to a preferred embodiment, the at least one sensor comprises a flow sensor located downstream of the sample injector, the data processing unit being configured to detect a decline of the fluid flow when the switching of the multiport valve starts and a subsequent rise of the fluid flow when the sample loop is introduced into the flow path.

[0035] According to a preferred embodiment, the actual injection event is used as a point of reference for the analysis of the compounds of the sample fluid.

[0036] According to a preferred embodiment, the actual injection event is used as a point of reference for determining respective retention times of the compounds of the sample fluid.

[0037] According to a preferred embodiment, the actual injection event is used as a point of reference for the operation of at least one of: the mobile phase drive, a detector unit, a collection unit, a thermostat, switching valves comprised in the system.

[0038] According to a preferred embodiment, the actual injection event is used as a point of reference for the operation of other system units, operation parameters of which are dependent on the progress of separation process.

[0039] According to a preferred embodiment, the fluid separation system further comprises a detector configured to detect separated compounds of the sample fluid.

[0040] According to a preferred embodiment, the fluid separation system further comprises a collection unit configured to collect separated compounds of the sample fluid.

[0041] According to a preferred embodiment, the fluid separation system further comprises a degassing apparatus for degassing the mobile phase.

[0042] According to embodiments of the present invention, a method of determining an actual injection event when a sample fluid is actually introduced into a fluid separation system is provided. The fluid separation system comprises a mobile phase drive, preferably a pumping system, configured to drive a mobile phase through the fluid separation system, and a separation unit, preferably a chromatographic column, configured for separating compounds of the sample fluid in the mobile phase. The method comprises measuring a physical property of at least one of the mobile phase and the sample fluid as a function of a reference variable, introducing the sample fluid into the mobile phase, and determining the actual injection event of the fluid sample from a course of the physical property, or from a value derived there from.

[0043] Embodiments of the present invention might be embodied based on most conventionally available HPLC systems, such as the Agilent 1290 Series Infinity system, Agilent 1200 Series Rapid Resolution LC system, or the Agilent 1100 HPLC series (all provided by the applicant Agilent Technologies -see jjgJ[entco -which shall be incorporated herein by reference).

[0044] One embodiment of an HPLC system comprises a pumping apparatus having a piston for reciprocation in a pump working chamber to compress liquid in the pump working chamber to a high pressure at which compressibility of the liquid becomes noticeable.

[0045] One embodiment of an HPLC system comprises two pumping apparatuses coupled either in a serial or parallel manner. In the serial manner, as disclosed in EP 309596 Al, an outlet of the first pumping apparatus is coupled to an inlet of the second pumping apparatus, and an outlet of the second pumping apparatus provides an outlet of the pump. In the parallel manner, an inlet of the first pumping apparatus is coupled to an inlet of the second pumping apparatus, and an outlet of the first pumping apparatus is coupled to an outlet of the second pumping apparatus, thus providing an outlet of the pump. In either case, a liquid outlet of the first pumping apparatus is phase shifted, preferably essentially 180 degrees, with respect to a liquid outlet of the second pumping apparatus, so that only one pumping apparatus is supplying into the system while the other is intaking liquid (e.g. from the supply), thus allowing to provide a continuous flow at the output. However, it is clear that also both pumping apparatuses might be operated in parallel (i.e. concurrently), at least during certain transitional phases e.g. to provide a smooth(er) transition of the pumping cycles between the pumping apparatuses. The phase shifting might be varied in order to compensate pulsation in the flow of liquid as resulting from the compressibility of the liquid. It is also known to use three piston pumps having about 120 degrees phase shift.

[0046] The separating device preferably comprises a chromatographic column providing the stationary phase. The column might be a glass or steel tube (e.g. with a diameter from 50 pm to 5 mm and a length of 1 cm to 1 m) or a microfluidic column (as disclosed e.g. in ffJj71Qj2_AI or the Agilent 1200 Series HPLC-Chip/MS System provided by the applicant Agilent Technologies, see e.g. For example, a slurry can be prepared with a powder of the stationary phase and then poured and pressed into the column. The individual components are retained by the stationary phase differently and separate from each other while they are propagating at different speeds through the column with the eluent. At the end of the column they elute one at a time.

During the entire chromatography process the eluent might be also collected in a series of fractions. The stationary phase or adsorbent in column chromatography usually is a solid material. The most common stationary phase for column chromatography is silica gel, followed by alumina. Cellulose powder has often been used in the past. Also possible are ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, though in EBA a flu idized bed is used.

[00471 The mobile phase (or eluent) can be either a pure solvent or a mixture of different solvents. It can be chosen e.g. to minimize the retention of the compounds of interest and/or the amount of mobile phase to run the chromatography. The mobile phase can also been chosen so that the different compounds can be separated effectively. The mobile phase might comprise an organic solvent like e.g. methanol or acetonitrile, often diluted with water. For gradient operation water and organic is delivered in separate bottles, from which the gradient pump delivers a programmed blend to the system. Other commonly used solvents may be isopropanol, THF, hexane, ethanol and/or any combination thereof or any combination of these with aforementioned solvents.

[0048] The sample fluid might comprise any type of process liquid, natural sample like juice, body fluids like plasma or it may be the result of a reaction like from a fermentation broth.

[0049] The fluid is preferably a liquid but may also be or comprise a gas and/or a supercritical fluid (as e.g. used in supercritical fluid chromatography -SF0 -as disclosed e.g. in US 4982597 A).

[0050] The pressure in the mobile phase might range from 2-200 MPa (20 to 2000 bar), in particular 10-150 MPa (100 to 1500 bar), and more particular 50-120 MPa (500 to 1200 bar).

[0051] The HPLC system might further comprise a sampling unit for introducing the sample fluid into the mobile phase stream, a detector for detecting separated compounds of the sample fluid, a fractionating unit for outputting separated compounds of the sample fluid, or any combination thereof. Further details of HPLC system are disclosed with respect to the aforementioned Agilent HPLC series, provided by the applicant Agilent Technologies, under wwwaqUenLcom which shall be in cooperated herein by reference.

[0052] Embodiments of the invention can be partly or entirely embodied or supported by one or more suitable software programs, which can be stored on or otherwise provided by any kind of data carrier, and which might be executed in or by any suitable data processing unit. Software programs or routines can be preferably applied in or by the control unit.

BRIEF DESCRIPTION OF DRAWINGS

[0053] Other objects and many of the attendant advantages of embodiments of the present invention will be readily appreciated and become better understood by reference to the following more detailed description of embodiments in connection with the accompanied drawing(s). Features that are substantially or functionally equal or similar will be referred to by the same reference sign(s). The illustration in the drawing is schematically.

[0054] Figure 1 shows a liquid separation system 10, in accordance with embodiments of the present invention, e.g. used in high performance liquid -10-chromatography (H PLC).

[00551 Figure 2 shows a sample injector comprising a switching valve and a sample loop, the switching valve being depicted in three different positions.

[0056] Figure 3 shows a chromatogram, i.e. a signal detected by the detection unit as a function of time.

[0057] Figure 4 illustrates the deviation between the estimated target injection time and the actual injection time.

[0058] Figure 5 shows a separation flow path with a pressure sensor located upstream of the sample injector, together with a diagram showing the pressure variations detected by said pressure sensor.

[0059] Figure 6 shows a separation flow path with a pressure sensor located in the sample loop, together with a diagram showing the pressure variations detected by said pressure sensor.

[0060] Figure 7 shows a separation flow path with a pressure sensor located downstream of the sample injector, together with a diagram showing the pressure variations detected by said pressure sensor.

[0061] Referring now in greater detail to the drawings, Fig. 1 depicts a general schematic of a liquid separation system 10. A pump 20 receives a mobile phase from a solvent supply 25, typically via a degasser 27, which degases the mobile phase and thus reduces the amount of dissolved gases in it. The pump 20 -as a mobile phase drive -drives the mobile phase through a separating device 30 (such as a chromatographic column) comprising a stationary phase. A sample injector 40 can be provided between the pump 20 and the separating device 30 in order to add (often referred to as sample introduction) a sample fluid into the mobile phase. Compounds of the sample liquid are separated on the stationary phase of the separating device 30. A detector 50 is provided for detecting separated compounds of the sample fluid. A fractionating unit 60 can be provided for collecting separated compounds of sample fluid.

[0062] While the mobile phase can be comprised of one solvent only, it may also be mixed from plural solvents. Such mixing might be a low pressure mixing and provided upstream of the pump 20, or in the pump 20, so that the pump 20 already receives and pumps the mixed solvents as the mobile phase. Alternatively, the pump 20 might be comprised of plural individual pumping units, with plural of the pumping units each receiving and pumping a different solvent or mixture, so that the mixing of the mobile phase (as received by the separating device 30) occurs at high pressure und downstream of the pump 20 (or as part thereof). The composition (mixture) of the mobile phase may be kept constant over time, the so called isocratic mode, or varied overtime, the so called gradient mode.

[0063] A data processing unit 70, which can be a conventional PC or workstation, might be coupled (as indicated by the dotted arrows) to one or more of the devices in the liquid separation system 10 in order to receive information and/or control operation.

For example, the data processing unit 70 might control operation of the pump 20 (e.g. setting control parameters) and receive therefrom information regarding the actual working conditions (such as output pressure, flow rate, etc. at an outlet of the pump).

The data processing unit 70 might also control operation of the solvent supply 25 (e.g. setting the solvent/s or solvent mixture to be supplied) and/or the degasser 27 (e.g. setting control parameters such as vacuum level) and might receive therefrom information regarding the actual working conditions (such as solvent composition supplied over time, flow rate, vacuum level, etc.). The data processing unit 70 might further control operation of the sample injector 40 (e.g. controlling sample injection or synchronization sample injection with operating conditions of the pump 20). The separating device 30 might also be controlled by the data processing unit 70 (e.g. selecting a specific flow path or column, setting operation temperature, etc.), and send -in return -information (e.g. operating conditions) to the data processing unit 70.

Accordingly, the detector 50 might be controlled by the data processing unit 70 (e.g. with respect to spectral or wavelength settings, setting time constants, start/stop data acquisition), and send information (e.g. about the detected sample compounds) to the data processing unit 70. The data processing unit 70 might also control operation of the fractionating unit 60 (e.g in conjunction with data received from the detector 50) and provides data back. -12-

[0064] In Figs. 2A to 2C, a sample injector 200 for a chromatography system is shown in more detail. The sample injector 200 is configured for introducing a volume of liquid sample into the separation flow path of a chromatography system. The sample injector 200 comprises a switching valve 205, which may for example be implemented as a 2-position/6-port multi-route valve. The switching valve 205 comprises a stator with six stator ports A to F which lie on a port circle. The switching valve 205 further comprises a disc shaped rotor element with three circumferential switching channels 210, 215, 220. The switching channels 210, 215, 220 are configured for providing a fluidic connection between two adjacent stator ports of the switching valve 205. By pivoting the rotor element around an axis of rotation 225, the switching valve 205 can be set to two different positions. The face of the rotor element extends perpendicular to the axis of rotation 225 and lies facewise adjacent to the face of the stator.

[0065] In Fig. 2A, the rotor element of the switching valve 205 is set to its first position. In the first position of the rotor element, the switching channel 210 provides a fluidic connection between stator ports A and B, the switching channel 215 provides a fluidic connection between stator ports C and D, and the switching channel 220 provides a flu idic connection between stator ports E and F. [0066] A sample loop 230 is connected between the stator ports B and E. In the first position of the switching valve 205, the sample loop 230 is included in the sample loading flow path. When the switching valve 205 is set to its first position, a volume 235 of liquid sample may be loaded into the sample loop 230. For this purpose, a supply unit 240 is fluidically connected to the stator port A. The supply unit 240 may for example comprise a plurality of vials 245 filled with different solvents and liquid samples. The supply unit 240 further comprises a needle 250 attached to an actuation device 255, which can be moved to different positions. The supply unit 240 further comprises a needle seat 260 that is flu idically connected with the stator port A. The sample loading flow path further comprises a waste outlet 265 flu idically connected to stator port F. For injecting a volume 235 of liquid sample into the sample loop 230, the needle 250 is moved to a vial containing the respective liquid sample. Then, a certain volume of the respective liquid sample is taken in by the needle 250. Then, the needle 250 is moved to the needle seat 260, and a certain volume of the liquid sample is injected into the sample loading flow path. Thus, the volume 235 of liquid sample is -13-supplied to the sample loop 230.

[00671 The setup shown in Fig. 2Afurther comprises a pumping unit 270 fluidically connected to stator port D, and a separation column 275 fluidically connected with the stator port C. The outlet of the separation column 275 is fluidically connected with an inlet of a detection unit 280. When the switching valve 205 is set to the first position, as shown in Fig. 2A, the stator ports D and C are fluid ically connected by the switching channel 215, and the sample loop 230 is not included in the sample separation flow path yet.

[0068] Next, it will be described how the rotor element of the switching valve 205 is rotated from the first position shown in Fig. 2A to the second position shown in Fig. 2C.

The rotor element may for example be driven by an electromechanical drive unit that is coupled with the rotor element. As soon as the electromechanical drive unit receives a trigger signal from the data processing unit 70 shown in Fig. 1, the rotor element starts rotating around the axis of rotation 225. For example, as indicated in Fig. 2B, the rotor element may start rotating in the clockwise direction, as indicated by arrow 285.

Accordingly, the switching channels 210, 215, 220 located at the front face of the rotor element start moving in the clockwise direction as well, as indicated by arrows 290.

[0069] In Fig. 2C, the rotor element of the switching valve 205 has arrived at its second position, and the switching channels 210, 215, 220 have arrived at their respective final positions. Now, the switching channel 210 provides a fluidic connection between the stator ports B and C, the switching channel 215 provides a fluidic connection between the stator ports D and E, and the switching channel 220 provides a fluidic connection between the stator ports F and A. [0070] In the second position of the switching valve 205, the supply unit 240 is fluidically connected, via the switching channel 220, with the waste outlet 265. Hence, the sample loop 230 is no longer included in the sample loading flow path. Instead, the sample loop 230 is now included in the sample separation flow path. The pumping unit 270 is fluidically connected, via the switching channel 215, with a first end of the sample loop 230, and the second end of the sample loop 230 is fluidically connected, via the switching channel 210, with the inlet of the separation column 275. The outlet of the separation column 275 is fluidically coupled with the detection unit 280. During -14-the separation process, the pumping unit 270 is configured for supplying a flow of mobile phase. Driven by the flow of mobile phase, the volume 235 of liquid sample contained in the sample loop 230 is moved to the inlet of the separation column 275 and starts traversing the separation column 275. During the passage through the separation column 275, the various compounds of the liquid sample are separated. At the detection unit 280, the various different compounds of the liquid sample can be detected as they leave the column. The various different compounds may e.g. be detected as a function of time. The detected signal can be registered as a function of time, or of delivered volume, or of any other reference variable.

[0071] Fig. 3 shows a chromatogram, i.e. a detected signal 300 as a function of time. Alternatively, the detected signal may be detected as a function of any other suitable reference variable, e.g. as a function of delivered volume. The detected signal 300 comprises a plurality of peaks 301, 302, 303, with each of the peaks corresponding to a respective component of the liquid sample. By analyzing the peaks 301, 302, 303 of the detected signal 300, the components contained in the liquid sample can be identified. Recording of signal intensity is started at a target injection time 304, which may for example be derived from a trigger signal that triggers operation of the electromechanical drive unit, the electromechanical drive unit driving the rotor element of the switching valve. The target injection time 304 is an estimated injection time. The target injection time 304 is set as the zero point of the time scale shown in Fig. 3. Accordingly, the retention time 305 of the first peak 301 is determined with respect to the target injection time 304, which is taken as a zero point of the time scale. Similarly, the retention time 306 of the peak 302 and the retention time 307 of the peak 303 are also determined relative to the target injection time 304, which is taken as a zero point of the time scale.

[0072J However, the target injection time 304 is only an estimated injection time, which may for example be derived from the trigger signal. The actual injection time, which is the point of time when the liquid sample is actually injected into the separation flow path, may differ from the target injection time.

[0073] Fig. 4 is a schematic diagram showing both the target injection time 400 and the actual injection time 401. In Fig. 4, the deviation between the actual injection time -15- 401 and the assumed target injection time 400 is denoted as At. The target injection time 400 may for example be derived from the trigger signal 402, said trigger signal being configured for triggering operation of the switching valve's electromechanical drive unit. For example, it may be assumed that there is a fixed time delay 403 between the trigger signal 402 and the expected target injection time 400.

[0074] The time delay At between the target injection time 400, which is taken as the zero point of the time scale, and the actual injection time 401 leads to errors of the retention times determined by the chromatographic separation system. For example, the actual injection time 401 is within an actual injection time window 404, which is also indicated in Fig. 4, and the actual injection time window 404 may be approximately 50-ma Hence, each of the retention times 305, 306, 307 can only be determined with an inaccuracy of about 50-60 ms. In Fig. 3, the actual injection time window404, which indicates the error of the zero point of the time scale, is also shown in Fig. 3.

[0075] Some time ago, chromatographic separations have been performed in time intervals of about 30 mm or even longer. Compared to these large time scales, an error of about 50-60 ms does not really affect the obtained results. However, the time intervals required for performing a chromatographic separation have been reduced continuously, and nowadays, a chromatographic separation may be performed within seconds. In this case, an error of up to about 50-60 ms cannot be neglected anymore. Hence, for obtaining accurate measurements of the retention time, it is desirable to determine the actual injection time 401, and to use said actual injection time 401 as a zero point of the time scale.

[0076] In Figs. 3 and 4, the detected signal is recorded and evaluated as a function of time, and the actual injection time is determined. Hence, in Figs. 3 and 4, time is used as a reference variable. Alternatively, the detected signal may be recorded and evaluated as a function of any other suitable reference variable. For example, the detected signal may be recorded and evaluated as a function of delivered volume.

[0077] Embodiments of the present invention are directed at determining the actual injection event by tracking a physical property of the liquid in the separation flow path.

Then, the actual injection event is derived from the course of the detected physical -16-property.

[00781 For example, a pressure sensor may be installed at some point of the separation flow path, the pressure sensor being configured for detecting a pressure of the liquid as a function of a reference variable. Then, the actual injection event may be derived from the pressure variations detected by the pressure sensor.

[0079] Alternatively, a flow sensor may be installed at some point of the separation flow path, the flow sensor being configured for detecting flow as a function of a reference variable. By analyzing variations of the flow detected by the flow sensor, the actual injection event can be derived from the flow variations.

[0080] Further alternatively, the actual injection event may for example be determined by evaluating an index of refraction of the liquid in the separation flow path.

When the liquid sample is injected into the separation flow path, the index of refraction of the liquid in the separation flow path is affected by the presence of the liquid sample. The resulting variations of the index of refraction can be detected and analyzed, in order to determine the actual injection event when the liquid sample is actually injected. For example, a sensor configured for determining the index of refraction as a function of a reference variable may be included at some point of the separation flow path.

[0081] Further alternatively, the actual injection event may be determined by tracking the conductivity of the liquid in the separation flow path as a function of a reference variable. When the switching valve is switched from its first position to its second position, the sample loop is switched into the separation flow path, and the presence of the liquid sample may induce a change of the conductivity of the liquid in the separation flow path. Hence, by detecting conductivity of the liquid, the actual injection event can be determined. According to a preferred embodiment, conductivity of the liquid is measured across the switching valve. In this embodiment, a first electrode is positioned upstream of the switching valve, and a second electrode is positioned downstream of the switching valve. By measuring conductivity between these two electrodes, the actual injection event when the sample loop is switched into the separation flow path can be detected with high accuracy. -17-

[0082] Further alternatively, the index of refraction of the liquid in the separation flow path may be detected as a function of a reference variable. When the liquid sample is injected into the separation flow path, the presence of the liquid sample will affect the index of refraction. Therefore, by tracking the index of refraction of the liquid in the separation flow path, the actual injection event of the liquid sample can be determined. Preferably, a sensor configured for detecting the index of refraction is positioned downstream of the switching valve.

[0083] As a further alternative, the temperature of the liquid in the separation flow path may be detected as a function of a reference variable. When the liquid sample is injected into the separation flow path, switching of the injection valve may induce pressure variations, and these pressure variations may cause corresponding temperature variations of the liquid. For example, a rise of pressure causes a temperature increase of the liquid, and a pressure drop causes a temperature decrease of the liquid. By tracking and evaluating temperature variations of the liquid in the separation flow path, the actual injection event can be determined. Therefore, a temperature sensor configured for detecting a temperature of the liquid in the separation flow path is well-suited for detecting the actual injection event.

[0084] In the following, it will be explained in more detail how pressure variations measured at some point of the separation flow path can be used for determining the actual injection point when the liquid sample is actually injected into the separation flow path.

[0085] As shown in Fig. 5A, a pressure sensor 500 is located downstream of the pumping unit 505, but upstream of the sample injector 510. The sample injector 510 may for example comprise a 2-position/6-port switching valve 515 and a sample loop 520 that is fluid ically connected between port B and port E of the switching valve 515, as shown in Figs. 2A to 2G. The separation flow path shown in Fig. 5A further comprises a separation column 525, which is located downstream of the sample injector 510, and a detection unit 530.

[0086] In Fig. SB, the pressure measured by the pressure sensor 500 is shown as a function of time. Alternatively, the pressure measured by the pressure sensor may as well be indicated as a function of any other suitable reference variable, e.g. as a -18-function of delivered volume. Initially, the switching valve 515 is set to its first position shown in Fig. 2A. In the switching valve's first position, the pumping unit 505 is fluidically connected with the separation column 525, but the sample loop 520 is not included in the separation flow path yet. Accordingly, during the time interval 540, the pressure detected by the flow sensor 500 is equal to the regular pressure in the chromatographic separation system.

[0087} Then, at the point of time 545, the switching operation of the switching valve 515 is started, and the rotor element of the switching valve 515 is moved from the position of Fig. 2A to the position of Fig. 2B. Accordingly, the switching channels 210, 215, 220 do no longer provide fluidic connections between the stator ports A and B, between the stator ports C and D and between the stator port E and F. As a consequence, the fluidic connection between the pumping unit 505 and the separation column 525 is disrupted. Nevertheless, during the time interval 550, the pumping unit 505 continues supplying a flow of mobile phase, but this flow of mobile phase is no longer forwarded to the separation column 525. As a consequence, a rise 555 of the pressure in the section of the flow path upstream of the switching valve 510 is detected by the pressure sensor 500.

[0088] At the point of time 560, the rotor element of the switching valve 515 arrives at its second position, which is shown in Fig. 2C. In this second position, the switching channels 210, 215, 220 provide fluidic connections between the stator ports B and C, between the stator ports D and E, and between the stator ports F and A. Accordingly, the sample loop 520 is now included in the separation flow path, and the pumping unit 505 is fluidically connected, via the sample loop 520, with the inlet of the separation column 525. The mobile phase contained in the section of the separation flow path upstream of the switching valve 510 is supplied to the separation column 525, together with the liquid sample.

[0089] At the point of time when the fluidic connections between stator ports B and C and between stator ports D and E are established, a sudden drop 565 of the pressure determined by the pressure sensor 500 is observed. This sudden drop 565 of the pressure is due to the sudden relaxation of the compressed fluid in the section of the separation flow path upstream of the sample injector 510. Now, the switching valve -19- 510 is in its second position, and the sample loop 520 is part of the separation flow path. During the time interval 570, the pressure detected by the pressure sensor 500 slowly approaches regular system pressure, and correspondingly, a slow rise 575 of the pressure detected by the pressure sensor 500 is observed.

[0090] The pressure variations shown in Fig. SB are detected by the pressure sensor 500 and analyzed by some kind of data processing unit. By analyzing the pressure variations detected by the pressure sensor 500, it can be determined that at the point of time 580, the liquid sample contained in the sample loop 520 is actually injected into the separation flow path, because at the point of time 580, the sudden drop 565 of the pressure is observed. The pressure drop 565 corresponds to the point of time when the fluidic connection between the pumping unit 505 and the separation column 525 is established. Hence, the point of time 580 is determined as the actual switching point.

[0091] In Fig. 5B, the pressure variations are detected and evaluated as a function of time. Alternatively, the pressure variations may be detected and evaluated as a function of any other suitable reference variable. For example, the pressure variations may be detected and evaluated as a function of delivered volume.

[0092] In Fig. 6A and 6B, a different embodiment of the invention is shown. Fig. 6A depicts the separation flow path which comprises a pumping unit 600, a sample injector 605, a separation column 610 and a detection unit 615. The sample injector 605 comprises a 2-position/6-port switching valve 620 and a sample loop 625 which is connected between the stator ports B and E, as shown in Figs. 2A to 2C. In the embodiment of Fig. 6A and 6B, a pressure sensor 630 is included in the sample loop 625, with the pressure sensor 630 being configured for determining the pressure of the liquid contained in the sample loop 625.

[0093] Fig. 6B shows the pressure detected by the pressure sensor 630 as a function of time. Alternatively, the pressure detected by the pressure sensor may as well be indicated as a function of any other suitable reference variable, e.g. as a function of delivered volume. During the time interval 635, the sample loop 625 is not part of the separation flow path yet, and accordingly, the liquid contained in the sample loop 625 is in a relaxed state. The pressure detected by the pressure sensor 630 is -20 -close to atmospheric pressure. At the point of time 640, switching of the switching valve 620 is started, and the rotor element of the switching valve starts rotating.

However, during the time interval 645, the rotor element of the switching valve 620 has not arrived at its second position yet, and accordingly, the sample loop 625 is not included in the separation flow path yet. Therefore, the pressure detected by the pressure sensor 630 remains quite low.

[0094] At the point of time 650, the rotor element of the switching valve 625 reaches the second position shown in Fig. 20, and the switching channels 210, 215, 220 provide flu idic connections between the stator ports B and C, between the stator ports D and F, and between the stator ports F and A, as shown in Fig. 2C. Now, the sample loop 625 is included in the separation flow path. The pumping unit 600 is fluidically coupled, via the sample loop 625, with the separation column 610. As a consequence, the liquid contained in the sample loop 625 is exposed to the regular system pressure of the chromatographic separation system, and a corresponding rise 655 of the pressure detected by the pressure sensor 630 is observed. The pressure detected by the pressure sensor 630 rises until the regular system pressure 660 is reached. The course of the pressure detected by the pressure sensor 630 may be analyzed by some kind of data processing unit. As a result of this analysis, the point of time 665 is determined as the actual switching point. At the point of time 665, the liquid sample contained in the sample loop 625 is actually injected into the separation flow path.

[00951 In Fig. 6B, the pressure variations are detected and evaluated as a function of time. Alternatively, the pressure variations may be detected and evaluated as a function of any other suitable reference variable. For example, the pressure variations may be detected and evaluated as a function of delivered volume.

[0096] In Fig. 7A and 7B, another embodiment of the present invention is shown.

Fig. 7A depicts a separation flow path of a chromatographic system comprising a pumping unit 700, a sample injector 705 with a switching valve 710 and a sample loop 715, a separation column 720 and a detection unit 725. In the embodiment of Fig. 7A, a pressure sensor 730 is located downstream of the sample injector 705 and upstream of the separation columns 720. The pressure sensor 730 is configured for detecting the pressure of the liquid downstream of the sample injector 705.

-21 - [0097] In Fig. 7B, the pressure detected by the pressure sensor 730 is shown as a function of time. Alternatively, the pressure detected by the pressure sensor may as well be indicated as a function of any other suitable reference variable, e.g. as a function of delivered volume. During the time interval 735,thepressuredetected bythe pressure sensor 730 is equal to the regular system pressure of the chromatographic separation system. Then, at the point of time 740, the switching operation of the switching valve 710 is started, and the rotor element of the switching valve 710 starts rotating. The rotor element is moved from its first position shown in Fig. 2A to the position shown in Fig. 2B, and accordingly, the fluidic connections between the stator ports A and B, between the stator ports C and D, and between the stator ports E and F are disrupted. As a consequence, the pumping unit 700 is no longer fluidically connected with the pressure sensor 720 and the separation column 720. The liquid contained in the section of the flow path downstream of the sample injector 705 continues moving in the direction towards the detection unit 725, but the supply of mobile phase from the pumping unit 700 is disrupted. Therefore, during the time interval 745, a decline 750 of the pressure measured by the pressure sensor 730 is observed.

[0098] At the point of time 755, the rotor element of the switching valve 710 has reached its second position, which is shown in Fig. 2C. Now, the switching channels 210, 215 and 220 provide fluidic connections between the stator ports B and C, between the stator ports D and E, and between the stator ports F and A. As a consequence, the sample loop 715 is included in the separation flow path, and hence, the fluidic connection between the pumping unit 700 and the separation column 720 is reestablished. As a consequence, a flow of mobile phase provided by the pumping unit 700 is supplied to the pressure sensor 730 and the separation column 720. Therefore, during the time interval 760, an increase 765 of the pressure detected by the pressure sensor 730 is observed, with the detected pressure slowly approaching regular system pressure of the liquid chromatography system.

[0099] The course of the pressure detected by the pressure sensor 730 may be analyzed by some kind of data processing unit. By analyzing the pressure variations, the point of time 770 is identified, which is the point of time when the sample loop 715 is switched into the separation flow path. Hence, the point of time 770 is the actual -22 -injection point, i.e. the point of time when the liquid sample is actually injected into the separation flow path.

[00100] In Fig. 7B, the pressure variations are detected and evaluated as a function of time. Alternatively, the pressure variations may be detected and evaluated as a function of any other suitable reference variable. For example, the pressure variations may be detected and evaluated as a function of delivered volume.

-23 -

Claims

CLAIMS1. A fluid separation system for separating compounds of a sample fluid, the fluid separation system comprising: a mobile phase drive, preferably a pumping system, configured to drive a mobile phase through the fluid separation system; a sample injector configured to introduce the sample fluid into the mobile phase; at least one sensor configured to measure a physical property of at least one of the mobile phase and the sample fluid as a function of a reference variable; a separation unit, preferably a chromatographic column, configured for separating compounds of the sample fluid; the fluid separation system or at least one of its units being configured to determine an actual injection event from a course of the physical property measured by the at least one sensor, or from a value derived there from.
2. The fluid separation system of claim 1, comprising at least one of: the reference variable is a variable indicating progress of a separation process; the reference variable is a variable indicating a progression of time; the reference variable is time; the reference variable is a delivered volume of fluid.
3. The fluid separation system of claim I or any one of the above claims, wherein the actual injection event corresponds to a certain value of the reference variable, said value of the reference variable being taken as a point of reference for a separation process.
4. The fluid separation system of claim 1 or any one of the above claims, wherein said value derived from the course of the physical property measured by the at least one sensor is: -24 -any specific feature, disruption or discontinuity in the course of the measured physical property, or a derivative of first or higher order that relates to a fluid property change caused by the actual injection event, or any mathematical function of one or more of: the measured physical property, fluid parameters, any specific feature, disruption or discontinuity in the course of the measured physical property, a derivative of first or higher order that relates to a fluid property change caused by the actual injection event, or any combination of one or more of: the measured physical property, fluid parameters, any specific feature, disruption or discontinuity in the course of the measured physical property, a derivative of first or higher order that relates to a fluid property change caused by the actual injection event, any mathematical functions derived from these properties.
5. The fluid separation system of claim 1 or any one of the above claims, comprising at least one of: the at least one sensor comprises a pressure sensor configured for measuring fluid pressure as a function of the reference variable; the at least one sensor comprises a flow sensor configured for measuring flow of the fluid as a function of the reference variable; the at least one sensor comprises a sensor configured for measuring conductivity ofthefluid; the at least one sensor comprises a sensor configured for measuring an index of refraction of the fluid; the at least one sensor comprises a sensor configured for determining a temperature of the fluid.
6. The fluid separation system of claim I or any of the above claims, the at least one sensor comprising at least one of: a sensor located within the mobile phase drive; -25 -a sensor located downstream of the mobile phase drive and upstream of the sample injector; a sensor located within the sample injector; a sensor is located downstream of the sample injector and upstream of the separation unit.
7. The fluid separation system of claim 1 or any of the above claims, wherein the sample injector comprises a multiport valve and a sample loop fluidically connected to ports of the multiport valve, wherein in a first position of the multiport valve, the sample loop is not included in a separation flow path, and wherein in a second position of the multiport valve, the sample loop is included in the separation flow path.
8. The fluid separation system of claim 1 or any of the above claims, wherein the sample injector comprises a set of valves which are configured and operated to switch a flow stream such that a sample loop is fluidically connected into the flow stream going to the separation unit.
9. The fluid separation system of claim 7 or any of the above claims, the at least one sensor comprising at least one of: a pressure sensor located upstream of the sample injector, the data processing unit being configured to detect a rise of pressure when the switching of the multiport valve starts, a pressure drop when the sample loop is introduced into the flow path, and a subsequent rise of the pressure; a pressure sensor located within the sample injector, the data processing unit being configured to detect a rise of the pressure in the sample loop when the sample fluid is introduced into the separation flow path; a pressure sensor located downstream of the sample injector, the data processing unit being configured to detect a decline of the pressure when the switching of the multiport valve starts and a subsequent rise of the pressure when the sample loop is introduced into the flow path.

-26 -
10. The fluid separation system of claim 1 or any of the above claims, the at least one sensor comprising at least one of: a flow sensor located upstream of the sample injector, the data processing unit being configured to detect a drop of fluid flow when the switching of the multiport valve starts, and a rise of the fluid flow when the sample loop is introduced into the flow path; a flow sensor located within the sample injector, the data processing unit being configured to detect a rise of fluid flow when the sample fluid is introduced into the separation flow path; a flow sensor located downstream of the sample injector, the data processing unit being configured to detect a decline of the fluid flow when the switching of the multiport valve starts and a subsequent rise of the fluid flow when the sample loop is introduced into the flow path.
11. The fluid separation system of claim 1 or any of the above claims, comprising at least one of: the actual injection event is used as a point of reference for the analysis of the compounds of the sample fluid; the actual injection event is used as a point of reference for determining respective retention times or retention volumes of the compounds of the sample fluid; the actual injection event is used as a point of reference for the operation of at least one of: the mobile phase drive, a detector unit, a collection unit, a thermostat, switching valves comprised in the system.
12. The fluid separation system of claim I or any of the above claims, further comprising at least one of: a detector configured to detect separated compounds of the sample fluid; a collection unit configured to collect separated compounds of the sample fluid; -27 -a degassing apparatus for degassing the mobile phase.
13. A method of determining an actual injection event when a sample fluid is actually introduced into a fluid separation system, the fluid separation system comprising a mobile phase drive, preferably a pumping system, configured to drive a mobile phase through the fluid separation system, and a separation unit, preferably a chromatographic column, configured for separating compounds of the sample fluid in the mobile phase, the method comprising measuring a physical property of at least one of the mobile phase and the sample fluid as a function of a reference variable; introducing the sample fluid into the mobile phase; determining the actual injection event of the fluid sample from a course of the physical property, or from a value derived there from.
14. A software program or product, preferably stored on a data carrier, for controlling or executing the method of claim 13, when run on a data processing system such asacomputer.-28 -