PURITY DETECTION OF SEPARATED SAMPLE PORTION AS BASIS FOR A POSITIVE OR NEGATIVE DECISION CONCERNING FURTHER SEPARATION

Abstract

A sample separation apparatus for separating a fluidic sample includes an initial dimension sample separation device configured for separating the fluidic sample, a subsequent dimension sample separation device configured for further separating separated fluidic sample received from the initial dimension sample separation device, a purity detector configured for detecting information indicative of a purity of a portion of the fluidic sample which has been separated by the initial dimension sample separation device, and a control unit configured for controlling, depending on the detected information, whether or not further separation of the portion of the fluidic sample which has been separated by the initial dimension sample separation device is carried out by the subsequent dimension sample separation device.

Claims

1. A sample separation apparatus for separating a fluidic sample, the sample separation apparatus comprising: an initial dimension sample separation device configured for separating the fluidic sample in accordance with a first separation criterion; a subsequent dimension sample separation device configured for further separating separated fluidic sample received from the initial dimension sample separation device in accordance with a second separation criterion; a purity detector configured for detecting information indicative of a purity of a portion of the fluidic sample which has been separated by the initial dimension sample separation device, wherein the purity is indicative whether the portion of fluidic sample has substantially only a single component or is composed of multiple different components which can be further separated; and a control unit configured for controlling, depending on the detected information, whether or not further separation of the portion of the fluidic sample which has been separated by the initial dimension sample separation device is carried out by the subsequent dimension sample separation device.

2. The sample separation apparatus according to claim 1, wherein the purity detector is configured for detecting whether the separated portion of the fluidic sample comprises only one pure component or is composed of multiple different components.

3. The sample separation apparatus according to claim 1, wherein the purity detector (50) is configured for detecting whether the separated portion of the fluidic sample comprises only one pure component or is composed of multiple components based on a detected chromatogram.

4. The sample separation apparatus according to claim 1, wherein the purity detector is configured for detecting whether the separated portion of the fluidic sample comprises only one pure component or is composed of multiple components based on an optical peak detected on the separated portion of the fluidic sample.

5. The sample separation apparatus according to claim 4, wherein the purity detector is configured for detecting the information by further analyzing the optical peak.

6. The sample separation apparatus according to claim 5, wherein the purity detector is configured for further analyzing the optical peak by recording and comparing a plurality of characteristic curves all relating to the optical peak and obtained by varying at least one physical parameter over time.

7. The sample separation apparatus according to claim 6, wherein the purity detector is configured for assuming purity of the portion of the separated fluidic sample if the plurality of characteristic curves differ concerning their shapes by less than a predefined threshold.

8. The sample separation apparatus according to claim 5, wherein the purity detector is configured for further analyzing the optical peak by recording at least one characteristic curve relating to the optical peak by varying at least one physical parameter over time, and by comparing at least one characteristic curve with at least one reference curve relating to a reference sample with pre-known properties.

9. The sample separation apparatus according to claim 1, wherein the purity detector is a non-destructive detector configured for analyzing the fluidic sample without destructing the fluidic sample.

10. The sample separation apparatus according to claim 1, wherein the purity detector comprises a spectral analysis detector configured for carrying out a spectral analysis with the portion of the fluidic sample.

11. The sample separation apparatus according to claim 1, wherein the purity detector comprises a mass spectrometry detector configured for analyzing part of the portion of the fluidic sample by mass spectrometry concerning purity, whereas another part of the portion of the fluidic sample is forwarded for further separation to the subsequent dimension sample separation device if the purity detector detects an insufficient purity level for the portion of the fluidic sample.

12. The sample separation apparatus according to claim 1, wherein the purity detector is configured for detecting components of the fluidic sample separated by the initial dimension sample separation device.

13. The sample separation apparatus according to claim 1, wherein the control unit is configured for triggering further separation of the separated portion of the fluidic sample in the subsequent dimension sample separation device if the detected information is indicative of the presence of a plurality of components in the detected portion of the fluidic sample.

14. The sample separation apparatus according to claim 1, wherein the control unit is configured for discharging the separated portion of the fluidic sample out of the sample separation apparatus without further separation of the separated portion of the fluidic sample in the subsequent dimension sample separation device if the detected information is indicative of a purity of the detected portion of the fluidic sample.

15. The sample separation apparatus according to claim 1, wherein the control unit is configured for controlling in-line whether or not further separation of the portion of the fluidic sample, which has been separated by the initial dimension sample separation device, is carried out or not by the subsequent dimension sample separation device.

16. The sample separation apparatus according to claim 1, wherein the control unit is configured for operating the sample separation apparatus in a heart-cutting mode.

17. The sample separation apparatus according to claim 1, comprising at least one of the following features: configured as a two-dimensional sample separation apparatus; configured as a as two-dimensional chromatographic sample separation apparatus; comprising at least one further dimension sample separation device configured for further separating the portion of the fluidic sample, which has been separated by the initial dimension sample separation device and by the subsequent dimension sample separation device, in at least one further separation dimension; configured as one of an analytic sample separation apparatus or a preparative sample separation apparatus; comprising a sampling valve at an interface between the initial dimension sample separation device and the subsequent dimension sample separation device, wherein the control unit is configured for switching the sampling valve depending on detected information to thereby control whether or not further separation of fluidic sample which has been separated by the initial dimension sample separation device is carried out by the subsequent dimension sample separation device; comprising a sampling valve at an interface between the initial dimension sample separation device and the subsequent dimension sample separation device, wherein the control unit is configured for switching the sampling valve depending on detected information to thereby control whether or not further separation of fluidic sample which has been separated by the initial dimension sample separation device is carried out by the subsequent dimension sample separation device, wherein the sampling valve comprises at least one sample accommodation volume, preferably a plurality of sample accommodation volumes, configured for temporarily accommodating a portion of the fluidic sample after separation by the initial dimension sample separation device and before separation by the subsequent dimension sample separation device; wherein the initial dimension sample separation device comprises an initial dimension fluid drive unit configured for driving mobile phase and the fluidic sample after injection in the mobile phase, and comprises an initial dimension sample separation unit configured for separating the fluidic sample upstream of the purity detector; wherein the subsequent dimension sample separation device comprises a subsequent dimension fluid drive unit configured for driving mobile phase and the separated fluidic sample after injection in the mobile phase, and comprises a subsequent dimension sample separation unit configured for further separating the separated fluidic sample downstream of the purity detector; wherein the subsequent dimension sample separation device comprises a subsequent dimension fluid drive unit configured for driving mobile phase and the separated fluidic sample after injection in the mobile phase, and comprises a subsequent dimension sample separation unit configured for further separating the separated fluidic sample downstream of the purity detector, wherein the subsequent dimension sample separation device comprises a subsequent dimension detector configured for detecting the further separated fluidic sample downstream of the subsequent dimension sample separation unit.

18. The sample separation apparatus according to claim 1, comprising at least one of the following features: at least one of the initial dimension sample separation device and the subsequent dimension sample separation device is configured for performing a separation in accordance with one selected from the group consisting of: liquid chromatography, high-performance liquid chromatography (H PLC), supercritical-fluid chromatography, gas chromatography, capillary electrochromatography, and electrophoresis; the sample separation apparatus is configured to analyze at least one physical, chemical, and/or biological parameter of at least one compound of the fluidic sample; the sample separation apparatus is configured to conduct the fluidic sample with a high pressure; the sample separation apparatus is configured to conduct the fluidic sample with a pressure in a range selected from the group consisting of at least 500 bar, at least 1000 bar, and at least 1200 bar; the sample separation apparatus is configured to conduct a liquid or a gas; the sample separation apparatus is configured as a microfluidic device; the sample separation apparatus is configured as a nanofluidic device.

19. A method of separating a fluidic sample, the method comprising: separating the fluidic sample in accordance with a first separation criterion; detecting information indicative of a purity of a portion of the separated fluidic sample, wherein the purity is indicative whether the portion of fluidic sample has substantially only a single component or is composed of multiple different components which can be further separated; and controlling, depending on the detected information, whether or not further separation of the portion of the separated fluidic sample will be carried out in accordance with a second separation criterion.

20. The method according to claim 19, comprising at least one of the following features: wherein the method comprises, preferably in an in-line process, further separating said portion of the fluidic sample if an insufficient purity level has been detected for said portion of the fluidic sample; wherein the method comprises draining off said portion of the fluidic sample away from a further separation path without a further separation if a sufficient purity level has been detected for said portion of the fluidic sample; wherein the method comprises forwarding at least one separated portion of the fluidic sample to a further separation path for carrying out a further separation and draining at least one other separated portion of the fluidic sample away from the further separation path without a further separation, depending on a respective detected purity level of said separated portions of the fluidic sample.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] 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 accompanying drawings. Features that are substantially or functionally equal or similar will be referred to by the same reference signs.

[0057] FIG. 1 illustrates a liquid chromatography system according to an exemplary embodiment.

[0058] FIG. 2 illustrates a multidimensional sample separation apparatus according to an exemplary embodiment.

[0059] FIG. 3 illustrates an absorption peak captured after a first separation dimension of a sample separation apparatus according to an exemplary embodiment.

[0060] FIG. 4 illustrates wavelength spectra captured at three temporal positions of the absorption peak of FIG. 3 for determining a purity of a separated fluidic sample portion according to an exemplary embodiment.

[0061] FIG. 5 illustrates a mass spectrometry diagram captured at a certain temporal position of the absorption peak of FIG. 3 for determining a purity of a separated fluidic sample portion according to an exemplary embodiment.

[0062] FIG. 6 illustrates another mass spectrometry diagram captured at a different temporal position of the absorption peak of FIG. 3, in comparison to FIG. 5, for determining a purity of a separated fluidic sample portion according to an exemplary embodiment.

[0063] FIG. 7 illustrates diagrams for explaining sample portion-specific decisions concerning multi-dimensional separations made individually for different fluidic sample portions based on a sample portion-specific purity analysis by means of chromatograms according to an exemplary embodiment.

[0064] FIG. 8 shows a fluidic interface region between a primary stage sample separation device and a secondary stage sample separation device according to an exemplary embodiment in which a modulator valve cooperates with two buffer valves each cooperating with a plurality of buffer volumes for temporarily storing a respective fluid packet.

[0065] The illustration in the drawing is schematic.

DETAILED DESCRIPTION

[0066] Before describing the figures in further detail, some basic considerations of the present invention will be summarized based on which exemplary embodiments have been developed.

[0067] According to an exemplary embodiment of the invention, an eluate from a first or primary separation stage of a sample separation apparatus may be made subject to a spectral analysis or mass spectrometry analysis for recording an eluate spectrum based on a chromatographic peak. Based on a time resolution of such a peak of such a chromatogram, the purity of the section of fluidic sample relating to said peak may be determined. For example, it is possible to record one or multiple optical spectra around the peak. Said one or multiple optical spectra may be compared with one or multiple predetermined reference spectra and/or potential changes of the spectra over time may be observed. On the basis of such analysis, it is possible to determine whether the sample section corresponding to the peak includes a pure substance or a mixture of different substances. If the sample section is pure, no further separation in the subsequent separation dimension is necessary, so that the sample section may be fractionated at an outlet of the first separation dimension. If the sample section is not pure but is still composed of multiple components, said sample section may be guided to the second separation dimension for further separation. By taking this measure, unnecessary further separation processes may be avoided, and the time needed for a precise sample separation may be reduced. Furthermore, hardware resources may be used more efficiently. For example, a spectral impurity of a peak may be used as a basis for cutting out a corresponding section of the fluidic sample separated in the first separation dimension in a heart cutting mode, i.e. making selectively such a fluidic sample section subject to a further separation. Highly advantageously, it may be possible to measure sample section purity online and decide live or in real-time whether a presently passing sample section should be directly guided to the subsequent separation dimension for further separation or should not be further separated in the subsequent separation dimension, since it is already pure or sufficiently pure.

[0068] In an embodiment, a purity-based multidimensional chromatography apparatus is provided which is configured for controlling sample separation to be carried out in a number of dimensions, which number is determined by a purity detection of the already separated sample separation in a preceding separation stage or dimension and before forwarding the separated sample for further separation into a subsequent separation stage or dimension. Such an embodiment, when configured for in-line operation, may overcome limitations of offline workflows, i.e. a potential loss of sample (for instance due to degradation, adsorption, etc.). Furthermore, such a purity-based multi-dimensional chromatography apparatus may speed-up the analysis, since it may prevent unnecessary further separation of an already completely (or sufficiently) separated fluidic sample. Furthermore, a purity detection at an interface between adjacent dimensions of a multidimensional sample separation apparatus may allow to gain information for improving control of a sample operation task. In particular, exemplary embodiments of the invention may result in an increased efficiency of multidimensional sample separation, in particular two-dimensional liquid chromatography (2D-LC) or two-dimensional gas chromatography (2D-GC).

[0069] A conventional peak-based operation lacks an access to relevant information. Comprehensive 2D-LC is often not sufficient for achieving required resolution.

[0070] An exemplary embodiment of the invention provides a sample separation apparatus, which may be preferably embodied as 2D-LC (or 2D-GC), with increased resolution by finding out if compounds have been separated sufficiently.

[0071] Generally, operation of a two-dimensional sample separation apparatus can be done in a heart-cutting mode or in a comprehensive mode. In a comprehensive mode, the entire eluent of the first separation dimension is injected to the second separation dimension for further or more refined separation. However, the analysis time is frequently short and may be too short for achieving superior resolution. Heart-cutting allows increasing this resolution, but is limited to one or a limited number of positions in the first dimension separation. For unknown samples, first dimension retention times are not known, or additional peaks may show up unexpectedly. In that case, peak-based operation can be applied.

[0072] However, it has been found by the present inventor that peak-based operation usually re-analyzes cuts in the second dimension based on the criterion “is there a peak” rather than based on the more relevant criterion “is there a peak with multiple compounds”, i.e. based on whether or not a sample portion relating to a peak is pure. For instance, a sample portion can be considered as pure if it includes only one component or fraction. Such information may be typically extracted after the separation, i.e. during data analysis and therefore off-line. Algorithms are in place for determining peak purity by using an ultraviolet detector or mass spectral information. It can be determined whether the spectrum changes within a peak, or whether different wavelengths are absorbed. It can further be determined whether different masses are measured.

[0073] According to an exemplary embodiment of the invention, a purity determination can be done within the firmware of a purity detector with spectral capabilities (for instance a diode array detector, a fluorescence detector, and/or a mass spectrometry detector) at an outlet of the first separation dimension, such that an on-line or in-line decision concerning a potential further analysis or separation in the second dimension may be performed. This may advantageously avoid (offline) re-injection and re-analysis with intermediate user interaction and data analysis. Advantageously, an exemplary embodiment may use a spectral analysis of peaks which does not influence or reduce the amount of sample.

[0074] More specifically, an exemplary embodiment of the invention provides a two-dimensional liquid chromatography apparatus, wherein a decision process which portions separated by the first dimension should go into the second dimension for further separation may be made on the basis of a purity detection. Hence, a gist of an exemplary embodiment of the invention is to analyze purity of peaks—detected in the first dimension—and to decide based on the result of such a purity analysis whether a further separation in the second dimension makes sense and shall be made.

[0075] Exemplary embodiments of the invention are particularly appropriate also for a multi-dimensional use. In other words, the principle described above can be applied to more than two dimensions, for instance ultimately doing or repeating separations until peaks are measured to be pure or at least sufficiently pure.

[0076] While an optimum resolution may be achieved by different and ideally orthogonal separation conditions in the first separation dimension versus the second separation dimension, an improved separation can also be obtained by using longer run times but the same mobile phase and/or stationary phase. Therefore, the described mechanism can be advantageously used for dynamically extending run times, in particular exactly whenever needed. Advantageously, an exemplary embodiment of the invention foresees online detection of peak purity and automated heart-cutting on the basis of an outcome of the purity detection.

[0077] Preferred embodiments of the invention relate to analytical workflows. However, other embodiments can be applied to on-line purification workflows for separation to pure components.

[0078] Referring now in greater detail to the drawings, FIG. 1 depicts a general schematic of a two-dimensional liquid separation system as an example for a sample separation apparatus 100 according to an exemplary embodiment of the invention. A first pump in form of a first fluid drive unit 20 receives a mobile phase (also denoted as fluid) from a first solvent supply 25, typically via a first degasser 27, which degases and thus reduces the amount of dissolved gases in the mobile phase. The first fluid drive unit 20—as a mobile phase drive—drives the mobile phase through a first sample separation unit 30 (such as a chromatographic column) comprising a stationary phase. A sampling unit or injector 40 can be provided between the first fluid drive unit 20 and the first sample separation unit 30 in order to subject or add (often referred to as sample introduction) a sample fluid (also denoted as fluidic sample) into the mobile phase. The stationary phase of the first sample separation unit 30 is configured for separating compounds of the sample liquid. The components of the separated fluidic sample can be detected by a detector 50. The detector 50 is provided for detecting separated compounds of the sample fluid.

[0079] Simultaneously, detector 50 functions as a purity detector configured for detecting purity of individual peaks in a chromatogram of the fluidic sample separated in first sample separation unit 30. Detector 50 is controlled by a control unit 70 and transmits detection signals to control unit 70. Members 25, 27, 20, 40, 30 and 50 relate to a first dimension sample separation device 102.

[0080] A second pump or second fluid drive unit 20′ receives another mobile phase (also denoted as fluid) from a second solvent supply 25′, typically via a second degasser 27′, which degases and thus reduces the amount of dissolved gases in the other mobile phase. By a fluidic valve 114, the first dimension (reference numerals 20, 30, . . . ) of the two-dimensional liquid chromatography system of FIG. 1 may be fluidically coupled to the second dimension (reference numerals 20′, 30′, . . . ). In a second sample separation unit 30′, the pre-separated components of fluidic sample from the first separation dimension may be further separated. The further separated fluidic sample may be detected in a further detector 50′ and may be optionally fractionated in a fractionator 60′. Members 25′, 27′, 20′, 30′, 50′, 60′ constitute a second dimension sample separation device 104.

[0081] The fluidic sample is separated into multiple components by the first dimension, and each component can be further separated into multiple sub-components by the second dimension, when the fluidic valve 114 is switched under control of control unit 70 to introduce the separated fluidic sample from the first dimension into the second dimension. However, it is also possible that the fluidic valve 114 is switched under control of control unit 70 to direct the separated fluidic sample from the first dimension to a fractionating unit 60 (or to a waste line) rather than for further separation in the second dimension. The fractionating unit 60 can be provided for outputting separated compounds of sample fluid. More specifically, if purity detector 50 detects that a sample section as an eluate of the first separation dimension only includes a single component and is therefore pure, the control unit 70 uses this detection result for switching fluidic valve 114 so that said sample section is directly fractionated rather than further separated. If however purity detector 50 detects that a sample section as an eluate of the first separation dimension is still a mixture of multiple components or sub-components and is therefore impure, the control unit 70 uses this detection result for switching fluidic valve 114 so that said sample section is further separated in the second separation dimension.

[0082] While each of the mobile phases can be comprised of one solvent only, it may also be mixed from plural solvents. Such mixing may be a low pressure mixing and provided upstream of the fluid drive units 20, 20′, so that the respective fluid drive unit 20, 20′ already receives and pumps the mixed solvents as the mobile phase. Alternatively, the fluid drive unit 20, 20′ may 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 respective sample separation unit 30, 30′) occurs at high pressure and downstream of the fluid drive unit 20, 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 over time, the so called gradient mode.

[0083] Control unit 70, which can be embodied as a data processing unit (e.g., a computing device) such as a conventional PC or workstation, may be coupled (as indicated by the dotted arrows) to one or more of the devices in the sample separation apparatus 100 in order to receive information and/or control operation. For example, the control unit 70 may control operation of the fluid drive units 20, 20′ (for example 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 control unit 70 may also control operation of the solvent supply 25, 25′ (for instance setting the solvent/s or solvent mixture to be supplied) and/or the degasser 27, 27′ (for instance setting control parameters such as vacuum level) and may receive therefrom information regarding the actual working conditions (such as solvent composition supplied over time, flow rate, vacuum level, etc.). The control unit 70 may further control operation of the sampling unit 40 (for instance controlling sample injection or synchronization of sample injection with operating conditions of the fluid drive unit 20). The respective sample separation unit 30, 30′ may also be controlled by the control unit 70 (for instance selecting a specific flow path or column, setting operation temperature, etc.), and send—in return—information (for instance operating conditions) to the control unit 70. Accordingly, the detector 50 may be controlled by the control unit 70 (for instance with respect to spectral or wavelength settings, setting time constants, start/stop data acquisition), and send information (for instance about the detected sample compounds) to the control unit 70. The control unit 70 may also control operation of the fluidic valve 114 (for instance in conjunction with data received from the detector 50) and provides data back.

[0084] The sample separation apparatus 100 illustrated in FIG. 1 may be operated for separating a fluidic sample selectively in one or two separation dimensions. More specifically, the sample separation apparatus 100 may be operated for deciding individually for each portion of fluidic sample separated in the first separation dimension whether the respective portion of fluidic sample is or is not to be further separated in the second separation dimension. For this purpose, it may be possible to detect data or information indicative of a purity of each individual separated portion of the fluidic sample at the outlet of the first separation dimension by detector 50. Furthermore, control unit 70 may control individually for each separated portion of the fluidic sample whether or not further separation of the separated fluidic sample will be carried out or not in the second separation dimension depending on detected purity information. For instance, a respective separated portion of fluidic sample having a purity above a predefined threshold value may be disabled to enter the second separation dimension. In contrast to this, a respective separated portion of fluidic sample having a purity below the predefined threshold value may be enabled to enter the second separation dimension for further separation.

[0085] In the following, referring to FIG. 2, a multidimensional liquid chromatography apparatus 100 according to an exemplary embodiment of the invention will be explained. The illustrated liquid chromatography apparatus 100 may be configured for analytic sample separation or for preparative sample separation.

[0086] The illustrated sample separation apparatus 100 is configured for separating a fluidic sample, in particular a liquid sample (or a gas sample, when the sample separation apparatus 100 is configured as gas chromatography apparatus). The shown sample separation apparatus 100 comprises a first dimension sample separation device 102 configured for separating the fluidic sample. In a second dimension sample separation device 104, it may be possible to further separate the separated fluidic sample received from the first dimension sample separation device 102. In an optional third dimension sample separation device 116 (which is shown only schematically), it may be possible to further separate the further separated fluidic sample received from the second dimension sample separation device 104, if desired or required. For example, construction of the third dimension sample separation device 116 may be the same or similar as construction of second dimension sample separation device 104.

[0087] As shown, the first dimension sample separation device 102 comprises a first dimension fluid drive unit 20 (such as a high-pressure mobile phase pump) configured for driving mobile phase (such as a solvent or a solvent composition) and the fluidic sample after injection by an injector 40 in the mobile phase. The injector 40 may comprise an injection valve 95 which may be switched into the flow path between first dimension fluid drive unit 20 and first dimension sample separation unit 30 for sample injection. The first dimension sample separation unit 30 (such as a chromatographic column) is configured for separating the fluidic sample in the mobile phase received from the first dimension fluid drive unit 20 and from the injector 40.

[0088] A detector 50 arranged downstream of the first dimension sample separation unit 30 fulfills a double function: On the one hand, detector 50 detects separated components of the fluidic sample in subsequent portions of the fluidic sample flowing through the conduits of the first dimension sample separation device 102. On the other hand, detector 50 is configured as purity detector for detecting information indicative of a purity of a respective portion of the fluidic sample separated by the first dimension sample separation device 102. Descriptively speaking, detector 50 therefore also delivers information to control unit 70 regarding whether a respective fluidic sample portion consists of only one component (and can thus be considered as pure) or is still a mixture of multiple different components at the outlet of the first dimension sample separation device 102 (and can thus be considered as impure). In other words, the purity detector 50 is configured for detecting whether each individual separated portion of the fluidic sample comprises only one pure component or is composed of multiple components. The purity detector 50 can make this conclusion by detecting and evaluating a chromatogram. Preferably, the purity detector 50 is a non-destructive detector configured for analyzing the fluidic sample without destroying the fluidic sample during the detection process. For this purpose, the purity detector 50 may advantageously comprise a spectral analysis detector configured for carrying out a spectral analysis with the portion of the fluidic sample (compare FIG. 3 and FIG. 4).

[0089] If the purity detector 50 is alternatively a destructive detector, i.e. destroys fluidic sample during the process of detection, the fluidic sample section may be split at a flow splitter (not shown, for instance a fluidic T-piece) into a first part which is directed to the purity detector 50 for purity detection and into a second part which may be used for fractionating or further separation of the second part of the fluidic sample portion. For example, a mass spectrometry detector configured for further analyzing the portion of the fluidic sample by mass spectrometry may be another appropriate choice for detector 50, although a part of the fluidic sample will be destroyed during purity detection. An example for a corresponding analysis is illustrated in FIG. 5 and FIG. 6 in combination with FIG. 3.

[0090] As already mentioned, detector 50 may be synergistically configured for detecting components of the fluidic sample separated by the first dimension sample separation device 102, apart from fulfilling the task of purity detection.

[0091] Control unit 70 is provided with the purity detection results of detector 50, i.e. with the detected purity data. Control unit 70 is configured for controlling whether or not further separation of fluidic sample separated by the first dimension sample separation device 102 shall be carried out or not by the second dimension sample separation device 104 depending on detected purity information. More specifically, the control unit 70 is configured for triggering further separation of the separated fluidic sample in the second dimension sample separation device 104 if the detected information is indicative of the presence of a plurality of components in the detected portion of the fluidic sample. Furthermore, the control unit 70 is configured for discharging the portion of fluidic sample out of the sample separation apparatus 100 without further separation of the separated fluidic sample in the second dimension sample separation device 104 if the detected information is indicative of a purity of the detected portion of the fluidic sample. In the latter scenario, the analyzed portion of the fluidic sample which has already been separated by the first dimension sample separation device 102 is not further separated in the second dimension sample separation device 104, but is in contrast to this directly forwarded into fractioning unit 60 or alternatively a waste container without further separation. Highly advantageously, the control unit 70 is thus configured for controlling in-line whether or not further separation of fluidic sample separated by the previous first dimension sample separation device 102 is to be carried out by the subsequent second dimension sample separation device 104. Thus, the fluidic sample remains within the flow paths of sample separation apparatus 100 during the processes of sample separation, purity detection and switching of sampling valve 114 (as described below). The decision about further separation in at least one additional separation dimension or discharging separated fluidic sample without further operation in an additional separation dimension can thus be taken in real time and without the need to involve a user into a cumbersome manual purity detection task. For instance, the control unit 70 may be configured for operating the sample separation apparatus 100 in a heart-cutting mode (preferably in a multiple heart-cutting mode) for selectively cutting out from a continuous stream of fluidic sample one or several discrete sections for additional analysis in an additional separation dimension, on the basis of detected purity information. Advantageously, additional sample separation may thus be limited to cases where purity of a fluidic sample section after a first dimension separation is not yet sufficient.

[0092] In order to establish the described logic of forwarding or not forwarding individual sample sections for further separation, sampling valve 114 may be arranged at a fluidic interface between the first dimension sample separation device 102 and the second dimension sample separation device 104 and may be switched or operated under control of control unit 70, wherein a switching scheme may be determined in accordance with the detected purity information. More specifically, the control unit 70 is configured for switching the sampling valve 114 depending on the detected purity information.

[0093] As shown in FIG. 2 as well, the second dimension sample separation device 104 comprises a second dimension fluid drive unit 20′ (such as a further high-pressure mobile phase pump) configured for driving further mobile phase (such as a further solvent or solvent composition) and the separated fluidic sample after injection via sampling valve 114 in the further mobile phase. A second dimension sample separation unit 30′ (such as a further chromatography column) is configured for further separating the separated fluidic sample received via sampling valve 114 from purity detector 50. Furthermore, the second dimension sample separation device 104 comprises a second dimension detector 50′ configured for detecting the further separated fluidic sample downstream of the second dimension sample separation unit 30′. As detector 50, also detector 50′ may detect purity of the further separated fluidic sample. A decision whether the further separated fluidic sample shall be introduced into the third dimension sample separation device 116 for carrying out yet another separation, or removing the further separated fluidic sample out of the sample separation apparatus 100 after the second dimension separation and into a further fractioning unit 60′ can be taken based on the results of the purity detection by second dimension detector 50′. This decision can be taken in a corresponding way as described above for detector 50. By taking this measure, it can be flexibly decided for each individual fluidic sample section whether a separation in one, two, three or even more separation dimensions shall be carried out. Proper separation accuracy can thus be synergistically combined with a fast and resource saving operation.

[0094] Next, construction and operation of sampling valve 114 will be described in further detail: FIG. 2 shows a first switching state of sampling valve 114. In this first switching state, an outlet of detector 50 is fluidically coupled via a first groove 140 in a rotor member of the rotary-type sampling valve 114 and via ports of a stator member of the rotary-type sampling valve 114 with a (first) sample accommodation volume 142 (here embodied as sample loop). Via a second groove 144 in the rotor member and via further ports of the stator member, the sample accommodation volume 142 is brought in fluid communication with fractionating unit 60 (or alternatively a waste container). In this first switching state, the second dimension fluid drive unit 20′ is fluidically coupled via a third groove 146 in the rotor member and via ports of the stator member with a further (or second) sample accommodation volume 148 (here embodied as further sample loop). Via a fourth groove 150 in the rotor member and via further ports of the stator member, the further sample accommodation volume 148 is brought in fluid communication with second dimension sample separation unit 30′ for further separation of a fluidic sample portion which has previously been buffered in further sample accommodation volume 148.

[0095] Thus, in the first switching state of sampling valve 114 illustrated in FIG. 2, a section of fluidic sample which has previously been introduced in the further sample accommodation volume 148 is presently separated in the second separation dimension. Another fluidic sample section is presently introduced into the first sample accommodation volume 142. After switching sampling valve 114 in a second switching state (not shown), which differs from FIG. 2 in that the rotor is rotated by 90°, a fluidic sample section in sample accommodation volume 142 may be separated in the second separation dimension, whereas the further sample accommodation volume 148 may be filled with a fresh fluidic sample section. With the shown configuration, a substantially continuous separation operation can be carried out without significant delay time.

[0096] However, when a fresh fluidic sample section flows out of detector 50 and in or through a respective sample accommodation volume 142, 148, it may be decided depending on a purity level of an individual fluidic sample section as just detected by detector 50 in combination with a proper switching of sampling valve 114 whether said individual fluidic sample section is further separated in the second dimension sample separation device 104 or is guided to the fractioning unit 60 or to the waste container without secondary separation. More specifically, control unit 70 receives the purity information from detector 50 and switches the sampling valve 114 so that only selected (i.e. not yet sufficiently pure) fluidic sample sections are further separated in the second dimension.

[0097] FIG. 3 illustrates an absorption peak 106 of a chromatogram captured by detector 50 according to FIG. 1 or FIG. 2 after a first separation by sample separation apparatus 100 according to an exemplary embodiment. FIG. 4 illustrates three wavelength spectra captured at three temporal positions t1, t2 and t3 of the absorption peak 106 of the chromatogram of FIG. 3 for determining a purity of a separated fluidic sample portion according to an exemplary embodiment.

[0098] FIG. 3 shows a diagram 160 having an abscissa 162 along which the time, t, is plotted. Along an ordinate 164, an absorption intensity, I1, is plotted. Absorption peak 106 in diagram 160 can be detected by detector 50 when a specific portion of fluidic sample passes detector 50. FIG. 4 shows a further diagram 170 having an abscissa 172 along which the wavelength of electromagnetic radiation, λ, is plotted in nanometers (nm). Along an ordinate 174, a signal intensity, I2, is plotted. Three characteristic curves 108, 109 and 110 corresponding to absorption peak 106 in diagram 160 can be detected by detector 50 when a specific portion of fluidic sample passes detector 50. Characteristic curve 108 shows the dependency of the signal intensity I2 from a wavelength of electromagnetic detection radiation at a point of time t1 defined in FIG. 3. Characteristic curve 109 shows the dependency of the signal intensity I2 from the wavelength of electromagnetic detection radiation at a point of time I2 defined in FIG. 3. Characteristic curve 110 shows the dependency of the signal intensity I2 from the wavelength of electromagnetic detection radiation at a point of time t3 defined in FIG. 3.

[0099] For obtaining diagram 160, the purity detector 50 may detect a chromatogram of the fluidic sample separated in the first separation dimension. This chromatogram includes the absorption peak 106 of FIG. 3 which relates to a specific portion or section of the fluidic sample to be separated. As shown in FIG. 3, the portion of the fluidic sample corresponds to the optical peak 106 which is here a single absorption peak. In addition, detector 50 is configured for further analyzing the optical absorption peak 106 by recording, for instance at the three temporal positions t1, t2 and t3 of the absorption peak 106 of FIG. 3, a respective wavelength spectrum, as shown in FIG. 4. Thus, the purity detector 50 is configured for detecting the purity information by further analyzing the optical peak 106 in terms of a spectral analysis. Each wavelength spectrum describes, for one specific point of time t1, t2 or t3, a dependency of a detected signal amplitude from a wavelength of electromagnetic radiation detected by detector 50. More generally, the purity detector 50 is configured for further analyzing the optical absorption peak 106 by recording the three (or any other appropriate number of) characteristic curves 108 to 110 relating to the optical peak 106 by varying the physical parameter “wavelength” over time.

[0100] Based on the diagram 170 in FIG. 4, it can be decided whether the portion of the fluidic sample includes only one component or multiple components, i.e. is pure or not.

[0101] If the already separated portion of the fluidic sample is pure, no further separation of this portion of the fluidic sample is necessary. If the already separated portion of the fluidic sample is not pure, further separation of the portion of the fluidic sample is necessary in a subsequent separation stage.

[0102] The purity information may be derived from diagram 170 in different ways. If the already separated fluidic sample comprises only one component or species and is therefore pure, the three characteristic curves 108 to 110 would only differ in height, but not in shape. In this scenario, the three characteristic curves 108 to 110 would have constant proportions, i.e. would only differ by a proportionality factor (and possibly by an offset). In the shown example, however, the shapes of the different characteristic curves 108 to 110 are fundamentally different, so that diagram 170 is the fingerprint of a portion of the fluidic sample which still has different components, fractions or species and needs further separation of said different components, fractions or species in a subsequent separation dimension. For instance, the purity detector 50 is configured for assuming purity of the portion of the separated fluidic sample if the plurality of characteristic curves 108 to 110 differ concerning their shapes by less than a predefined threshold. By taking this measure, relatively small shape differences between the various characteristic curves 108 to 110, which have their origin not in different species in the assigned fluidic sample portion, but in measurement artifacts will not result in an incorrect classification of a pure fluidic sample portion as impure.

[0103] Alternatively, a respective one of the characteristic curves 108 to 110 may be compared with a number of preknown reference curves for determining whether one or more characteristic curves 108 to 110 indicate the presence of one or multiple species. For instance, a best match of a characteristic curves 108 to 110 with one of multiple reference curves stored in a database may be searched. When each reference curve of the database is correlated with a certain number (in particular one and more than one) of species in an assigned sample, the found best match may provide purity information.

[0104] FIG. 5 and FIG. 6 illustrate different mass spectrometry diagrams 180, 190 captured at two different temporal positions t2, t3 of the absorption peak 106 of FIG. 3 for determining a purity of a separated fluidic sample portion according to an exemplary embodiment.

[0105] Each of diagrams 180, 190 has an abscissa 182 along which the mass-electric charge ratio (m/z) is plotted. Along an ordinate 184, a relative abundance is plotted in percent. Two characteristic curves 109 and 110 corresponding to absorption peak 106 in diagram 160 of FIG. 3 can be detected by detector 50, which is here embodied as mass spectrometer detector, when a specific portion of fluidic sample passes detector 50. Characteristic curve 109 shows the dependency of the relative abundance from the mass-electric charge ratio at a point of time t2 defined in FIG. 3. Characteristic curve 110 shows the dependency of the relative abundance from the mass-electric charge ratio at a point of time t3 defined in FIG. 3. Since the analyzed fluidic sample portion comprises various constituents, the peak ratios in diagrams 180, 190 are different from each other. Thus, purity information can also be derived from a comparison of the diagrams 180, 190.

[0106] FIG. 7 illustrates diagrams 200, 210, 220 plotted for explaining sample portion-specific decisions concerning single- or multi-dimensional separations made individually for different fluidic sample portions based on a sample portion-specific purity analysis by means of chromatograms according to an exemplary embodiment.

[0107] FIG. 7 shows diagrams 200, 210, 220 each having an abscissa 162 along which the time, t, is plotted. Along an ordinate 164, an absorption intensity, 11, is plotted, as in FIG. 3. Absorption peaks 106(1), 106(2), 106(3), 106(4), 106(5), in diagram 200 can be detected by detector 50 at an outlet of a first dimension sample separation device 102 when five subsequent (or successive) portions of fluidic sample pass detector 50 one after the other. For each of the absorption peaks 106(1), 106(2), 106(3), 106(4), 106(5), an analysis as shown in FIG. 4 and/or an analysis according to FIG. 5 and FIG. 6 can be carried out for determining purity information individually for each of the five subsequent (or successive) portions of the fluidic sample. In the shown embodiment, analysis of the absorption peaks 106(2), 106(3), 106(4) provides the information that the three corresponding fluidic sample portions are all pure, i.e. each contains only a single component. Consequently, no further separation of these three fluidic sample portions in a subsequent second separation dimension is carried out, which is shown schematically by reference signs 230. In contrast to this, analysis of the absorption peaks 106(1) and 106(5) provides the information that the two corresponding fluidic sample portions are not pure, i.e. each contain multiple different components. Consequently, a further separation of these two fluidic sample portions in the subsequent second separation dimension is carried out, which is shown schematically by reference signs 240.

[0108] Absorption sub-peaks 106(11), 106(12) (which both correspond to absorption peak 106(1)) in diagram 210 can be detected by detector 50′ at an outlet of the second dimension sample separation device 104 when the two sub portions of fluidic sample pass further purity detector 50′ one after the other. For each of the absorption peaks 106(11), 106(12), an analysis as shown in FIG. 4 and/or an analysis according to FIG. 5 and

[0109] FIG. 6 can be carried out for determining purity information individually for each of the two subsequent sub portions of the fluidic sample. In the shown embodiment, analysis of the absorption peak 106(11) provides the information that the absorption peak 106(11) is pure, i.e. contains only a single component. Consequently, no further separation of the absorption peak 106(11) in a subsequent third separation dimension is carried out, which is shown schematically by reference sign 250. In contrast to this, analysis of the absorption peak 106(12) provides the information that the corresponding further separated fluidic sample portion is still not pure, i.e. still contains multiple different components. Consequently, a further separation of this fluidic sample portion in a subsequent third separation dimension is carried out, which is shown schematically by reference sign 260. A similar analysis can be made with absorption sub-peaks 106(51), 106(52) which both correspond to absorption peak 106(5), see diagram 220.

[0110] Thus, individual sub portions may be made subject to a third, fourth, etc. separation, and so on.

[0111] FIG. 8 shows a fluidic interface region between a primary stage sample separation device 102 and a secondary stage sample separation device 104 according to an exemplary embodiment in which a modulator valve 114 cooperates with two buffer valves 130, 132 each cooperating, in turn, with a plurality of buffer volumes 134, 136 for temporarily storing a respective fluid packet. FIG. 8 hence shows an alternative sampling valve configuration, compared with FIG. 2, in which one modulator valve 114 cooperates with two packet parking valves 130, 132 (substituting sample accommodation volumes 142, 148). Each of the packet parking valves 130, 132 serves six buffer volumes 134, 136 (see numbers 1 to 6 at the buffer valves 130, 132). Hence, any desired number of buffer volumes 134, 136 can be implemented following the principle of FIG. 8, so that basically any adaptation of a larger flow rate of the primary stage as compared to a smaller flow rate of the secondary stage is possible.

[0112] It should be noted that the term “comprising” does not exclude other elements or features and the term “a” or “an” does not exclude a plurality. Also elements described in association with different embodiments may be combined. It should also be noted that reference signs in the claims shall not be construed as limiting the scope of the claims.