Patent Publication Number: US-2005121392-A1

Title: Chromatographic system with mobile phase recycling ability

Description:
The present invention relates to chromatographic systems.  
     BACKGROUND OF THE INVENTION  
      Liquid chromatography, for example high performance liquid chromatography (HPLC) is a widespread separation technique, wherein a mixture of different components to be analyzed is dissolved in a liquid mobile phase. The mobile phase is applied to a stationary phase, normally a column, where the different components can interact in a different way with the stationary phase so the components elute from the column at different times, (when a solvent is used). Often a gradient elution, including a mobile phase gradient wherein the composition of the mobile phase is changed overtime, has to be run in order to elute the different components from the column. The separated components of interest, often called analytes, leaving the stationary phase are normally detected using a suitable detector, for example a UV or mass spectrometer (MS) detector. The representation of the detector signal as a function of time is called chromatogram. The chromatogram shows so-called (signal) peaks, which represent different components.  
      In preparative HPLC the operation of a chromatographic system normally requires a considerable amount of user input. This especially accounts for the direction of flow of the mobile phase after the mobile phase has passed the column. A mobile phase containing components of interest (analytes) typically is directed into a fraction collector for further analysis. Mobile phases containing constituents of no further interest are frequently drained into waste, whereas mobile phases with exactly the same composition as the phase at the beginning are often directed back into their original container for recycling purposes. A mobile phase switch, for example a valve, normally directs the flow of the mobile phase. The position of this valve is changed depending on the composition of the mobile phase in order to direct the flow of the mobile phase into the different containers mentioned above.  
      The Japanese patent application publication JP 10197506 A discloses a chromatographic system with solvent recycling abilities. A solvent recycling system including a microcomputer with a level setting section is part of the detector. In response to the signal of the detector exceeding a certain threshold level, i.e. a (signal) peak is detected, the valve is changed so that the mobile phase is directed into a specified collection vessel or into waste. In response to the signal not exceeding the threshold level, the solvent is considered to have the original composition of the solvent applied to the stationary phase and is redirected into the original reservoir of the original phase for recycling purposes.  
     SUMMARY OF THE INVENTION  
      It is an object of the present invention to provide a new and improved chromatographic system.  
      In order to operate the chromatographic system according to embodiments of the invention, a stationary phase is loaded into a container; alternatively, an already pre-packed stationary phase in an HPLC column might be provided and connected to the other components of the system via the mobile phase flow line. Furthermore a mobile phase, immiscible with the stationary phase, for performing a distribution equilibrium of analytes of interest between the mobile and stationary phases is provided.  
      In the chromatographic system according to certain aspects of the invention, a controlling system receives data from a detector (e.g. information about composition changes of the mobile phase and the presence of components eluted from the stationary phase e.g. analytes of interest or impurities) and data from the mobile phase supply system (e.g. information about the mobile phase flow rate and the composition of the mobile phase applied to the stationary phase). After processing these data, the controlling system directs the flow of the mobile phase passed through the stationary phase into different locations depending on the data. Since the controlling system is adapted to control the mobile phase switch, it automatically directs the flow of the mobile phase into different locations, depending on the composition of the mobile phase and the presence of components eluted from the stationary phase. Therefore, minimal user input is required for the chromatographic system of the invention.  
      Advantageously the mobile phase supply system includes a pump that is controlled by the controlling system.  
      In another embodiment of the chromatographic system of the invention, the mobile phase switch comprises a valve. Depending on the number of desired flow directions, the valve includes a plurality of ports with openings in the valve for different flow paths. Therefore, such a valve includes plural openings, i.e., ports, for directing the flow to different locations. Typical valves include six ports or even ten-ports.  
      In a further embodiment of the chromatographic system, the valve includes flow passages and port openings that might differ in their geometrical dimensions to address specific physical demands of the flow system.  
      Advantageously the detector and the mobile phase supply system include microprocessors for communication with the controlling system. The microprocessors process data recorded at the mobile phase system or the detector in order to ease the processing of these data within the controlling system.  
      Preferably the mobile phase flow line comprises at least one of the following components: tubes, capillaries and sample injector. Tubes and capillaries for high performance liquid chromatographic systems (HPLC systems) are especially designed and manufactured to fulfill their different individual chromatographic needs. The tubes and capillaries can be identical or different in length and inner diameter.  
      In preferred embodiments, a diode array based UV detector (DAD) measures the total spectrum of the mobile phase composition passed through a stationary phase and the spectrum of components eluted from the stationary phase at any time during a run, to monitor the mobile phase and any components in the mobile phase having a different origin from the mobile phase. For example the diode array based UV detector can simultaneously monitor the spectra of the components eluted at wavelengths of e.g. 254 nm, 210 nm and 260 nm/280 nm. These three different, individually defined wavelengths allow the simultaneous detection of aromatic compounds (254 nm), of peptides (210 nm), and other substances, e.g. nucleic acids (260 nm/280 nm). The composition of the mobile phase is typically monitored over the entire spectral range, starting from 190 nm or 200 nm, depending on the optical quality of the detector. Once the controlling system has determined that the composition of the mobile phase passing the stationary phase has exactly or nearly the same spectrum (e.g. UV spectrum) as the initial mobile phase composition that was applied to the stationary phase, this mobile phase is easily redirected back to the original storage container for recycling.  
      This is possible because a direct flow line connection between the mobile phase switch and the storage container is part of the system. In the case of components eluted from the stationary phase being monitored by the detector, the mobile phase containing such components is directed into (1) waste if the components are impurities or (2) a fraction collector for further analysis if the components are analytes of interest.  
      Advantageously the controlling system is adapted to combine (1) data of the composition of a mobile phase before the mobile phase enters the stationary phase and (2) those data that have been detected after the mobile phase composition has just passed the detector. Data (1) and (2) are preferably received from the mobile phase supply system and the detector, respectively.  
      The combination of data, the composition of the eluent flow with/without injected samples results in (1) and (2) and easy further processing of all the data from the detector and the mobile phase supply system within the controlling system.  
      Preferably the mobile phase supply system provides data about the mobile phase flow rate and composition per time unit and the detector provides at least one or a combination of the following data: change in refractive index, UV absorption, fluorescence intensity, m/z ratio in mass spectroscopy, light scattering intensity for determining the composition of the mobile phase and the presence of components eluted from the stationary phase at a specific time during a run.  
      In the case of analyte destroying detectors being used, e.g. mass spectrometers for determining the m/z ratio of analytes, a flow-splitter is advantageously provided. This flow-splitter divides the mobile phase flow line into first and second separate flow lines: the first flow line is for the majority of the mobile phase leading to the mobile phase switch and the second flow line is for a small fraction of the mobile phase flowing to the analyte destroying detector for analysis. The small fraction of the mobile phase in the second flow line is analyzed by the detector and the mobile phase switch connected to the first flow line is subsequently controlled according to the information received from the analysis and/or the mobile phase supply system.  
      The detector can also include different types of spectrometers, for example a UV spectrometer and a refractive index (RI) detector to provide an easier determination of the composition of the mobile phase by using and combining different detection methods.  
      Preferably the mobile phase flow rate generated by the mobile phase supply system is variably adjustable by the controlling system according to data received from the detector. Variable adjustment of the mobile phase flow rate, for example, allows an easy adaptation of the mobile phase flow rate and composition of the mobile phase, which passes through the stationary phase according to the composition of the mobile phase that is leaving the stationary phase and is monitored by the detector.  
      Therefore the chromatographic system can be adapted, for example, to enhance the flow rate and/or its mobile phase composition once a certain event has taken place (e.g. the substance of interest has been eluted from the stationary phase and has been detected in the detector).  
      Thus, an enhanced mobile phase flow rate accelerates the equilibration of the stationary phase with a new mobile phase composition for the following chromatographic run in order to reduce the overhead time necessary to bring the total system back to the starting conditions.  
      Advantageously, the controlling system comprises a computer system connected to the controlling system. The computer system processes and analyzes the data received from the detector and the mobile phase supply system. The computer system preferably is adapted to control the mobile phase switch, the valve, and the mobile phase supply system. The computer system can include a stationary system, e.g., a desktop computer or a handheld control system that monitors chromatographic run (see for example  FIGS. 2 and 3 ) and has manual inputs responsive to a user.  
      Furthermore, the mobile phase switch can comprise a second detector for a test compound, wherein the mobile phase switch provides data about the test compound to the controlling system.  
      The second detector for the test compound can comprise a photo-diode for detection of a dye in the test compound. For example, a blue emitting dye can be detected by using a red laser photo-diode built into the mobile phase switch, e.g. the valve. The dye serves as a test compound, which enables the controlling system to determine the delay volume between the UV detector and the mobile phase switch.  
      In the case of a blue emitting dye in the test compound, the controlling system receives data from the detection system about the time the dye in the test compound is detected (1) by the detector and (2) at the mobile phase switch. This embodiment of the chromatographic system of the invention allows a very precise determination of the delay time and thus the delay volume between the detector and the mobile phase switch. A detection system for determining the delay time is also disclosed in the U.S. Pat. No. 6,106,710, which is hereby incorporated in its entirety.  
      In other cases, where an exact determination of the dead volume is not necessary, the controlling system simply determines the dead volume between the mobile phase supply system and the mobile phase switch by using data about the mobile phase flow rate and the volume available for the mobile phase between the mobile phase supply system and the mobile phase switch. A detection system for a test compound in the mobile phase switch is, for example, shown in  FIG. 3 .  
      In another advantageous embodiment of the chromatographic system of the invention, a mobile phase mixer is part of the system. The mobile phase mixer might generates a mobile phase gradient by actively bringing at least two different initial eluents together and mixing them, as for example shown in  FIG. 3 . In this case the controlling system controls the mobile phase mixer, thereby enabling the mixing of at least two different initial eluents resulting in a mobile phase to be applied onto the stationary phase.  
      In another embodiment of the chromatographic system of the invention, the mobile phase supply system combines two different initial eluents to a mobile phase. In this case the mobile phase mixer is part of the mobile phase supply system as shown in  FIG. 1 . The mobile phase mixer then actively mixes (e.g. by using a stirrer blade) or passively mixes (e.g. by using beads) the already combined initial eluents ensuring complete mixing.  
      Using the different mobile phase mixers and mobile phase supply systems, mobile phases of different composition are generated in order to run gradients with linear and/or non-linear mobile phase compositions. Typically two different eluents are mixed together to form the mobile phase of interest. Normally the two different eluents have different compositions, differing in at least one component of the eluent. The two different eluents are e.g. acetonitrile and water. The two different eluents might also comprise the same solvents, but differ in the concentration of a salt.  
      A gradient mixer, typically controlled by the mobile phase pump or the controlling system, allows more complex chromatographic procedures, for the separation of substances when using a mobile phase gradient.  
      In a chromatographic system of the invention, which is able to run a gradient elution, the controlling system is preferably adapted to direct various mobile phases of different compositions, passed through the stationary phase via the mobile phase switch into at least one container, and the controlling system is adapted to determine the composition of the new mobile phase in the at least one container by using data received from the mobile phase supply system and the detector. In this embodiment, the chromatographic system is adapted to determine the composition of the mobile phase passed through the stationary phase and direct mobile phases of different composition having no or only minor contaminations after passing the stationary phase into at least one container. For example, mobile phases without impurities varying from 70% acetonitrile: 30% water to 90% acetonitrile: 10% water are collected in a container, resulting in a new mobile phase with a different composition. Furthermore the controlling system is adapted to allow the calculation of the thus formed new composition of the new mobile phase. The controlling system receives data from the detector and the mobile phase supply system about the mobile phase flow rate, the composition of the mobile phase at the beginning of the gradient, the composition of the mobile phase at the end of the gradient, the purity of the mobile phase, the steepness(es) of the gradient(s) and its (their) duration and the occurrence time of the mobile phase at the detector. The controlling system can use all these data in order to determine the composition and the purity of the new mobile phase in the container. In the case, of two or even more gradients being run during one chromatographic procedure, the mobile phases of each gradient passed through the stationary phase can be collected in one container, the container being different each time, so that two or even more mobile phases with new compositions can be generated by the chromatographic system of the invention.  
      Preferably an electronic microprocessor for direct data communication between the mobile phase supply system and the detector is part of the system. The electronic microprocessor can be part of at least the mobile phase supply system or the detector or both. Such a microprocessor can enable a direct and easy communication between the mobile phase supply system and the detector as, for example, shown in  FIG. 3 . The communication between the mobile phase supply system and the detector via their microprocessors can also involve local area network systems, for example, a communication local area network (CAN).  
      The operating method according to embodiments of the present invention allows automatic direction of the mobile phase to different locations depending on the composition of the mobile phase and the presence or absence of analytes or impurities.  
      Preferably at least one of the following data the mobile phase that has passed the stationary phase (and, if present, compounds eluted from the stationary phase) is received from the detector: current refractive index, UV spectrum, florescence spectrum, TIC mass spectrum, or light scattering capability at a particular time when passing the detector.  
      A further aspect of the invention relates to a method of operating a chromatographic system, the system including a mobile phase supply system that delivers a mobile phase, a stationary phase, a sample injector, a detector and a mobile phase switch that directs the flow of a mobile phase to different locations, all being connected via a mobile phase flow line. The method comprises (A) applying an initial mobile phase to the stationary phase using the mobile phase supply system by mixing together at least two different eluents in a variable manner resulting in the initial mobile phase being applied to the stationary phase, while the stationary phase is running a gradient elution and/or a step gradient elution, (B) determining the composition and purity of the mobile phase after the mobile phase has passed the stationary phase; step (B) is performed by using data received from the detector band data and from the mobile phase supply system; (C) directing the mobile phase passed through the stationary phase into a container depending on the data received in step (B) by using the mobile phase switch whereby the mobile phases of different compositions are directed to at least one container resulting in a new mobile phase; and (D) determining the composition of the new mobile phase by using data received in step (B).  
      Furthermore in step (B) at least one of the following data is received from the mobile phase supply system: flow rate of the mobile phase applied to the stationary phase, composition of this mobile phase, duration of delivery of this mobile phase at a particular composition and thus the mobile phase gradient steepness.  
      Such data received from the detector and the mobile phase supply system enable a very simple automated determination of the composition of the mobile phase and its purity after the composition has passed the stationary phase.  
      Advantageously in step (A), at least two different initial eluents are mixed together in a timely variable manner resulting in a mobile phase for application to the stationary phase, running a linear gradient elution and/or a step gradient elution. In this embodiment, the composition of the mobile phase is determined in step (B) by using additional data received from the mobile phase supply system. Preferably at least one of the following additional data is received from the mobile phase supply system: composition of the mobile phase at the beginning of the gradient, composition of the mobile phase at the end of the gradient and duration of the gradient.  
      Such an embodiment of the method of the invention enables an automatic determination of the composition of the mobile phases passed through the stationary phase during gradient elution and/or step gradient elution procedures.  
      It is also possible to recycle a mobile phase having a constant composition during an isocratic chromatographic run.  
      Preferably in step (C) mobile phases of different composition are automatically directed to at least one container, resulting in a new mobile phase. In step (D) the composition of this new mobile phase is automatically determined using data received in step (B) from the mobile phase supply system and the detector.  
      This embodiment of the method of the invention allows the generation of freshly adjusted mobile phases by mixing mobile phases of different compositions passed through the stationary phase to a completely new composition and thus provides an enhanced and simplified mobile phase recycling. This embodiment enables recycling of mobile phases with defined lower contamination than is obtained by performing the gradient elution prior to use. A chromatographic system that is able to be operated using such a method is shown in  FIG. 3 . This operating method of the invention allows the recycling of used mobile phases with variant compositions in one container, so that a new mobile phase is generated with a new composition, which can automatically be determined by the system.  
      Preferably at least one container for storage of the mobile phase is used. In this case it is possible to automatically direct, in step (C), the mobile phase that is passed through the stationary phase with the same composition as the mobile phase that is applied to the stationary phase in the at least one container. This method of the invention allows direct recycling of mobile phases without major impurities back into the storage container, which is connected to the mobile phase switch via a mobile phase flow line (see e.g.  FIG. 1 ). This method of the invention enables an immediate reuse of the recycled mobile phases because the container can also be connected to the mobile phase supply system.  
      In a further variant the delay time of the mobile phase between the mobile phase switch and either the mobile phase supply system (in the case of an isocratic run) or the mobile phase mixer (in the case of a gradient run) is automatically determined by using at least one of the following data: refractive index, UV spectrum, florescence spectrum, TIC mass spectrum, light scattering received from the detector for determination of the composition change of the mobile phase at a particular time of the mobile phase in the detector, information about the mobile phase flow rate received from the mobile phase supply system, and the physical volume of the mobile phase flow line connecting the detector and the mobile phase switch.  
      Such variants allow the determination of the delay volume and therefore the delay time of the mobile phase between the mobile phase supply system and the mobile phase switch. The delay time or delay volume between the mobile phase supply system and the mobile phase switch basically includes two different delay volumes. The first delay volume is the delay volume between the mobile phase supply system or the mixer and the detector and the second delay volume is the delay volume between the detector and the mobile phase switch (see for example  FIG. 1 ). The delay volume between the mobile phase supply system and the detector can be determined by using data about the mobile phase flow rate and the composition of the mobile phase delivered from the mobile phase supply system and the data received from the detector indicating the composition of the mobile phase that passed the stationary phase and the time of occurrence of the mobile phase at the detector. The delay volume between the detector and the mobile phase switch is determined by using information about the physical volume of the mobile phase flow line connecting the detector and the mobile phase switch and information about the mobile phase flow rate.  
      Embodiments of the invention thus enable automatic running of the chromatographic system under chromatographic modes different from the isocratic mode. Furthermore any problems occurring during the chromatographic process can be indicated, such as wear of mechanically permanent moving parts. An automatic chromatographic procedure can be provided, wherein the mobile phase is automatically directed into waste, into the original container or into a fraction collector, depending on the mobile phase composition and the presence or absence of analytes in the mobile phase.  
      In the following the invention will be described in more detail by the figures and embodiments. All figures are just simplified schematic representations presented for illustrative purposes only. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  is a schematic diagram of one preferred embodiment of a chromatographic system including the controlling system of the invention.  
       FIG. 2  is a schematic diagram of another embodiment of the chromatographic system of the invention for isocratic chromatographic runs.  
       FIG. 3  is a schematic diagram of a chromatographic system of the invention able to run a gradient elution.  
       FIG. 4  is a chromatogram of the type produced by the detectror of  FIG. 3 . 
    
    
     DETAILED DESCRIPTION OF OF THE DRAWING  
       FIG. 1  is a schematic representation of a chromatographic system  1  able to run a gradient elution. Solid lines in  FIG. 1  and all the other figures represent the mobile phase flow line  10 , whereas dashed lines represent data communication lines  40 . The chromatographic system comprises a mobile phase supply system  20 , which in this case is a pump with a mobile phase mixer  45 , a stationary phase  5 , for example a column, a detector  15  and a mobile phase switch  25  which are all in flow communication through a flow line  10 . The pump  20  is connected via the mobile phase flow line  10  to two containers  2 A and  2 B each container having a different eluent (e.g. water and acetonitrile). The pump  20  is adapted to deliver the mobile phase via the mobile phase flow line  10  to the stationary phase  5  and is also able to combine the different eluents in the containers  2 A and  2 B in order to generate a gradient. The gradient mixer  45  can mix the already combined eluents completely thereby avoiding concentration differences in the mobile phase. After having passed the stationary phase  5 , the mobile phase can flow through the detector  15  and the mobile phase switch  25 . The chromatographic system also comprises a controlling system  30  that comprises a microprocessor  30 A in data communication with a data processor  30 B, which can process the data that are received from the controlling system. The microprocessor is in data communication with the pump  20  and with the detector  15 . D/A converters  30 D and A/D converters  30 E are present in between the data communication lines between the microprocessor  30 A and the pump  20  and between the microprocessor  30 A and the detector  15 . The microprocessor  30 A receives data from the pump  20 , for example, information about the composition of the mobile phase driven into the chromatographic system, the time of first supply of the mobile phase and the mobile phase flow rate. The microprocessor also receives data from the detector  15 , for example information about the composition of the mobile phase detected at the detector at a particular time. The detector might comprise an UV or IR detector, a mass spectrometer or an apparatus for determination of the refractive index or any kind of combination thereof.  
      The microprocessor  30 A is adapted to process the data received from the detector  15  and the pump  20  using the data processor  30 B. The microprocessor  30 A can send controlling signals to the detector  15  and the pump  20  and may also be adapted to control the mobile phase switch  25  via the mobile phase switch driver  30 C. The reference numeral  100  represents the delay volume between the pump  20  and the detector  15 . Reference numeral  110  depicts the delay volume between the detector  15  and the mobile phase switch  25 .  
      During a chromatographic run compounds including samples to be analyzed can be injected via a sample injector  12  into the mobile phase, that is pumped by pump  20  through the mobile phase line  10 .  
      On its way through the mobile phase line  10 , the injected sample enters for example an HPLC column tube  15 , completely filled with stationary phase. The injected compounds can then interact with the stationary phase resulting in different duration times.  
      These compounds can be eluted via the mobile phase using the pump  20  through the stationary phase and into the detector  15 . The detector  15  as well as the pump  20  sends data to the microprocessor  30 A for further processing and analysis. The microprocessor  30 A is then adapted to control the mobile phase switch  25  via the mobile phase switch driver  30 C thereby changing the direction of flow into different locations. If the information received by the pump  20  and detector  15  indicates that pure initial mobile phase having passed through the stationary phase has reached the mobile phase switch  25 , the controlling system redirects the mobile phase via the mobile phase switch  25  back into its respective container  2 A or  2 B where it came from.  
      In the case, that the data received by the detector  15  and the pump  20  indicate that a mobile phase contaminated by impurities has passed the stationary phase  5 , the controlling system can direct this mobile phase into the waste container  26 . Compounds of interest that are eluted from the stationary phase can be directed for example into a fraction collector  27  for further action.  
       FIG. 2  is a diagram of another embodiment of a chromatographic system for isocratic runs, using just one premixed mobile phase in a container  2 . In comparison to the chromatographic system shown in  FIG. 1 a  computer system  90  in data communication with the controlling system  30  is part of the chromatographic system. The computer system  90  enables a manual user input for control of the chromatographic system and allows monitoring the chromatographic run. Additionally microprocessors  20 A and  15 A are respectively part of the pump  20  and the detector  15 . These microprocessors can simplify the data connection and processing between both components and the controlling system  30 .  
       FIG. 3  is a diagram of another variant of a chromatographic system suitable to run gradient elution procedures. In this chromatographic system, the gradient mixer  45  combines and mixes the two eluents originating from the containers  2 A and  2 B. The gradient mixer  45  might be a part of the pump  20  or might be a separate component of the chromatographic system.  
      The gradient mixer  45  is in data flow communication with the two storage containers  2 A and  2 B containing different eluents A and B for mixing a mobile phase. Again a computer system  90  is part of the chromatographic system. The mobile phase switch  25  additionally comprises a test compound detection system  15 , which includes a photodiode  25 A. As mentioned above detection system  15  is also in data communication with the control system  30 ; detection system exactly determines the delay volume between the detector  15  and the mobile phase switch  25 . To determine this delay volume, the stationary phase  5  has to be replaced with a mobile phase flow line  10 , because the test compound tends to interact with the stationary phase. The mobile phase switch  25  that, for example, comprises a valve can be controlled by the controlling system  30 . Depending on the information received by the control system  30 , the mobile phase switch  25  selectively directs the flow of the mobile phase into a waste container  26 A or into a fraction collector  27 . Since the data controlling system  30  is able to determine the composition and purity of mobile phases that pass the stationary phase  5  at any time, the control system can also direct the flow of mobile phases of different compositions into a container  26 B. This results in the above-mentioned new mobile phase, the composition of which can be determined by the controlling system  30  using data received from the detector  15  and the pump  20 . Additionally the microprocessors  20 A and  15 A, which are part of the pump  20 , and the detector  15  can also establish a direct data communication and processing between these components as indicated by the dashed line.  
       FIG. 4  is a chromatogram recorded by the detector  15 . When the detector  15  is a diode array-based detector, comprising a plurality of different diodes, signals at different wavelengths can be detected at the same time. The ordinate  50  denotes the absorption, for example the UV absorption of the mobile phase at a certain, predefined wavelength, and—if present—compounds in the mobile phase, indicated by line  60 , the so-called baseline.  
      The abscissa  55  indicates the time course of the chromatographic run. The line  150  shows the flow rate and the line  160  the mobile phase composition profile at a certain time and UV absorption at a set wavelength during the chromatographic run. The chromatogram shows the changing mobile phase composition at a predefined wavelength, caused by the presence of an injected sample mix into the sample injector  12 , the result of the separation power of the separation device  5  and the gradient composition of eluent A and eluent B, preformed by the mixer  45 .  
      For example, at the beginning of the chromatogram pure eluent A or a defined composition of eluent A and B is detected by the detector  15 , whereas at the end of the chromatographic run indicated by G, pure eluent B or a defined composition identical or different from the beginning composition might be detected.  
      In the chromatogram shown in  FIG. 4  the area denoted as  60 A consists of pure eluent A which can easily be directed back into the container for eluent A when a chromatographic system, for example as shown in  FIG. 3 , is used (container  2 A for eluent A). The area of the chromatogram denoted as  60 B contains analytes eluted from the stationary phase, which are directed into the waste container in a case of being of no interest in the case of the analyte being directed into the fraction collector  27 .  
      The area  60 C denotes a peak in the chromatogram. This peak indicates that at least one compound of interest becomes eluted from the stationary phase while the concentration of eluent B relative to eluent A is increased in the gradient as shown in line  160 .  
      This area  60 C can automatically be directed to a fraction collector  27 , e.g., using the chromatographic system of  FIG. 3 . The area  60 D contains a pure mixture of a well known composition between eluent A and eluent B and can therefore automatically be directed to a third storage container for reuse (for example container  26 B in  FIG. 3 ). The peak in the area  60 E indicates impurities eluted from the stationary phase while the concentration of eluent B is further increased. These impurities can also be directed automatically into a waste container (for example container  26 A in  FIG. 3 ). The area  60 F contains pure eluent B that can be directed back into the original container (e.g. container  2 B for solvent B in  FIG. 3 ). Area  60 G contains a mixture of eluent B and the original starting condition identical to area  60 A (eluent A), while equilibrating the column with the original starting condition. This area  60 G might be directed into the waste container.  
      Area  60 A again contains pure eluent A which is applied to the stationary phase for preparation of the next chromatographic run. The solvent in this area  60 A can be directed back into the container  2 A, shown in  FIG. 3  for eluent A. The flow rate  150  might be held constant, while eluting the components of interest from the stationary phase. Afterwards the flow rate can be increased as shown in  FIG. 4  in order to accelerate the equilibration of the stationary with the original starting conditions.  
      The scope of the invention is not limited to the embodiments shown in the figures. Indeed variations of the chromatographic system including any kind of combination of the different features shown in the figures are also possible. Further variations are possible concerning the numbers of storage containers and fraction collectors, which are connected to the mobile phase switch. Moreover, the controlling system can be part of the detector, the pump, the mobile phase switch, or a separate component of the chromatographic system of the invention.