Patent Application
20140284279
This application claims priority to European Patent Application No. EP13160714.5 filed on Mar. 22, 2013, the entire contents of which are incorporated herein by reference.
The present invention relates to instrumentation for flash chromatography and in particular to apparatus for connecting destructive detectors to flash chromatography instruments and fraction collectors.
It is well-known in the art of high-performance liquid chromatography (HPLC) to use various non-destructive detectors (such as UV detectors) and destructive mass detectors (such as Evaporative Light Scattering Detectors (ELSD) or mass spectrometers (MS)) to detect the separated molecules in the effluent from the chromatography column and direct an automated fraction collector to collect the separated molecules in separate vials based on signals from the detectors.
Zeng and co-workers (Zeng, 1998) developed a mass-spectrometry-based HPLC system that permitted automated purification of compound libraries using a mass spectrometer to signal fraction collection.
U.S. Pat. No. 6,106,710 disclose a fraction collection system for liquid chromatography wherein the sample stream can be subject to destructive detection without consuming the sample, and wherein the time a sample component is detected at the destructive detector is used to predict when the stream containing that sample component should arrive at the fraction collector.
U.S. published patent application 2001/0038071 disclose a system for separation and analysis comprising a chromatography column, a fraction collector and a mass spectrometer. In order to control the flow to the mass spectrometer, a transfer module is introduced. The transfer module directs a small fraction of the effluent flow from the chromatography column into the mass spectrometer in a controlled and easily adjustable manner. Transfer modules such as the one described in U.S.2001/0038071 are also referred to in the art as “active splitters”.
Flash purification is a technique developed by W.C. Still that uses a column or cartridge filled with an insoluble solid support (stationary phase) and elution solvent mixture (mobile phase) to separate and purify a mixture of organic compounds (Still, 1978). The stationary phase and the mobile phase typically have very different polarities, which work in tandem to separate compound mixtures. The separated molecules can then by means of a fraction collector be collected in a purified state for use in a subsequent synthesis or as a final product. Usually, a non-destructive detector, such as an UV detector, is used to detect the separated molecules in the effluent from the column and the detection signal from the UV detector is used to monitor or trigger the fraction collection.
In one aspect the present invention aims to provide an apparatus that can be used to connect a flash chromatography instrument to a fraction collector and a mass detector to perform mass-directed flash chromatography, wherein the flow rate out of the chromatography column can be in the range of 1-200 ml/min. This is achieved by providing a by-passable delay loop in the fluid path leading to the fraction collector, downstream of a splitting of the effluent flow from the flash chromatography column into a primary fluid flow to the fraction collector and a secondary flow to the mass detector.
In a further aspect, the invention aims to provide an integrated system for flash chromatography and mass. spectrometry, wherein a user can conveniently analyse a sample using a mass detector and use the obtained data for mass-directed flash chromatography. This is achieved by introducing a separate inlet to the fluid path leading from the flash chromatography column to the mass detector.
In a further aspect, the invention aims to provide an integrated system for flash chromatography and mass spectrometry comprising an active splitter, wherein the active splitter can be by-passed during equilibration of the flash chromatography column. This is achieved by introducing an alternative fluid path bypassing the active splitter.
In a further aspect, the invention relates to a method for separation of a sample into components and collection of at least one of said components in at least one separate fraction, comprising the steps
In a further aspect, the invention relates to a computer program comprising instructions inducing a computer to perform a method for controlling a fraction collector in a chromatography system comprising a chromatography column, a fraction collector, a destructive mass detector and a branched fluid path from said column to the fraction collector and the destructive mass detector, respectively, said method comprising the steps:
The invention is as set out in the appended independent claims and preferred embodiments are set forth in the dependent claims.
The term “mass detector” is used to denote detectors capable of detecting the mass of a molecule or ion. Examples of mass detectors usable in the present invention are mass spectrometers and mass ion detectors. It is currently preferred to use a mass spectrometer, in particular an Electrospray Ionization Mass Spectrometer (ESI-MS).
The term “mass-directed chromatography” is used to denote chromatography wherein detection of the mass of an analyte in an effluent stream from a chromatography column is used to direct a fraction collector to collect fractions of the effluent stream in separate vials.
The term “active splitter” refers to a device that actively transfers a portion of one fluid stream to a second fluid stream.
The term “coupling module” denotes an apparatus that can be used to connect a flash chromatography instrument to a fraction collector and a mass detector to perform mass-directed flash chromatography.
The present inventors have identified a number of problems with existing technology relating to mass directed chromatography, in particular mass-directed flash chromatography.
Mass detectors require very small amounts of samples and flow rates in the range of μl/min or nl/min. Flash chromatography instruments, on the other hand, may operate at a wide range of flow rates, from 1 ml/min up to 100 or even 200 ml/min. This is several orders of magnitude higher flow rates than used in HPLC (1-1000 μl/min), which in turn may be several orders of magnitude higher than what is tolerated by a mass detector.
The high rate fluid flow from the flash chromatography column can be diverted to a secondary flow having a significantly lower flow rate by means of an active splitter, as is known in the art from e.g. U.S. 2001/0038071, whereby the secondary flow goes to the mass detector and the primary flow goes to a fraction collector.
However, the flow rate in the primary flow cannot be allowed to go above a certain flow rate where a fraction comprising a component that the user of the system wishes to collect arrives at the fraction collector before it is detected by the mass detector. This upper limitation on the flow rate is set by the dimensions of the fluid conduits in the system and by the maximum flow rate into the mass detector.
The upper limitation of the flow rate could be raised by making the fluid path from the flow splitter to the fraction collector longer or wider. This would however entail undesired band broadening at lower flow rates, making the collection of specific compounds less reliable.
The current inventors have solved this problem by providing a coupling module for connecting a flash chromatography instrument to a mass detector and a fraction collector, said coupling module comprising an active splitter and having a by-passable delay loop between the active splitter and the outlet to the fraction collector. The flow from the chromatography column is directed through the delay loop when the flow rate is such that a fraction comprising a component of interest would arrive at the fraction collector before it would arrive at the mass detector, if the flow was to go directly to the fraction collector.
On the other hand, when the flow rate is such that a component of interest would be detected by the mass detector before the corresponding flow fraction comprising the component of interest would arrive at the fraction collector, and providing sufficient time for signal processing and automatic operation of the fraction collector, the delay loop is bypassed and band broadening minimized.
A further aim of the present inventors is to simplify the use of mass detectors in combination with flash chromatography and to provide integration of flash chromatography instruments and mass detectors.
This is done by the present invention by providing the above mentioned coupling module with a fluid inlet situated between the inlet from the flash chromatography column and the active splitter. This provides the user of the system with easy access to injection of an unseparated aliquot of a sample into the system. It also minimizes direct user interaction with the mass detector, facilitating the mass detector to be entirely controlled by the system.
Columns for use in flash chromatography are packed with a chromatography medium, such as silica or a polymeric resin. Before using a flash chromatography column, the column is commonly equilibrated by running 3-5 column volumes of solvent through the chromatography media packed in the column.
This equilibration process may cause fine particles of the chromatography media to be washed out of the column by the solvent. This is not a problem in standard flash chromatography instruments using a non-destructive detector for fraction collection, wherein the fluid conduits are relatively wide and solvent used for equilibration can be directed to the waste container of the fraction collector.
However, due to the minute flows into mass detectors, it is desirable to have fluid conduits with relatively small diameters connecting the mass detector to the column. The fine particles released from the flash chromatography column during equilibration may cause clogging of the narrower fluid conduits used in connection with a mass detector, requiring cleaning or exchange of the conduits. Furthermore, fine particles of chromatography media should not be allowed to pass to the mass detector as they may damage the mass detector.
In one embodiment of the present invention, this problem is solved by providing an alternative fluid path that bypasses the active splitter and thereby the mass detector. The alternative fluid path can be used during equilibration or in any other situation when it is desirable to bypass the active splitter and/or the mass detector. One such situation may be that the user of the system wishes to use only a non-destructive detector, provided as standard in the flash chromatography instrument, for triggering or monitoring fraction collection, without physically disconnecting the coupling module.
One aspect in which flash chromatography differs from High Performance Liquid Chromatography (HPLC) is that the amounts and concentrations of sample are generally about an order of magnitude higher in flash chromatography compared to HPLC. Equipment used for HPLC is therefore not directly applicable to use in flash chromatography, as the high amounts of sample may cause blockage.
One way according to the present invention to avoid blockage is to dilute the effluent flow from the flash chromatography cartridge when it is split to the secondary flow towards the mass detector. The ratio of dilution may be 1:100 to 1:1000, such as 1:250, 1:500 or 1:750. A presently preferred ratio is 1:500.
One way according to the present invention to avoid blockage is to introduce a filter for removing particulate material in a fluid path before the mass detector. Such a filter may have a pore size of 1-5 μm, preferably 2 μm, to prevent particles from entering the mass detector.
In a mass-directed flash chromatography system according to the present invention, a detection signal is sent from the mass detector to a processor when a compound of a certain mass-to-charge ratio is detected. The processor in turn determines, based on instructions provided by the user and/or the provider of the instrument, if this corresponds to an analyte of interest that should be collected and sends a signal to the fraction collector to collect the portion of the effluent fluid stream comprising the analyte or send it to waste.
In order to correlate the detection of an analyte by the mass detector with a specific portion of the effluent fluid stream, the processor may use information of the current flow rates in, and volume of, the respective fluid paths to calculate when the analyte reach the fraction collector. An aliquot of the sample stream containing the analyte of interest can be selectively collected by causing the fraction collector to collect an aliquot from the sample stream at the expected arrival time of the analyte at the fraction collector; or, where the sample collector is continually collecting aliquots in the sample stream, the particular aliquot that is collected at the expected arrival time can be identified as containing the analyte. The correlation of detection at the mass detector and arrival at the fraction collector can be done in various ways as known in the art and is not itself part of the present invention.
In a further aspect, the present invention relates to an integrated system for mass-directed flash chromatography. The system according to this aspect facilitates easy pre-chromatography analysis of a sample by the mass detector, automated transfer of acquired analytical data from the mass detector to a data processing unit, and use of the acquired analytical data to control the collection of fractions of the effluent flow from the chromatography column. Historically, systems for mass-directed chromatography have had separate user interfaces for the chromatography part and the mass detector part of the system, respectively. The configuration of the system according to the invention allows a user to control the entire system and workflow through a single user interface making the operation of the system easier.
The system comprises a coupling module as described above, a flash chromatography instrument, a destructive mass detector and a fraction collector. Destructive mass detectors and flash chromatography instruments, optionally including a fraction collector, as well as stand-alone fraction collectors are commercially available and may be modified for use with a coupling module according to the invention to realize an integrated system according to the invention. A currently preferred flash chromatography system including a fraction collector is commercially available from Biotage AB (Sweden) under the tradename Isolera™ Spektra.
In a further embodiment, the invention relates to a method for separation of a sample into and collection of at least one of said components in at least one separate fraction, comprising the steps
The method according to this aspect facilitates easy pre-chromatography analysis of a sample by the mass detector, automated transfer of acquired analytical data from the mass detector to a data processing unit, and use of the acquired analytical data by the data processing unit to control the collection of fractions of the effluent flow from the chromatography column.
In a further embodiment, the invention relates to a computer program for use in the data processing unit of the above described system, said computer program comprising instructions inducing a computer to perform a method for controlling a fraction collector in the system comprising a chromatography column, a fraction collector, a destructive mass detector and a branched fluid path from said column to the fraction collector and the destructive mass detector, respectively, said method comprising the steps:
In order to present a user of the system or method according to the invention with accessible information regarding the constituents of an unseparated sample injected into the fluid path of the coupling module described above, it may be advantageous to process the detector signal from the mass detector to do e.g. peak integration, noise subtraction, Base peak chromatogram (BPC), and Extracted ion chromatogram (EIC). Standard software solutions for this are available, generally from the providers of the mass detector.
A quadropole mass analyzer, as optionally used in the present invention, produce a time-resolved Total Ion Current (TIC) signal for a number of mass-to-charge ratios (m/z-ratios), the number depending on the resolution of the detector. When a sample is injected into the coupling module secondary flow, the intensity measured at the mass detector varies over time and appears as a peak in time of the TIC intensity signal The TIC signal is also sensitive to low intensity evenly distributed noise and to high intensity random spikes.
One aspect of the invention relates to an improved method for processing detector signal data from a mass spectrometer and presenting it to a user, comprising the steps -obtaining a plurality of signal intensity values for a plurality of m/z ratios from a mass spectrometer;
In one embodiment N=5 and the value presented to the user is the lowest value stored in the information storage buffer.
This method takes care of problems described above. Since the maximum intensities are collected it is not necessary to synchronize the measurement of the spectrum in time with when the maximum intensity appears at the mass detector. Measurement can be started early and ended late. The low level noise evens out over time and the displayed spectrum stabilizes at the actual signal plus the maximum low level noise amplitude. The spikes are not affecting the displayed spectrum since they are random, unless the number of spikes at an individual mass point is greater than or equal to the size of the buffer.
The method described in this aspect may be implemented as a computer program to run on the data processing unit 500, as shown in
The computer programs according to the invention may be configured to perform the method aspects of the present invention and to realize the computer controllable aspects of the apparatus and system aspects of the present invention. The method aspects of the invention may be implemented using the product aspects of the invention.
Turning to the figures,
The first and second fluid paths pass an active splitter 13. A suitable active splitter is commercially available from Rheodyne (Rohnert Park, Calif., U.S.) under the trade name MRA®. This splitter not only splits the fluid flow from fluid path (10) to fluid paths (10) and (20), but preferably also dilutes the fluid in fluid path (10) in the fluid of fluid path (20). The ratio of dilution may be 1:100 to 1:1000, such as 1:250, 1:500 or 1:750. A presently preferred ratio is 1:500. The dilution can be controlled by the data processing unit (500), shown in
The fluid path 10 continues downstream of the active splitter and includes two parallel fluid paths; one path 10′ leading directly to the outlet 15 and one longer delay loop 14. A fluid flow in the fluid path 10 can thus be directed to go either directly to the outlet, along the shorter fluid path 10′, or along the longer delay loop 14, but directed to both the shorter fluid path 10′ and the delay loop 14 at the same time. In a preferred embodiment shown in
The fluid path 10 also comprise a further inlet 12, which preferably is adapted for direct manual injection of a sample, optionally dissolved or diluted in a suitable solvent.
In
The coupling module is further connected to a solvent reservoir 600 that supplies suitable solvent or solvents to arrange the fluid flow through the second fluid path of coupling module 100 to the mass detectors 300. In one embodiment, the solvent reservoir comprises three solvent subreservoirs for containing different solvents, such as one acidic solvent, one basic solvent and one neutral solvent. In such a case, a set of valves may be configured in the fluid paths from the subreservoirs to the inlet 21 of coupling module 100 so that at each time only one type of solvent is delivered to inlet 21. Such valves may be arranged physically within or outside the coupling module 100, and are preferably under control of the data processing unit 500.
The system further comprises a mass detector 300 in fluid connection with the outlet port 22 of the second fluid path of the coupling module 100. A suitable mass detector is an electrospray ionization mass spectrometer, such as available from Microsaic Systems plc (Woking, UK) under the product name 3500 MiD.
The system further comprises a fraction collector 400 in fluid connection with the inlet 21 of the second fluid path 20 of the coupling module 100.
The system further comprises a data processing unit 500 in data communication with the other components of the system through data communication links 5100, 5200, 5300 and 5400, respectively, shown in dotted lines in
The data processing means 500 is configured to receive detector signals from the mass detector 300 through the data communication link 5300, and information representing the dimensions of, and/or flow rate in, the fluid paths 10, 20 connected to the fraction collector 400 and mass detector 300, respectively. This information may be stored in an information storage unit (not shown) or entered by a user of the system, or monitored by the system, or any combination thereof, and provided to the data processing unit.
When in use, the mass detector 300 generates a detector signal in response to a compound being detected. The data processing unit 500 is configured to compute, based on the information above, a time period between the time of detection of the compound at the mass detector 300 and arrival of the compound at the fraction collector 400, and generate a collector signal to direct the fraction collector 400 to collect a fraction of a fluid stream comprising said compound.
The data processing unit may further be configured to generate a control signal to valve 17 (shown in
In a preferred embodiment, the system comprises a single user interaction interface 700 for normal interaction with a user 1 as shown in
The components of the system shown in
The present invention is not limited to the above-described preferred embodiments. Various alternatives, modifications and equivalents may be used. Therefore, the above embodiments should not be taken as limiting the scope of the invention, which is defined by the appending claims.
Still, W. (1978). Rapid Chromatographic Technique for Preparative Separations with Moderate Resolution. 43(14), pp. 2923-2925.
Zeng, L. e. (1998). Automated analytical/preparative high performance liquid chromatography-mass spectrometry system for the rapid characterization and purification of compound libraries. 794, 3-13.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13160714.5 | Mar 2013 | EP | regional |
