WO2004111632A1

WO2004111632A1 - Chromatography separation methods and apparatus

Info

Publication number: WO2004111632A1
Application number: PCT/GB2004/002556
Authority: WO
Inventors: Mark Jones; Niels Waleson; Mark Portsmouth
Original assignee: Combipure Ltd
Priority date: 2003-06-13
Filing date: 2004-06-14
Publication date: 2004-12-23
Also published as: GB0313732D0

Abstract

A method and apparatus for isolating a component of a sample using chromatography are described. The method includes carrying out a first chromatography run using a part of the sample. A time window based on a retention time for a target component of the sample is determined. Then a second chromatography run is carried out using the sample and the output of the chromatography device is collected during the time window. The apparatus includes a chromatography device for separating the sample into component parts, and a detector connected downstream of the chromatography device to detect the component parts. A data processor is configured to determine a retention time of the target component and to calculate a time window during which the target component can be collected.

Description

CHROMATOGRAPHY SEPARATION METHODS ND APPARATUS

The present invention relates to analytical methods and apparatus, and in particular to methods and apparatus relating to chromatography based analysis or purification of samples.

Chromatography is a method by which the components of a sample can be separated. One particular application of chromatography can be to allow the different components of a sample to be isolated, for example to purify the sample so as to remove unwanted components, or to allow the sample to be analysed, e.g. by determining properties or the identities of the different component parts. As will be appreciated, chromatography can be a powerful analytical tool. One particular area in which purification or analysis can be particularly useful is in the field of drug discovery programs or in analysing or purifying chemical libraries. As will be appreciated in these areas, it is often necessary to process large number of samples, e.g. tens of thousands or more. Hence, it would be advantageous to be able to provide a method for isolating the component parts of chemical samples in a simple and efficient manner.

According to a first aspect of the present invention, there is provided a method for isolating a component of a sample using a chromatography device, comprising carrying out a first chromatography run using a part of the sample, determining a time window based on a retention time for a target component of the sample, carrying out a second chromatography run using the sample and collecting the output of the chromatography device during the time window.

By carrying out a first run using a small portion of the sample to determine when the target component elutes, it is possible to carry out a simpler second run in order to reliably collect the isolated target component of interest. The second run uses less resources than the first run, such as detectors, and so can provide a more efficient way of isolating significant amounts of the target component, compared with carrying out a single run using all the sample. The method can further comprise measuring the components of the sample downstream of the chromatography device during the first run.

The method can include using a detector to determine purity. The detector can be an evaporative light scattering device, or preferably a UV detector. In this way a measure of the purity of the sample or components of the sample can be obtained

The method can include using a mass spectrometer. The mass spectrometer can be used to determine the mass of the components of the sample. The mass spectrometer can be used during the first run only. Hence, the mass spectrometer can be used for other purposes during the time of the second run and so this expensive resource is freed for other uses. For example, the mass spectrometer can be used to carry out quality control measurements on collected purified samples. For example, the mass spectrometer can be interfaced with an analytical hplc system.

A sample of the components of the sample can be supplied to the mass spectrometer. The sample can be supplied to the mass spectrometer occasionally. Preferably a sample is supplied periodically to the mass spectrometer. A sample of the components of the sample can b₍e supplied over at least a part of the duration of the first chromatography run.

The method can be automated and preferably the method is automated under computer control. More preferably, at least some of the computer control is effected over a network. Automation of the method can include using a timing controller. The timing controller can be used to control the duration and period of control signals causing a sample of the components of the sample to be supplied to a mass spectrometer.

The method can further comprise determining a plurality of time windows based on a plurality of retention times for a plurality of respective target components of the sample and collecting the separate outputs of the chromatography device during the respective time windows. Hence more than one target component can be isolated from the same sample. The sample can be a compound or pooled sample comprising portions of more than one different actual sample for processing.

Preferably, the first chromatography run and the second chromatography are carried out using the same chromatography device.

A third chromatography run using a part of a further sample can be carried out at least partially while the second chromatography run is being carried out. Hence, in this way there can be parallel processing of the sample and further sample. The first screening run for the further sample can be carried out at least partially while the collection or isolation run is being carried out for the preceding sample.

The sample can comprise a pooled sample, the first chromatography run can be carried out using a chromatography device, a plurality of time windows can be determined based on a plurality of respective retention times for a plurality of target components of the sample, a second chromatography run can be carried out for each of the plurality of target components using the same chromatography device, and the outputs of the further second chromatography runs can be collected separately during the respective time windows.

The method can include carrying out the first chromatography run and second chromatography runs for a first pooled sample using a first chromatography device, and carrying out the first chromatography run and second chromatography runs for a second pooled sample using a second chromatography device. The first chromatography run can at least partially overlap with the second chromatography run on the other chromatography device.

According to a further aspect of the invention, there is provided a chromatography system for isolating a target component of a sample, the system comprising a chromatography device for separating the sample into component parts, a detector connected downstream of the chromatography device to detect the component parts, a data processor configured to determine a retention time of the target component and to calculate a time window during which the target component can be collected. The system can further include control circuitry which can generate a control signal corresponding to the time window. The system can further including a valve at an output of the system, the valve being electronically actuable responsive to the control signal.

The system can further include a sampling valve downstream of the chromatography device and actuable to be in fluid communication with the detector to pass a portion of the sample stream to the detector.

The system can further include a timing controller in communication with a sampling valve and configured to cause the sampling valve to periodically pass a portion of the sample stream to the detector during at least a part of the duration of a chromatography run.

The detector can be any device that can determine the mass of the components of the sample. Preferably the detector is a mass spectrometer.

The system can further comprise a UN detector downstream of the chromatography device. The UN detector can be used to provide an indication of the purity of the sample or component parts of the sample.

The system can further comprise a further chromatography device for separating the sample into component parts, an electronically actuable valve connected to an output of the further chromatography device and a controller in communication with the valve and configured to operate the valve during the time window to allow the target component to be collected from a chromatography run carried out using the further chromatography device.

The system can further comprise a plurality of further chromatography devices for separating a plurality of pooled samples into component parts, a plurality of electronically actuable valves each connected to a respective output of a one of the plurality of further chromatography devices and a controller in communication with the valves and configured to operate the valves during respective time windows to allow a plurality of target components to be collected from respective chromatography runs carried out using the further chromatography devices.

The system can further include an autosampler for introducing samples into the chromatography device sample streams. The system can further comprise a robotic arm configured to automatically place empty receptacles adjacent an output of the valves to collect target components and to remove receptacles holding a target component.

An embodiment of the invention will now be described, by way of example only, and with reference to the accompanying drawings, in which:

Figure 1 shows a schematic block diagram of a chromatography system including a sampling arrangement according to the invention;

Figures IA and IB show schematic diagrams illustrating a sample introducing part of the system of figure 1 ;

Figure 2 show a schematic block diagram of an embodiment of a sampling arrangement according to the invention;

Figure 3 shows a flow chart illustrating at a high level a method of operation of the system shown in Figure 1 ; Figure 4 shows a flow chart illustrating in greater detail a first part of the method illustrated in Figure 3;

Figure 5 shows a flow chart illustrating in greater detail a second part of the method illustrated in Figure 3;

Figure 6 shows a flow chart illustrating operations carried out by a computer control system and corresponding generally to the method illustrated in Figure 4;

Figure 7 shows a flow chart illustrating operations carried out by a computer control system and corresponding generally to the method illustrated in Figure 5;

Figure 8 shows a schematic block diagram of another embodiment of a chromatography system according to the invention; Figure 9 shows a flow chart illustrating at a method of operation of the system shown in Figure 8 according to the invention; Figure 10 shows a flow chart illustrating a method for seleting samples to include in a pooled sample; and

Figure 11 shows a timing diagram illustrating the method of operation of the system shown in Figure 8..

Similar items in different Figures share common reference numerals unless indicated otherwise. Unless indicated otherwise, arrows without reference signs represent local fluid flow in the system.

With reference to figure 1 there is shown a chromatography system, generally designated 100, according to the present invention and operating according to a method aspect of the invention. The chromatography system and method can be used in the general investigation or analysis of samples of chemical compounds and are particularly suited for use in the isolation of components of a sample or the purification of a sample, e.g. in order to extract a particular target chemical compound. The system and method are particularly suited for use in purifying samples as part of a drug discovery program, but are not limited to that particular application. The range of application of the system 100 and principles underlying the method of operation of the system will be apparent to a person of ordinary skill in the art in light of the following discussion of the general principles underlying the invention.

Chromatography system 100 includes a chromatography subsystem 102 comprising various chromatography related parts, apparatus and detectors, i.e. the 'wet chemistry' parts of the system, together with an electronic control subsystem 104.

The chromatography subsystem includes a pumping system 106, including first 106' and second 106" pumps, which are connected by tubing to a source of solvent, or eluent, for use during a chromatography run. By way of example, a suitable pump for pumps 106' and 106" would be a PU-2086 model available from Jasco (UK) Ltd. The solvent can comprise an aqueous solution of an organic liquid and can include separate sources of water 108 and the organic component 110. Examples of suitable organic components would include, acetonitrile and methanol. The outputs of pumps 106', 106" are connected via a mixer 107 and can be used to carry out a gradient chromatography method in which the proportion of organic component in the solvent stream passing through the chromatography column is increased during the chromatography run. Gradient chromatography methods are well known to persons of ordinary skill in the art and will not be described in further detail herein.

The output of pumping system 106 is connected by a length of tubing to a sample introduction part 112 of the system. The sample introduction part 112 includes a valve 116 to which the pumps are connected via a first input port. The sample introduction part 112 also includes an injector port 114 into which a sample can be injected. A sample loop 118 is connected across a third and fourth port of the valve. The valve 116 has a waste output port connected to a length of tubing providing a waste line 122, and a further main output port 120.

As illustrated in Figures 1 A and IB, valve 116 actuable in use to provide to modes of operation. In a first loading mode shown in Figure 1 A, the sample loop is connected to the injection port 114 and waste line 122 and the pump and a chromatography column are connected. Hence a sample can be injected into sample loop 118. In a second injection mode shown in Figure IB, the sample loop is connected to the pump line and the chromatography column so that the sample can be introduced into the eluent stream, and the injection port is connected to the waste line. A suitable sample injector device is the 215 model as provided by Gilson Inc.

The main output port 120 of valve 116 is connected to a chromatography column 124 which provides a chromatography device allowing different components of a sample to be separated based on their relative affinities for the column. By way of example, a suitable chromatography column is the Genesis model as provided by Argonaut Technologies, Inc. That column is approximately 150 mm long with an internal diameter of approximately 10 mm and is packed with 7 micron silica micro-spheres which provide the chromatography medium. However, the details of the chromatography device will depend on the application of the system, the sample compounds and the performance required. The output of the chromatography column 124 is connected by a further length of tubing to a UN detector 128. By way of example, a suitable UN detector is the MWD device as provided by Agilent, Inc. The UN detector is provided mainly for diagnostic purposes, i.e. to monitor the column performance. However, the UN detector could also be used to trigger component collection, either alone or in conjunction with the mass spectrometer to be described below. The UN detector 128 is provided downstream of the chromatography column and in use receives the separated components of the sample in sequence. The different components of the sample are removed from the chromatography column as the concentration of the organic component in the eluent increases.

An input port of a second valve 130 is connected by a length of tubing to an output of the UN detector and is provided downstream of the chromatography column 124 and UN detector 128. A first output port of valve 130 is connected by a third length of tubing, including a T-junction 131, to a pressure controlling or regulating device 132. The output of the pressure regulator 132 has a length of tubing connected to it providing a further waste line 136. The third limb of the T-junction is connected by a length of tubing to an input of a mass spectrometer 134. Byway of example, a suitable mass spectrometer is a G1946 as provided by Agilent Inc. Mass spectrometer 134 provides a detector which can detect and measure the masses of the components of the sample.

As will be described in greater detail below, valve 130 is actuated to divert a sample of the main sample stream toward the mass spectrometer 134, and pressure regulator 132 ensures that the mass spectrometer is not exposed to a fluid pressure likely to over load or cause damage to the mass spectrometer. Sampling arrangement 138 will be described in greater detail with reference to figure 2 below.

A further output port of valve 130 is connected by a line of tubing to an input of a third valve 140. A first output of valve 140 has a line of tubing attached thereto providing an output line 142 from which an isolated component or components can be collected. A second output of valve 140 has a further section of tubing attached thereto providing a waste line 144. In one embodiment, and by way of example only, a suitable valve for valves 130 and 140 is a 3-way solenoid controlled, isolation valve, such as the 100T3 model as provided by Bio-Chem Naive, Inc.

The chromatography and detector parts of the chromatography subsystem 102 are controlled by the electronic control subsystem 104. The control subsystem includes a computer 150, such as a conventional personal computer, including a data processing device and primary and secondary storage devices as will be apparent to person of ordinary skill in the art. In figure 1, the dashed lines indicate control lines along which control signals and/or data can be transmitted between the control subsystem 104 and the parts of the chromatography subsystem. Computer 150 has a network device 152 in communication therewith providing a local network over which data and control instructions can be transmitted to various parts of the chromatography system. Pump 106 is connected by a serial communication line to a serial interface of computer 150. The sample injector is also connected by a serial communication line to a serial interface of computer 150 (not shown). UN detector 128 and mass spectrometer 134 are in communication with the computer 150 over network 152. The control subsystem also includes a controller 154 for controlling the timing of various events during operation of the system as will be described in greater detail below. In particular, controller 154 is connected to the network 152 and includes control lines to communicate signals to and from valves 116, 130 and 140.

For ease of reference, in the following, valve 116 will be referred to as the sample or injection valve, valve 130 will be referred to as the sampling valve and valve 140 will be referred to as the collection or output valve.

With reference to figure 2, there is shown an example embodiment of the sampling arrangement 138. The sampling arrangement includes the sampling valve 130 and the pressure monitor or regulator 132. As illustrated, sampling valve 130 is a solenoid valve. When a gate signal is applied to the solenoid 152, the valve is actuated to connect the input 154 to the first output 156 so as to pass a part of the sample stream toward the mass spectrometer 134. In the absence of a gating signal applied to solenoid 152, the sampling valve passes the sample stream from input 154 to output 158. The pressure regulator 132 is configured to prevent the mass spectrometer from being exposed to a fluid pressure sufficiently large to damage the mass spectrometer. There are a number of mechanisms by which this can be accomplished. In the illustrated example, the pressure regulator 132 is provided by a two port pressure relief valve 172. Ifthe fluid pressure experienced by the valve 172 exceeds the selected safe threshold level for the mass spectrometer 134, then the pressure release valve is actuated, under action of the sample fluid pressure, and the sample is passed to the waste line 136 and ejected, thereby reducing the fluid pressure to which the mass spectrometer would otherwise experience at T-junction 131. Byway of example only, a suitable pressure relief valve is the 075RN model as provided by Bio-Chem Naive, Inc. This pressure release valve operates at approximately 20 psi and hence will prevent the mass spectrometer 134 from being exposed to fluid pressures in excess of 20 psi. However, other pressure thresholds can be used depending on the detector device.

The pressure regulator 132 acts to reduce the flow rate of solvent to the mass spectrometer. The sampling valve sprays the whole of the sample stream into tube 160 and the fluid flow rate is mainly determined by the amount of time for which the sampling valve is 'open' and diverting the sample stream into conduit 160. Diverting a part of the whole of the sample stream on an occasional basis, provides a more accurate and reliable component detection by the mass spectrometer an is not affected by gradient changes during a run. Further, it helps to avoid blockages forming in the tubing which changes the impedance of the tubing thereby reducing the reliability and accuracy of the mass spectrometer measurements. Further more, it is easy to control the rate of fluid flow to the mass spectrometer by simply changing the time for which the sampling valve is open to divert the sample stream.

Other mechanisms can be used to provide pressure regulation. In the foregoing and following, pressure regulation merely means that the device prevents the mass spectrometer connected downstream of it from being exposed to a pressure in excess of a safe pressure value. It does not mean that a fluid sample must be supplied to the mass spectrometer at a specific or fixed level. There are various mechanisms by which a pressure regulator can be realised and the foregoing is by way of example only. For example in a further embodiment the pressure regulator 132 could include a solenoid valve and a pressure sensor to detect the pressure that the mass spectrometer is being exposed to and to actuate the solenoid valve to redirect fluid to waste ifthe pressure exceeds the safe pressure threshold value.

As will be appreciated, the sampling arrangement 138 provides a simple mechanism to ensure that a detector device attached to it is not exposed to a fluid pressure likely to damage the device. The use of the sampling arrangement is not limited to use with a mass spectrometer 134 but can be used with any detector or device which is pressure sensitive and which can be used in as a detector in a chromatography system. Further, use of the sampling arrangement is not considered to be limited to the specific chromatography system shown in figure 1 and that other applications of the sampling device in chromatography systems will be apparent to persons of ordinary skill in the art in light of the foregoing.

With reference to figure 3, there is shown a flowchart 300 illustrating at a high level, a method of operation of the system shown in figure 1 according to an aspect of the invention. A first, pre-screen, chromatography run is carried out at step 302 using a portion of the sample in order to determine a time window during which a target component of the sample of interest would be available for collection at the output of the chromatography system. A second, preparation, chromatography run is then carried out using the remainder of the sample and the target compound of interest is then collected during the collection window as determined in the previous run. Hence, the mass spectrometer is used only during the pre-screen run and does not need to be used during the second preparation run.

The purification method 300 will now be described in greater detail with reference to figure 4. Figure 4 shows a flowchart 308 illustrating operations carried out during the first step of method 300 and corresponds generally to method step 302. At a first step 310, the sample to be analysed is prepared, by dissolving the chemical sample to provide a solution for use in the chromatography runs. For example, a suitable solvent is dichloromethane. Approximately 5 microlitres of the sample solution is used for injection in the first run. At step 312, the sample is injected into the sample loop 118. Then, at step 314, the UN detector 128 and mass spectrometer 134 are started, a gradient run is initiated by starting the pump system 106. Then at step 316, valve 116 is actuated to connect the sample loop 118 into the eluent stream and the sample stream carries the sample into chromatography column 124.

The initial components of the sample leave the chromatography column 124 and pass through detector 128 where the components are detected for mainly diagnostic purposes. The UN detector can be used to monitor the progress of the separation or column performance and typically responds to all components of a sample, some of which may not be detected by the mass spectrometer. The components then pass through sampling valve 130 which is periodically actuated at step 320 so as to obtain a small portion, or sample, of the main sample stream and which is directed toward mass spectrometer 134. The amount of the sample can easily controlled by varying the sampling frequency and duration of the sample. Provided the fluid pressure is sufficiently low, the sample of the main sample stream is passed through pressure regulator 132 to the mass spectrometer 134 where any component in the sample of the sample stream is detected and measured by the mass spectrometer. The output valve 140 connects the sample stream to waste line 144 and the sample stream is run off to waste as indicated by step 322. During the gradient run, the proportion of organic component in the eluent is increased and the components of the sample are sequentially removed from the chromatography column. The components pass through the system and a sample of the components is diverted to the mass spectrometer on a periodic basis, e.g., once every second.

After the first run has been completed, the mass spectrometer 132 will have recorded data indicative of the start time and the stop time of the first, pre-screen, chromatography run and the mass of the components detected during that run, and the time at which those components were detected in the sample stream. Knowing the time at which a component corresponding to a target component of interest, e.g. a pharmaceutical compound to be isolated, it can be determined at what time the component would be present at the output of the chromatography system for collection. Based on this retention time of the target component, i.e. the time from the beginning of the run after which the target component would be available for collection, a window of time extending slightly before and slightly after the retention time can be determined and used in a second run to collect the target component only.

Figure 5 shows a flowchart 330 illustrating various operations carried out during the second, preparative chromatography run and corresponding generally to method step 304 of figure 3. At step 332, the remainder of the sample solution is used and all, or most, of the remaining sample solution is then injected into sample loop 118 at step 334. At step 336, the second preparative chromatography run is begun and pump 106 is started together with the UN detector and mass spectrometer 134. Naive 116 is also operated to pass the eluent stream through sample loop 118 so as to introduce the sample into the main chromatography sample stream. Sampling valve 130 does not need to be periodically activated during this second run, but merely connects directly to the output valve 140 as indicated by method step 338. Sampling valve 130 can be considered to be 'open' from the perspective of the collection valve 140.

Up to the beginning of the collection window, valve 140 is actuated to connect to the waste line 144 and the eluent and any non-target components of the sample are run off to waste 340. At the time corresponding to the beginning of the collection window, collection valve 140 is actuated to connect the sample stream to the product collection line 142 and the target component is collected at step 342. At the end of the collection window, collection valve 140 is actuated again to connect the sample stream to waste line 144 and the remainder of the sample stream can be run off to waste at step 344. Hence, it is possible to isolate the target component of interest during the second chromatography run, using the collection window identified during the first pre-screen run.

It will be appreciated that the apparatus and method are particularly suited for use in purification of a sample, i.e., to extract a single target component having a particular mass from a sample. Alternatively, the system and method can be used to isolate different target components from the same sample by determining temporally separated collection windows for each of the target components present in the initial sample. The collection valve 140 is then operated for each collection window in order to allow the target components to be collected in different receptacles.

Having described the method of operation of the chromatography subsystem 102, the method will be described again, but with reference to the operations carried out by the electronic control subsystem 104.

Figure 6 shows a flowchart 350 illustrating operations carried from the perspective of the control subsystem 104 during the first chromatography run. As described previously, computer 150 can send and receive control signals to various components of the chromatography subsystem 102 and transmit and receive data to and from the components of the chromatography subsystem. Computer 150 can control pump 106 and injector 114 over serial communication lines. Naive 116, sampling valve 130 and collection valve 140 are all connected to a controller 154 which communicates with computer 150 over network 152. Controller 154 includes a microcontroller having a microprocessor together with local memory for storing instructions to control operation of the controller and to store data representing a timetable of events and control signals to be issued to the valves corresponding to those events. The UN detector 128 and mass spectrometer 134 can also include onboard control systems including microprocessor devices and memories into which control and timetable data can be downloaded over network 152 by computer 150. UN detector 128 and mass spectrometer 134 can record and store data locally during operation and subsequently upload the detector experimental data to computer 150 over network 152 for subsequent processing and analysis.

At step 352, prior to beginning the first pre-screen run, computer 150 downloads timetables to the timing controller 154, pump 106, UN detector 128 and mass spectrometer 134. As explained above, the timetables include data indicating sequences of events and the timing of those events to be carried out by the devices. The pump, detectors and valves are also initiated during step 352. At step 354, the pre-screen run is begun. The pump is operated to begin the gradient chromatography run, sample valve 116 is operated to connect the sample loop 118 into the eluent stream, the mass spectrometer and the UV detector are started, and a clock signal is reset to t = 0 and then started. The UN detector 128 detects the intial components as they are released from the chromatography column and, knowing the start time of the chromatography run, records measured data from the UN detector as a function of the time of detection of the various components.

The timetable for operation of the sampling valve 130 includes an initial delay period (e.g. from t=0 to t=0.5 mins) to allow for the finite amount of time before a first component of the sample is likely to have passed through the chromatography column. The timing controller 154 knows the start time of the chromatography run and when its internal clock determines that the delay has expired (e.g. after 0.5 minutes) then the controller inspects the timetable data to determine a duration for a gating signal and a period for the frequency of applying that gating signal to the solenoid 152 of the sampling valve 130. The timetable also includes an indication of the duration of time during which the gating signal is periodically applied to the solenoid of the sampling valve 130. For example, the duration of the gating signal can be 40 milliseconds and the gating signal can be applied once every minute up until between three to five minutes after expiry of the initial delay.

Hence, at step 358, a gate signal is applied to the solenoid of sampling valve 130 which directs a sample of the main sample stream to mass spectrometer 134 for 40ms. Naive 130 then reconnects to collection valve 140 for 960ms until the next gating pulse is applied. The gating pulse is periodically applied until it is determined at step 360 that the sampling time has expired.

Provided the fluid pressure of the sampled part of the sample stream does not exceed the safety pressure threshold for the mass spectrometer 134, then pressure regulator 132 is not actuated and the sample is passed to mass spectrometer 134. The mass spectrometer knows the start time of the sample run, the elapsed time and determines the mass of any components detected in the sample and the time of detection of the components.

After a fixed time, during which all components of interest should have been released by the chromatography column, then the timetables indicate to each component to cease operation and the chromatography run completes at step 362. The UV experimental data can then be transferred over the network 152 and stored on computer 150 and similarly with the mass spectrometer experimental data. At step 364 a collection window or windows are determined for each target component or components of interest. The total ion current from the mass spectrometer is integrated and the centre of the peaks for each component is identified to provide a retention time for that component. The retention time so determined corresponds to the time at which the mass spectrometer actually detected the component. Any time offsets (positive or negative) to account for the fact that the component of interest may be received at collection valve 140 at a time before or after the component was actually detected by the mass spectrometer (e.g. because of different fluid path lengths) are then determined. The start and end times for a collection window spanning the corrected retention time is then determined.

For example, if a component having a mass corresponding to a target compound of interest is detected, then the centre of the total ion current peak for that component is determined and may be e.g. 3.5 minutes. That component may be expected to arrive at the collection valve 140 approximately 0.1 minutes before the component would be detected by the mass spectrometer, e.g. as the fluid path to the collector valve is shorter, and therefore the corrected retention time would be 3.4 minutes. In order to ensure that the whole of the target component is collected, an, e.g., 24 second duration collection window may be used. Therefore, the collection window would start time would be at 3.2 minutes from the start of the chromatography run (t = 0) and stop time would be 3.6 minutes from the start of the chromatography run. Hence the collection window timings have been determined and can be used in the second preparative run.

These operations can be carried out by an application executing on computer 150 or can be determined manually or partially automatically and partially manually. It will be appreciated that the temporal location and duration of the collection time window will vary depending on the specific details of the chromatography apparatus being used and the above timings are by way of example only.

Figure 7 shows a flowchart 370 illustrating operations carried out by the control subsystem 104 during the second preparative chromatography run. At step 372, timetable data is downloaded to the timing controller 154 and also to pump 106 in order to carry out a gradient chromatography run substantially identical to the run used during the pre-screen run. At step 374, the preparative run is begun by initiating the gradient, introducing the sample into the sample stream and starting the clock at t=0. There is no need to sample the sample stream during the preparation run as it is not necessary to detect the target component in the sample stream. Therefore the control system waits at step 376 until the elapsed time of the chromatography run is equivalent to the start time of the collection window, in this example, t=3.2 minutes. At this time, valve timing controller 154 applies a control signal to the solenoid of collection valve 140 to 'open' valve 140 to direct the sample stream to collection line 142 for collection in a receptacle. The timing control 154 then determines whether the elapsed time of the chromatography run has reached the stop time of the collection window, in this example t=3.5 minutes, and then removes the gating signal from the solenoid of valve 140 which 'closes' to direct the sample stream to waste line 144 at step 380. The second preparative chromatography run can then be ended at step 382.

With reference to figure 8, there is shown a further embodiment of a chromatography system 400 according to the present invention. The chromatography system 400 comprises four chromatography systems, similar to that shown in figure 1, but with some modifications in order to improve automation and throughput of the system. A control subsystem (not shown in figure 8) is also provided, again similar to that shown in figure 1, but adapted for the four column system shown in figure 8. The system of figure 8 provides four chromatography channels which can each operate independently, and in parallel, in order to improve the throughput of samples by the system, and utilisation of the mass spectrometer 134.

The system includes an automated sample handler 404 which includes a plurality of well plates 406 having a plurality of wells each holding a different sample in solution for analysis by the system. The sample handling device 404 can, in one embodiment, be an autosampler. The sample handling device includes a needle 408 mounted on a track 410 by which the needle can be moved to take a sample from any of the sample wells in well plate 406 and inject the sample into a port for each of sample introduction part 112A-D, each corresponding generally to sample introduction part 112 as shown in figure 1.

Each chromatography channel 412, 414, 416, 418 includes a separate chromatography column 124A-D, UV detector 128A-D and sampling valve 130A-D and collection valve 140A-D. System 400 also includes an automated collection tube handling system 420. The collection system 420 includes a rack 422 for holding empty tubes and tubes holding collected samples 421 A-D. The system also includes a robotic arm 424 which can move as indicated by arrow 426 to place an empty tube adjacent to a collection line 142A-D and to collect tubes holding collected samples and place them in the tube holder 422. Hence the tube handling system 420 can automatically ensure that there is an empty tube available for collecting the output from each channel and to replace filled tubes with an empty tube as required. Each sampling valve 130A-D has an output connected to an electronically controllable valve 430, which is connected to a further valve 432, and valves 430 and 432 can be actuated to allow the output of a one of the sampling valves 130A-D to be connected to the mass spectrometer 134 and pressure regulator 132.

A method of operation of the chromatography system 400 will now be described with reference to flowchart 430 of figure 9. Reference will also be made to figure 11 which shows a graphical representation 500 of the chromatography runs (illustrated by arrows) carried out by the different columns (channel numbers 1-4) at different times and the samples being processed by each column.

At a first step 432, the control system initiates the pumps, UV detectors and mass spectrometer and the sample handling 404 and sample collection system 420. At step 434, auto sampler 404 collects 5 microlitres of a first, second, third and fourth sample from the well plate 406 and injects the pooled sample into the port for the first chromatography channel at step 436. As will be appreciated, the sample present in the first channel 412 can be considered to be a pooled sample as it comprises four separate actual chemical samples, samples 1, 2, 3 and 4 in this example. It is possible to handle pooled samples, provided the target components for each of the actual samples have sufficiently different masses. This is to ensure that the target components can be discriminated between by the mass spectrometer. Preferably the different samples in a pool are each separated by at least 6 atomic mass units from all the other samples in the pool.

In one embodiment of the system, the computer part of the control subsystem includes a sample pooling application to select suitable samples to pool into a single sample. For example, a plurality of samples can be provided each having a unique identifier for the target compound, a molecular weight for the target compound and an identifier for the well in the well plate in which the sample is located. This data can be presented in a spreadsheet format and ordered by molecular weight. The application can then execute an algorithm in order to select groups of three samples which are sufficiently separated by mass.

Figure 10 shows a flow chart 480 illustrating processes carried out by the sampling pooling application. The process 480 initiates 482 and then determines 484 the number of samples to be used in each pool, x, which in this example is set at four. The process then determines at step 486, the total number of samples that need to be processed, y, and which for the purposes of this example can be 96. Then at step 488, the number of pooled samples that will need to be injected in order to process all the samples is determined, and is given by n=y/x, which in this example is 24. It will be appreciated that an integer number of pooled samples will need to be injected, and so any remainders are rounded up and the last 'pooled' sample may actually include only a single sample, or less than the number of samples in the other pooled samples. Then at step 490, the samples are ordered by mass.

At steps 492, 493 and 496 create the pooled samples. Tthe first pooled sample is created at step 494, whose members will, in general, be the group comprising: sample p, sample p+n, sample p+2n, sample p+3n, etc. In this example, as n=24, the members of the first pooled group (i.e. for p=l) will be the 1^st, 25^th, 48^th and 73^rd samples by mass. Process flow loops and the next pooled group is then created (i.e. for p=2), and comprises the 2^nd , 26^th , 49^th and 74^th samples by mass. Hence, 24 groups of four samples are obtained.

At step 436, the pre-screen run is started for the first chromatography channel 412 which includes the first, second, third and fourth samples as illustrated by arrow 502 in figure 10. At the end of the pre-screening run, the collection windows for the first to fourth samples are determined at step 438. Then, at step 440 the preparative run 504 is started for a one of the samples, e.g. the first, using the sample chromatography column, i.e. channel 1. The method then collects the next pooled sample at step 442 and a pre-screen run 506 is carried out for the 5^th to 8^th samples using the second channel. At any time during, or after run 506, the method can at step 444 obtain another sample and start its preparative run, e.g. run 508 for the second sample in channel 1, or the robotic arm can automatically collect the isolated sample at step 446 and place an empty tube in place ready for the next sample. Similarly, at any appropriate time, the method can determine the collection windows at step 448 for a pre-screen run that has completed.

If it is determined at step 450 that all the samples have been processed, then the method can terminate at step 452. If it is determined at step 450 that samples remain to be processed, then the method generally loops and more preparative runs are carried out and more pre-screen runs are carried out using the channels in sequence until all four channels are being used in parallel. Although the arrows in figure 11 show synchronisation, in practice, synchronisation is not necessary and the processing of pre-screen and preparative runs in different channels will overlap in different ways. As illustrated in Figure 11, channel 1 carries out a pre-screen run for the first pooled sample and then preparative runs 504, 508, 510, 512 for each of the samples, before carrying out a further pre-screen run 514 for a further pooled sample, the fifth, comprising the 17^th to 20^th samples. Similarly, channel 2 carries out a pre-screen run 506 for the second pooled sample and then preparative runs 516, 518, 520, 522 for each of the samples. Similarly, channels 3 and 4 carry out a pre-screen run 524, 526 for the third and fourth pooled samples and then preparative runs for each of the individual samples. It will be appreciated that the same chromatography device is used to carry out the pre-screen and preparative runs. Hence, it will be appreciated that by operating a chromatography column which carries out the pre-screen and preparative runs in parallel with at least one other column carrying out pre-screen and preparative runs, a significant throughput can be achieved for the system. It will be further appreciated that more or fewer than four channels can be used, and that more or fewer than four samples can be pooled. Preferably, the number of channels is the same as the number of samples in a pooled sample.

The target component present in the collected sample can be further analysed, e.g. using hplc, in order to verify that the target component has been isolated. It has been found that the collected sample can stratify in the collection tubes. Therefore, it is preferred if a sample for quality control purposes is collected at different heights throughout a collection tube, e.g. at three different heights, toward the bottom, toward the middle and toward the top of the sample, in order to ensure that the target component is not missed as can occur if a sample from only a single location in the collection tube is taken for quality control purposes.

Further in relation to quality control measurements, the height of the measured UV peak for a target component can be used as an indicator of the amount of the target component in a collected sample to be presented to a detector so as to help avoid saturating the detector. For example, ifthe UV detector peak for the target component as measured during a preparation run is determined to exceed a first threshold, e.g. 1000 arbitrary units, then a first amount of the collected sample can be injected into an analytical LC- UV-MS system, e.g. 2 microlitres. Ifthe UV detector peak is in excess of a second threshold value, e.g. more than 2000 arbitrary units, then a reduced amount of the collected target component, e.g. 1 microlitre, can be injected into the analytical system in order to verify the molecular weight of the target component. Hence by using the height of the UV peak as an indicator of the amount of target component present in the sample, the amount of sample presented to a quality control detector can be varied to ensure that the detector is neither saturated nor that the resultant response will be too low for good quantification. In another embodiment of the system, rather than collecting the sample during the collection window in a receptacle, in which case the target component is mixed up with the eluent preceding and following it, the sample is collected in a relatively long piece of tubing, for example having an internal storage volume of approximately 20ml. In this way the target component remains in the same position within the collected sample stream. It is possible that the target sample eluted at a different time during the preparative run compared to the pre-screen run. As UV data is collected during the preparative run, it is possible to determine at exactly what time, the target component actually eluted during the preparative run. Therefore it is possible to determine with reasonable accuracy where in the sample collection tubing the target component will be. Hence, the eluent preceding and following the target component can be run off to waste and a more concentrated sample can be obtained from the collection tubing.

Also it is possible that a narrow collection window would miss or collect only a part of the target compound if there is a significant discrepancy between the time of elution of the target compound in the pre-screen and preparative runs. Therefore, a longer collection window can be used, the sample collected in the collection tubing and data from the UV detector used to determined exactly when the target component did elute and hence to extract the target component from the tubing. Also there may be circumstances in which a target component and another component are too close together to be easily separated using the collection valve only. Collecting the sample during the collection window in a narrow piece of tubing effectively increases the separation between the components and so makes it easier to extract the target component only. Hence, the use of a length of collection tubing or hose can improve the sensitivity, accuracy and reliability of the method and apparatus.

Although various embodiments of the invention have been described in detail above, it will be appreciated that they are by way of illustration only and that the invention is not considered to be limited to the specific systems or methods described above. In particular, some of the operations mentioned in the flowcharts may be omitted and/or their sequence of operation changed while still falling within the general teaching of the invention. Further, the flowcharts are by way of illustration only and, unless the context requires otherwise, should not be considered limitative of the sequence of operations carried out. Further, it will be apparent to a person of ordinary skill in the art, in light of the general teaching of the foregoing, how to amend or otherwise modify the systems or methods in order to arrive at different embodiments as indicated above.

Claims

CLAIMS:

1. A method for isolating a component of a sample using a chromatography device, comprising: carrying out a first chromatography run using a part of the sample; determining a time window based on a retention time for a target component of the sample; carrying out a second chromatography run using the sample; and collecting the output of the chromatography device during the time window.

2. The method as claimed in claim 1, further comprising measuring the components of the sample downstream of the chromatography device during the first run.

3. The method as claimed in claim 2, wherein a UV detector is used.

4. The method as claimed in claim 2, wherein a mass spectrometer is used.

5 The method as claimed in claim 4, wherein the mass spectrometer is used during the first run only.

6. The method as claimed in claim 4 or claim 5, wherein a sample of the components of the sample is supplied to the mass spectrometer.

7. The method as claimed in claim 6, wherein a sample is supplied periodically to the mass spectrometer over at least a part of the duration of the first chromatography run.

8. The method as claimed in any preceding claim, wherein the method is automated.

9. The method as claimed in claim 8, wherein the method is automated under computer control.

10. The method as claimed in claim 9, wherein at least some of the computer control is effected over a network.

11. The method as claimed in claim 8, wherein a timing controller is used.

12. The method as claimed in claim 11, wherein the timing controller is used to control the duration and period of control signals causing a sample of the components of the sample to be supplied to a mass spectrometer.

13. The method as claimed in claim 1 , further comprising: determining a plurality of time windows based on a plurality of retention times for a plurality of respective target components of the sample; and collecting the separate outputs of the chromatography device during the respective time windows.

14. The method as claimed in claim 13, wherein the sample is a compound sample comprising more than one different actual sample for processing.

15. The method as claimed in any preceding claim 1 , in which the first chromatography run and the second chromatography run are carried out using the same chromatography device.

16. The method as claimed in any of claims 1 to 14, in which the first chromatography run and the second chromatography run are carried out using different chromatography devices.

17. The method as claimed in claim 16, further comprising: carrying out a third chromatography run using a part of a further sample at least partially while the second chromatography run is being carried out.

18. The method as claimed in claim 1, and wherein the sample comprises a plurality of actual samples, the first chromatography run is carried out using a chromatography device, a plurality of time windows are determined based on a plurality of respective retention times for a plurality of target components of the sample, a second chromatography run is carried out for each of the plurality of target components using the same chromatography device, and collecting the outputs of the second chromatography runs separately during the respective time windows.

19. The method as claimed in claim 18, and further comprising carrying out a further chromatography run using a further sample at least partially during the second run by at least a one of the further chromatography devices.

20. The method as claimed in claim 19, wherein the further chromatography run is carried out using the first chromatography device.

21. The method as claimed in claim 19 or 20, wherein the further chromatography run is carried out at least partially during the second run of all of the further chromatography devices.

22. A chromatography system for isolating a target component of a sample, the system comprising: a chromatography device for separating the sample into component parts; a detector connected downstream of the chromatography device to detect the component parts; a data processor configured to determine a retention time of the target component and to calculate a time window during which the target component can be collected.

23. The system as claimed in claim 22, and further including control circuitry which can generate a control signal corresponding to the time window.

24. The system as claimed in claim 23, and further including a valve at an output of the system, the valve being electronically actuable responsive to the control signal.

25. The system as claimed in claim 22, and further including a sampling valve downstream of the chromatography device and actuable to be in fluid communication with the detector to pass a portion of the sample stream to the detector.

26. The system as claimed in claim 25, and further including a timing controller in communication with the sampling valve and configured to cause the sampling valve to periodically pass a portion of the sample stream to the detector during at least a part of the duration of a chromatography run.

27. The system as claimed in any of claims 22 to 26, wherein the detector is a mass spectrometer.

28. The system as claimed in any of claims 22 to 27, and further comprising a UV detector downstream of the chromatography device.

29. The system as claimed in any of claims 22 to 28, and further comprising: a further chromatography device for separating the sample into component parts; an electronically actuable valve connected to an output of the further chromatography device; and a controller in communication with the valve and configured to operate the valve during the time window to allow the target component to be collected from a chromatography run carried out using the further chromatography device.

30. The system as claimed in any of claims 22 to 28 and further comprising: a plurality of further chromatography devices for separating the sample into component parts; a plurality of electronically actuable valves each connected to a respective output of a one of the plurality of further chromatography devices; and a controller in communication with the valves and configured to operate the valves during respective time windows to allow a plurality of target components to be collected from respective chromatography runs carried out using the further chromatography devices.

31. The system as claimed in claim 30, and further including an auto-sampler for introducing samples into the chromatography device sample streams.

32. The system as claimed in claim 30 or claim 31 and further comprising a robotic arm configured to automatically place empty receptacles adjacent an output of the valves to collect target components and to remove receptacles holding a target component.

33. The method as claimed in any of claims 1 to 21 , further comprising, collecting the sample during the collection window in a length of tubing and using detector data collected during the second chromatography run to determine the location of the target component in the length of tubing.

34. A method for isolating a component of a sample using a chromatography device substantially as hereinbefore described.

35. A chromatography system for isolating a target component of a sample substantially as hereinbefore described.