The present invention relates to the area of separation, such as liquid chromatography. More specifically, the invention relates to a mass-directed method of efficiently separating components of a liquid or solid sample; an automated such method and a computer capable of executing the method; as well as a system for mass-directed separating components of a liquid or solid sample.
Many diagnostic and preparative methods includes the detection of organic molecules from liquid samples obtained e.g. from a subject subjected to testing, such as a blood or urine sample, or obtained from a process of synthesis and/or manufacture. Liquid chromatography is a well-known and widely applied method for any preparative or diagnostic separation.
Flash chromatography is a chromatographic technique that has been used for decades, which uses a column or cartridge filled with an insoluble solid support, known as the stationary phase, and elution solvent mixture, known as the mobile phase, to separate and purify a mixture of organic compounds. The stationary phase and the mobile phase typically have very different polarities, which work in tandem to separate compound mixtures.
It is well-known in flash chromatography to use various non-destructive detectors, such as UV detectors, and/or destructive mass detectors, such as Evaporative Light Scattering Detectors (ELSD) or mass spectrometers (MS), to detect the separated molecules in the mobile phase and direct an automated fraction collector to collect the separated molecules in separate vials based on signals from the detectors. The separated organic compounds may find subsequent use in various applications, such as synthesis or as a final product.
When mass spectrometers are used as detectors in automated flash chromatography, a total ion current (TIC) chromatogram is commonly generated and represents the summed intensity across the entire range of masses being detected at every point in the analysis. Alternatively, an extracted-ion chromatogram (XIC, sometimes denoted EIC) is generated, wherein one or more m/z values representing one or more analytes of interest are recovered from the entire data set for a chromatographic run. The total intensity or base peak intensity, within a mass tolerance window around a particular analyte's m/z value is plotted at every point in such XIC (EIC) analysis.
However, in complex samples, the information for individual species may be obscured by background noise primarily caused by the solvent in the resulting chromatogram. Thus, there is a need in this area to provide new methods with improved resolution. In conventional mass-directed flash chromatography, a preparation step preceding the actual separation is commonly performed in order to identify each mass of interest. However, this is a user intensive and time-consuming step.
US 2014/0312216 (Advion Inc.) relates to signal processing for mass-directed fraction collection, and more specifically to a method for removing noise from a mass spectrometer signal in order to improve collection of fractions of an eluent from a chromatography column. The method is described as broadly applicable to any form of liquid chromatography, as exemplified by reverse phase chromatography, normal phase flash chromatography and supercritical fluid chromatography. The patent application also describes a system for performing fraction collection wherein a raw signal is obtained by analyzing an eluent from a chromatography column by mass spectrometry; a baseline signal is calculated based on said raw signal; and a processed signal, which is used in the subsequent fraction collection, is generated by subtracting the baseline signal from the raw signal. Thus, the described noise reduction is achieved by an analysis performed on the eluent from the chromatography column and directly preceding the actual chromatographic separation of analytes, i.e. in the same chromatographic run. Thus, the proposed method is proposed for isocratic elution, and does not appear to be operable for gradient chromatography. Further, as the method presupposes that the noise reduction calculation is completed on pure solvent, it will be important to be aware of exactly when the analyte breakthrough into the eluent occurs. Consequently, the method proposed in US 2014/0312216 may be difficult to successfully apply to small scale chromatography columns.
The present invention relates to a method of mass-directed separation of two or more components, which may provide improved resolution and hence improved fraction collection.
More specifically, the present invention may substantially reduce or even eliminate noise which is commonly caused by baseline drift due to solvent effects.
This may be achieved by a method wherein a solvent gradient defined for an intended separation is passed to a mass spectrometer in a step preceding an actual sample separation in order to determine a solvent noise level, which will be specific for each such solvent gradient.
Thus, the present invention may eliminate the need for commonly used time-consuming preparation steps for determination of the individual masses of each potentially interesting compound present in the sample.
In addition, in the method according to the present invention, the risk of sample impact on the detection of noise has been substantially eliminated.
A TIC (Total Ion Chromatogram) is a chromatogram created from the summed intensities across the entire range of masses being detected at every point in an MS analysis, as a plot of the total ion current vs. retention time. In conventional use, a TIC includes background noise as well as sample components.
In an extracted-ion chromatogram (XIC or EIC), one or more m/z values representing one or more analytes of interest are extracted from the entire data set for a chromatographic run.
In a first aspect, the present invention relates to a method for automated mass-directed separation of two or more components of a sample by flash chromatography. A system suitable for executing the method according to the invention is schematically illustrated in
The method is illustrated in the flowchart of
The predetermined range of m/z values are typically set by the manufacturer of the system for automated mass-directed separation and typically relate to the characteristics of the mass spectrometer, for example the resolution in m/z. The range comprises of a predetermined number of discrete sections in m/z. The discrete sections in m/z may be of equal length in m/z, but it could also be envisaged to have sections of different length in m/z, if for example a portion of in the m/z range is believed to carry more information than other portions. As indicated the predetermined range of m/z values is typically part of the basic configuration of the system for automated mass-directed separation, however a skilled user of the system may be given the option to modify the range of predetermined range of m/z values including the discrete sections in m/z to further tailor and optimize a separation process. Whether the predetermined range of m/z values including the discrete sections in m/z is part of the basic configuration or modified by the user the predetermined range of m/z values including the discrete sections in m/z must be kept the same in the step b) determining a solvent noise level and the following steps d-f relating to fractioning the sample.
The step b) of determining a solvent noise level comprises the substeps of:
b:1) Enter a pre-run phase;
b:2) directing a portion of the defined solvent gradient to the mass spectrometer;
b:3) for each discrete section in m/z:
b:4) determine a scalar value tn representing the noise level for the defined solvent gradient in a corresponding discrete section in m/z. According to one embodiment of the invention the scalar value tn is chosen as the maximum value in intensity for the corresponding discrete section n in m/z. According to one embodiment the scalar value tn is chosen a predetermined value or predetermined percentage above the maximum value in intensity. This would be to ensure that absolutely no noise from the defined gradient solvent could affect for example a TIC. It could also, for certain application wherein some noise could be acceptable, be suitable to choose a scalar value tn a predetermined value or predetermined percentage below the maximum value in intensity. Common measures to treat experimental data, for example to identify and exclude obvious outliers or the like may also be utilized.
b:4) exit the pre-run phase and entering a chromatography phase.
The step b) of determining a solvent noise level may be seen as pre-run. According to one embodiment of the method of the invention it is a fully automatic comprising that the step of directing b:1) is performed by controlling a selector to mix the defined solvent gradient with the mass spectrometer solvent and feed it to the mass spectrometer, whereby during the pre-run phase the chromatograph is bypassed.
In step e), a total value of all intensities received may be used to create a total ion chromatogram (TIC). Alternatively, a selection of signals is used to create an extracted-ion chromatogram (XIC/EIC). As the skilled person will appreciate, resulting signals can never be negative, hence the criteria of (t′1−t1)≥0, (t′2−t2)≥0, (t′3−t3)≥0 etc. must be fulfilled.
The solvent gradient of step a), which will include a gradually changing ionic strength, will be easily defined by the skilled person in this area following well known principles in the area of chromatography, including considerations related e.g. to the refraction index of the sample, the packing of the column used etc. Examples of solvents commonly used in flash chromatography include ethyl acetate, hexane, dichloromethane, acetone etc.
Further, the gradient signals generated in step b) may be used as such as a linear gradient, in which case in step c), a solvent gradient defined according to step a) i.e. identical, or substantially identical to the defined gradient, is passed across the column.
Alternatively, in step c), only a part of the defined gradient may be used in a shorter linear gradient or in a non-linear gradient, such as a step-wise gradient.
The slope of the gradients used according to the invention may be anything from a steep gradient to a substantially flat gradient, depending on the intended separation.
The separation according to the invention may be liquid chromatography, such as FLASH chromatography. The chromatography column used may be any commercially available and conventionally packed column, such as a silica column in case of FLASH chromatography. The skilled person is able to select the appropriate materials and methods for an intended separation.
The sample may be applied to the column in advance or just before the separation, and it may be a dry sample, or a liquid sample. More specifically, the sample may be applied as neat, simply applied as is onto the column; in solution in a suitable solvent; or deposited dried sample. Chromatography columns are available which have allocated spaces of suitable size for such sample application.
The method according to the invention is advantageously used for combined identification and purification of substances and/or compounds. The present invention may be used in relation to any target which may successfully be subjected to mass spectrometry, such as organic molecules including nucleic acids and peptides of suitable sizes. As flash chromatography is commonly used in the detection of substances in biological samples, such as disease markers or drugs, the sample may originate from blood, urine, saliva etc.
As used in the present context, the term “automated” separation means that at least one step is automated. Advantageously, all steps are automated, and the method of the invention may be referred to as “fully automated”. One or more steps of the present method may be controlled by a computer.
Thus, in a second aspect, the present invention relates to software i.e. a computer program, which may be provided on any suitable data carrier, and which is capable of executing at least one automated step of the present method, such as all steps. The invention also embraces a computer programmed to perform one or more steps of the method according to the invention.
A computer program according to the invention may comprise computer-executable instructions for causing a system to perform a method according to the invention. Thus, more specifically, a computer program product is provided according to the present invention, which comprises a memory and/or a computer-readable storage medium, the computer-readable storage medium having the computer program described above embodied therein. The memory and/or computer-readable storage medium referred to herein may comprise of essentially any memory, such as a ROM (Read-Only Memory), a PROM (Programmable Read-Only Memory), an EPROM (Erasable PROM), a Flash memory, an EEPROM (Electrically Erasable PROM), or a hard disk drive.
In a third aspect, the present invention relates to a system for performing automated mass-directed separation of two or more components of a sample, such as liquid chromatography, e.g. flash chromatography.
A system according to the invention may comprise a chromatography column, configured to hold any kind of chromatography packing, as discussed above. A system according to the invention may further comprise one or more containers configured to hold the appropriate solvent(s) for providing a liquid gradient of changing composition of said solvent(s). In order to facilitate the preparation of a liquid gradient of solvents, a system according to the invention may further comprise a selector configured to receive the solvent(s), and to pass on the appropriate proportions thereof to the column. The selector may e.g. be in the form of a manual valve or an actuator valve controllable by a control signal.
A system according to the invention may further be linked to, or comprise, a mass spectrometer (MS). As the skilled person will appreciate, the present invention may utilize any conventional mass spectrometer which is commonly used in combination with liquid chromatography, such as FLASH chromatography. The MS used according to the present invention may e.g. be a quadropole mass spectrometer.
The skilled person is also capable of combining the mass spectrometer with a conventional fraction collector in order to collect one or more targeted sample components, which according to the present invention will be obtained with higher purity than any of the prior art methods.
Thus, the present invention uses conventional elements linked to a conventional mass spectrometer, but suggests for the first time to reduce noise caused by solvents in the mass spectrometer by determining the noise level resulting from a specific gradient in a step preceding an actual separation of components of a sample. Differently worded, according to the present invention, a defined solvent gradient is subjected to mass spectrometry in a pre-run in order to define the signals it generates during mass spectroscopy. As such gradient signals would constitute noise if applied in a subsequent step for eluting targeted components from a chromatography column, gradient signals are proposed according to the invention to be subtracted from the sample signals obtained in a subsequent step of chromatography.
As the skilled person will appreciate, the pre-run according to the invention is used to obtain signals for the relevant concentrations used in a gradient, while there is no requirement for using the same flow rate in the pre-run as in a subsequent chromatography step. Thus, an advantage of the present pre-run is that it may be quicker than traditional ‘calibration runs’ performed on the relevant packed chromatography column.
As also appreciated by the skilled person, a pre-run according to the invention may run a gradient in either direction; either starting with the same composition as planned for a subsequent chromatography step, or, alternatively, by starting with the planned final composition and run until the planned starting composition is achieved.
As the skilled person will appreciate, a splitter type of valve may advantageously be used downstream of the column in order to combine an eluent from the column, or a liquid gradient which has bypassed the column, with conventional mass spectrometer solvent. The valve or splitter should be controllable by a control signal. An illustrative valve useful as a splitter device in the method and system according to the invention is described in U.S. Pat. No. 5,803,117.
Thus, more specifically, the present invention may be a system for performing automated mass-directed separation of two or more components, which system, schematically illustrated in
A system according to the invention may further comprise a processor configured to (i) subtract a set of gradient signal, obtained when the downstream valve is set to direct solvent flow via the bypass loop towards the splitter device, which is set to combine said flow with MS solvent and to direct the combined flow to the MS, from a set of sample signals, obtained when the downstream valve is set to pass eluate exiting the chromatography column to the splitting device, which is set to direct at least a part of said eluate towards the MS and a part of said eluate to the fraction collector; and to (i) instruct the operation of said fraction collector based on a spectrum generated using the resulting signals.
More specifically, a control unit used according to the present invention may be an Electronic Control Unit (ECU) or an on-board computer. The present control unit may comprise a processor communicatively coupled to a transceiver for wired or wireless communication. In one example, the processor may be any one of a selection of processing circuitry and/or a central processing unit and/or processor modules and/or multiple processors configured to cooperate with each-other. Further, the control unit may advantageously comprise a memory. The memory may contain instructions executable by the processor to perform any one of the herein described or indicated methods. The processor may be communicatively coupled to a selection of any of the transceiver and the memory.
A system according to the invention is advantageously used for combined identification and purification of compounds, such as the ones discussed above.
Number | Date | Country | Kind |
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17161347.4 | Mar 2017 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/SE2018/050247 | 3/14/2018 | WO | 00 |