Patent Application
20140312216
Liquid chromatography (LC) is a chromatographic technique used to physically separate mixtures of compounds in many areas of research including synthetic organic chemistry and biochemistry. Liquid chromatography can be used to isolate, purify, identify and quantify individual components. These components can be measured on line by a variety of detectors, such as UV and Mass Spectrometers, and can be also be isolated and collected by fraction collection devices that are triggered by the detector. There are different types of liquid chromatography that are used depending on the properties of the sample that is being separated, and a wide range of flow rates that are encompassed, depending on the quantity of compound to be separated. These chromatographic techniques include reverse phase liquid chromatography (often called HPLC, UHPLC or prep-LC), normal phase flash chromatography (NPFC) and supercritical fluid chromatography (SFC).
A system and removing noise from a mass spectrometer signal for fraction collection of LC eluent is described herein.
In some aspects, a method of performing fraction collection, the method comprising delivering an eluent containing a substance of interest from a liquid chromatography system, directing the eluent through a splitter device to cause a first portion of the eluent to be directed to a mass spectrometer and a second portion of the eluent to be directed to a collection device, analyzing the eluent using the mass spectrometer to obtain a raw signal, processing the raw signal in real time to generate a processed signal prior to the eluent corresponding to the processed signal reaching the collection device, the processed signal removing at least some noise from the raw signal, and selecting portions of the eluent to collect by the collection device based on the processed signal.
Embodiments can include one or more of the following.
Processing the raw signal to generate a processed signal can include during a first time period, calculating a baseline signal based on the raw signal and during time periods after the first time period, subtracting the baseline signal from the raw signal to generate the processed signal.
Processing the raw signal to generate a processed signal can include applying a triangle filter to the raw signal to generate the processed signal.
Processing the raw signal to generate a processed signal can include applying a box filter to the raw signal to generate the processed signal.
Processing the raw signal to generate a processed signal can include applying a Gaussian filter to the raw signal to generate the processed signal.
Processing the raw signal to generate a processed signal can include applying a Savitzky Golay filter to the raw signal to generate the processed signal.
The method can also include providing the processed signal to the fraction collection device.
Providing the processed signal to the fraction collection device can include providing the processed signal to the fraction collection device within 5 seconds of generating the raw signal.
Providing the processed signal to the fraction collection device can include providing the processed signal to the fraction collection device within 10 seconds of generating the raw signal.
Processing the raw signal to generate a processed signal can include processing the raw signal in real time.
Processing the raw signal to generate a processed signal can include concurrently processing portions of the raw signal while the mass spectrometer is collecting later portions of the raw signal.
Like reference symbols in the various drawings indicate like elements.
When system 10 is used in a liquid chromatography/mass spectrometry (LC/MS) fraction collection mode, an LC system 12 (in this example an SFC system) is coupled to a fraction collection device 16, which interfaces with a mass spectrometer 14 that performs spectral analysis. In use, an effluent provided by the LC system 12 is directed through a column 19 using a methanol pump 11 and a carbon dioxide pump 13. The column 19 retains individual components by the stationary phase differently and separates the different components from each other while they are running at different speeds through the column with the eluent. At the end of the column 19 they elute one at a time (e.g., the analytes of interest enter the fraction collection device 16 one at a time). More particularly, components of the sample move through the column 19 at different velocities, which are function of specific physical interactions with the sorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. Thus, by using a chromatography process the eluent is directed from the column 19 in a series of fractions.
The composition of the eluent flow is monitored using the mass spectrometer 14, which analyzes each fraction for dissolved compounds. In order to collect the desired fractions, the eluent is split post column 19 by a splitter device 18 such that a portion is directed to an interface to the mass spectrometer 14 while the remainder of the split eluent is collected at time segments into collection devices in the fraction collector such as flasks, tubes, multi-well plates. The splitting device may be a simple passive split where the split ratio is defined by the resistance to flow on either side of a ‘T’, or it might be an active splitting device such as a repetive switching valve (e.g., valve 17 shown in
In system 10, the mass spectrometer 14 generates a signal and sends the signal to the fraction collection device 16. The fraction collection device 16 uses the received signal in order to identify when the fraction of interest should be collected. Thus, by identifying a peak in the signal from the mass spectrometer 14, the fraction collection device 16 can determine when to collect the fraction of interest. More particularly, the fraction collection device 16 can divert the liquid into a collection container upon identifying the rising edge of the signal from the mass spectrometer and can stop diverting the liquid into the collection container upon identifying the falling edge of the signal from the mass spectrometer (e.g., the liquid can then be diverted to a waste container or other collection device). As shown in
The systems and methods described herein include software and processes for removing noise from the signal generated by the mass spectrometer 14 in real time, such that a processed signal 28 can be sent to the fraction collection device 16 and used to determine when to collect the desired sample. In some examples, the processed signal can be slightly delayed as compared to the signal generated by the mass spectrometer as indicated by arrow 27. For example, the filtered signal can be delayed by less than 10 seconds (e.g., less than 10 seconds, less than 8 seconds, less than 5 seconds, less than 4 seconds) from the acquisition time of the signal by the mass spectrometer. As such, the signal is received by the fraction device within 10 seconds (e.g., within 10 seconds, within 8 seconds within 5 seconds, within 4 seconds) from the measurement of the unprocessed/raw signal by the mass spectrometer. Thus, while filtering the signal does introduce a small amount of delay in providing the signal to the fraction collection device 16, this delay does not hamper collection of the desired fraction because the fluid path between the splitter 18 and the collection receptacle is designed to introduce a longer delay between the splitter and the output than the amount of time used to process the signal collected by the mass spectrometer 14. Triggering based on the processed signal 28, rather than the originally collected signal 26 can provide the benefit of improving the recovery rate for purification because the system can start and stop collection more accurately.
In the example shown in
Referring to
The LC system 12 begins delivery of the fluid through the splitter 18 and a portion of the fluid is directed to the mass spectrometer 14 (34). When the mass spectrometer begins measuring a signal, an internal clock can be initialized to track the timing for the received time window. The mass spectrometer 14 collects and caches the mass spectrometry signal (36). For each scan of the acquisition, a raw analog output signal value is calculated from the TIC or XIC specification, as requested by the user. The total ion current (TIC) chromatogram represents the summed intensity across the entire range of masses being detected at every point in the analysis. In an extracted ion chromatogram (XIC or EIC), one or more m/z values representing one or more analytes of interest are recovered (‘extracted’) from the entire data set. The TIC or XIC signal includes any noise that is measured by the mass spectrometer 14. To generate the processed signal, the mass spectrometer 14 sets the processed signal equal to zero for times prior to the end of the time window used for baseline determination (38). When the mass spectrometer has collected the mass spectrometry signal for the entire time of the user-defined baseline collection time window, the mass spectrometry system 14 calculates a baseline signal (40). The baseline signal can be calculated as the average of the signal values during the user-defined time window. For signals collected subsequent to the end of the user-defined time window, the system calculates a value for the processed signal value by subtracting the baseline signal value from the measured signal value (42). Thus, by subtracting the baseline signal value the entire processed signal curve is shifted to remove/zero out the contribution of the baseline signal.
Based on the process 30 above, the processed signal includes three portions. In a first portion which includes retention times before the specified baseline calculation start time, the processed signal is set to zero. For a second portion which includes retention times after the specified baseline calculation start time, all cached values having retention times within the specified window are averaged to determine the baseline value. The value for the processed signal during this time period is also set to zero. The third portion which includes retention times after the specified baseline calculation time, the processed signal equals the raw/measured signal minus the calculated baseline value. If the result is negative, the processed signal is set to zero.
In some examples, the same compound is repeatedly purified. In such examples, the timing of multiple, different peaks that are observed as the eluent is delivered from the column 19 could be generally known. In such cases, because the baseline signal can drift or vary over time, a new baseline signal can be calculated and subtracted from the acquired signal multiple different times. For example, at time between adjacent peaks, the baseline can be recalculated such that a new baseline signal would be subtracted from the acquired signal.
The LC system 12 begins delivery of the fluid through the splitter 18 and a portion is directed to the mass spectrometer 14 (54). When the mass spectrometer begins measuring a signal, an internal clock can be initialized to track the timing for the signal smoothing. The mass spectrometer 14 collects and caches the mass spectrometry signal (56). For each scan of the acquisition, a raw analog output signal value is calculated from the TIC or XIC specification, as requested by the user, and cached. The total ion current (TIC) chromatogram represents the summed intensity across the entire range of masses being detected at every point in the analysis. In an extracted ion chromatogram (XIC or EIC), one or more m/z values representing one or more analytes of interest are recovered (‘extracted’) from the entire data set. The TIC or XIC signal includes any noise that is measured by the mass spectrometer 14.
After acquiring the TIC or XIC signal, the mass spectrometer 14 processes the signal to remove a baseline signal if desired (58). For example, the baseline signal can be removed using a process such as the process described above in relation to
In one particular example, for each scan with a retention time before a specified smoothing time span, a smoothed value of zero is cached. For the first scan with retention time after the specified smoothing time span, the number of completed scans is noted and rounded down to the next odd integer. This is the filter width, or number of samples to be used when performing filtering. For each scan with a retention time after the specified smoothing time span (having scan index num), a Triangular Filter is applied over the previous width samples. This produces a smoothed signal value corresponding to scan index num-(width−1)/2. This value is cached for scan index num, effectively producing a signal delay of roughly half the requested smoothing time span. The appropriate cached value (processed or smoothed) is assigned to the electronics which produce an output voltage corresponding to the assigned value.
In the example above a triangle filter was used to generate the smoothed signal, however, other filters can be used. For example, a boxcar filter, simple averaging, a Gaussian filter, or a Savitzky Golay filter could be used. In general, the processed signal is produced in close to real time. For example, the processed signal is produced and provided to the fraction collection device with a signal delay of approximately half of the smoothing timespan.
Embodiments of the subject matter and the functional operations described in this specification can be implemented in digital electronic circuitry, in tangibly-embodied computer software or firmware, in computer hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. Embodiments of the subject matter described in this specification can be implemented as one or more computer programs, i.e., one or more modules of computer program instructions encoded on a tangible non-transitory program carrier for execution by, or to control the operation of, data processing apparatus. The computer storage medium can be a machine-readable storage device, a machine-readable storage substrate, a random or serial access memory device, or a combination of one or more of them.
The term “data processing apparatus” refers to data processing hardware and encompasses all kinds of apparatus, devices, and machines for processing data, including by way of example a programmable processor, a computer, or multiple processors or computers. The apparatus can also be or further include special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit). The apparatus can also optionally include, in addition to hardware, code that creates an execution environment for the computer programs in question, e.g., code that constitutes processor firmware, a protocol stack, a database management system, an operating system, or a combination of one or more of them.
A computer program (which may also be referred to or described as a program, software, a software application, a module, a software module, a script, or code) can be written in any form of programming language, including compiled or interpreted languages, or declarative or procedural languages, and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. A computer program may, but need not, correspond to a file in a file system. A program can be stored in a portion of a file that holds other programs or data, e.g., one or more scripts stored in a markup language document, in a single file dedicated to the program in question, or in multiple coordinated files, e.g., files that store one or more modules, sub-programs, or portions of code. A computer program can be deployed to be executed on one computer or on multiple computers that are located at one site or distributed across multiple sites and interconnected by a communication network.
The processes and logic flows described in this specification can be performed by one or more programmable computers executing one or more computer programs to perform functions by operating on input data and generating output. The processes and logic flows can also be performed by, and apparatus can also be implemented as, special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit).
Computers suitable for the execution of a computer program include, by way of example, can be based on general or special purpose microprocessors or both, or any other kind of central processing unit. Generally, a central processing unit will receive instructions and data from a read-only memory or a random access memory or both. The essential elements of a computer are a central processing unit for performing or executing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks. However, a computer need not have such devices. Moreover, a computer can be embedded in another device, e.g., a mobile telephone, a personal digital assistant (PDA), a mobile audio or video player, a game console, a Global Positioning System (GPS) receiver, or a portable storage device, e.g., a universal serial bus (USB) flash drive, to name just a few.
Computer-readable media suitable for storing computer program instructions and data include all forms of non-volatile memory, media and memory devices, including by way of example semiconductor memory devices, e.g., EPROM, EEPROM, and flash memory devices; magnetic disks, e.g., internal hard disks or removable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.
Although a few implementations have been described in detail above, other modifications are possible. In addition, the logic flows depicted in the figures do not require the particular order shown, or sequential order, to achieve desirable results. Other steps may be provided, or steps may be eliminated, from the described flows, and other components may be added to, or removed from, the described systems. Accordingly, other implementations are within the scope of the following claims.
