Patent Application
20140138526
Mass spectrometry (MS) is an analytical methodology used for quantitative elemental analysis of samples. Molecules (often referred to as analytes) in a sample are ionized and separated by a spectrometer based on their respective masses. The separated analyte ions are then detected and a mass spectrum of the sample is produced. The mass spectrum provides information about the masses and in some cases the quantities of the various analyte particles that make up the sample. In particular, mass spectrometry can be used to determine the molecular weights of molecules and molecular fragments within an analyte.
One type of mass spectrometry is time-of-flight (TOF) mass spectrometry. TOF mass spectrometry is used to form a mass spectrum for ions contained in a sample of interest. In a TOF mass spectrometer, ion packets are provided to a flight region of the instrument. The ions separate according to their mass-to-charge ratios, m/z, and their arrival times Ti are recorded. According to a known TOF method, a sample is divided into packets of ions that are launched along the propagation path using a pulse-and-wait approach. In releasing packets, one concern is that the lighter and faster ions of a trailing packet will pass the heavier and slower ions of a preceding packet. Using this so-called pulse-and-wait approach, the release of an ion packet is timed to ensure that the ions of a preceding packet reach the detector before any overlap can occur. Thus, the periods between packets are relatively long. If ions are being generated continuously, only a small percentage of the ions are actually detected. A significant amount of sample material is thereby wasted. The loss in efficiency and sensitivity can be reduced by storing ions that are generated between the launching of individual packets, but the storage approach carries some disadvantages.
The inefficiencies of the known pulse-and-wait methods have led to various techniques of sending ions in pulses from a stream of ions for a sample to the detector. In one known method, the ions are pulsed using a pseudo-random pulsing sequence. Because the pulsing sequence is known, the mass spectrum can be recovered by an inverse transform. One example of pseudo-random pulsing of ions in TOF mass spectrometry applications is dithered multi-pulsing, in which a small interval is added randomly to a nominal pulse interval. Measured peaks can be assigned to their respective pulses of origin by statistical analysis. Another example is Hadamard transform TOF, in which a continuous ion beam is modulated between two detectors according to a pulse sequence typically running at 10 kHz-1 MHz. This results in a so-called 100% duty-cycle measurement. It is possible, however, to modulate the continuous ion beam “on” and “off” at a single detector with 50% duty cycle. The resulting spectrum looks like noise, but the original mass spectrum can be recovered using the inverse Hadamard transform.
While these other known methods and attendant components do provide an improvement in efficiency over the pulse-and-wait methods, they generally require additional hardware and significant data processing resources. This results in a more complex TOF MS instrument. Moreover, by these known pulsing methods, artifacts may be introduced in the process of reconstructing lost information. As such, a unique solution is not guaranteed, and several different recovered mass spectra may be consistent with the measured data, which can result in reduced instrument accuracy.
What is needed, therefore, is a method and apparatus for analyzing ions that overcomes at least the drawbacks of the known methods described above.
The present teachings are best understood from the following detailed description when read with the accompanying drawing figures. The features are not necessarily drawn to scale. Wherever practical, like reference numerals refer to like features.
It is to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The defined terms are in addition to the technical and scientific meanings of the defined terms as commonly understood and accepted in the technical field of the present teachings.
As used in the specification and appended claims, the terms ‘a’, ‘an’ and ‘the’ include both singular and plural referents, unless the context clearly dictates otherwise. Thus, for example, ‘a device’ includes one device and plural devices.
As used in the specification and appended claims, and in addition to their ordinary meanings, the terms ‘substantial’ or ‘substantially’ mean to with acceptable limits or degree. For example, ‘substantially cancelled’ means that one skilled in the art would consider the cancellation to be acceptable.
As used in the specification and the appended claims and in addition to its ordinary meaning, the term ‘approximately’ means to within an acceptable limit or amount to one having ordinary skill in the art. For example, ‘approximately the same’ means that one of ordinary skill in the art would consider the items being compared to be the same.
In the following detailed description, for purposes of explanation and not limitation, representative embodiments disclosing specific details are set forth in order to provide a thorough understanding of the present teachings. Descriptions of known systems, devices, materials, methods of operation and methods of manufacture may be omitted so as to avoid obscuring the description of the example embodiments. Nonetheless, systems, devices, materials and methods that are within the purview of one of ordinary skill in the art may be used in accordance with the representative embodiments.
In accordance with representative embodiments described in more detail below, a method of determining the mass-to-charge (m/z) ratios of ions in a sample is disclosed. The method comprises providing a calibration sample comprising ions of differing m/z ratios to a TOF mass analyzer. The ion species in the calibration sample typically span a range of m/z ratios similar to what may be expected to be present within an unknown sample to be analyzed. The calibration data may also be extrapolated to allow measurements of unknown samples over a wider m/z range. The calibration sample often comprises chemical species chosen for long shelf life, low fragmentation during the mass spectrometer measurement, or other desirable characteristics. Moreover, the calibration sample may comprise molecules chosen to have properties similar to the molecules in the unknown sample (for example, similar collision cross sections as a function of m/z). Data from the calibration sample are acquired until an ion of the calibration sample having a greatest time of flight is incident on the detector. A peak width is determined for each of the ion species of the calibration sample after several TOF measurements of the calibration sample are summed, to provide calibration data of the peak width versus flight time for the calibration sample. Next, a data acquisition time is determined. As described more fully below, the data acquisition time is a fraction of the greatest time of flight. After determining the data acquisition time, ions from a continuous ion beam of a sample are provided to the TOF mass analyzer at pulse intervals having a duration equal to the predetermined fraction of the greatest time of flight. Next, a peak width and a flight time value are determined for each of the ion species in the sample after summing the data acquired during several pulse intervals. Notably, a scaled background spectrum of known contaminant ions may optionally be subtracted before determining the peak width values of the ion species in the sample. The measured flight time values are corrected by correlating the measured peak width values with the calibration data of peak width versus flight time, and the corrected flight time values are converted into the m/z ratios. As described below, the present teachings contemplate instantiation of the method in a non-transitory computer-readable medium, or in hardware, or in firmware, or a combination thereof.
The mass spectrometer 100 is generally a TOF mass spectrometer and comprises an ion source 101, an ion accelerator 102, a mass analyzer 103 and a detector 104. The mass spectrometer 100 also comprises an ion pulser (“pulser”) 105 coupled to the ion accelerator 102. Pulsed voltages are applied in the pulser 105, and ions are pulsed from the ion accelerator 102 to the mass analyzer 103 at desired pulse intervals, as described more fully below. The mass analyzer 103 is a TOF mass analyzer. The ion source 101, ion accelerator 102, mass analyzer 103 and detector 104 are known components used in TOF mass spectrometry. For example, many of the components of the mass spectrometer 100 are provided in instruments commercially available from Agilent Technologies. Inc. Santa Clara, Calif. (USA). As such, many of the details of these known components are not presented herein to avoid obscuring the description of representative embodiments.
The mass spectrometer 100 also comprises a controller 106 that receives an input 107 and commands from the controller 106, and drives the output of the pulser 105. Ultimately, the controller 106 sets the duration and frequency of ion pulses provided to the mass analyzer 103 and detector 104 according to aspects of the methods of representative embodiments. The controller 106 may be a dedicated component, or the controller of the instrument embodying the mass spectrometer.
The mass spectrometer 100 also comprises a processor 108 and a memory 109. The processor 108 is configured to determine the m/z ratios according to the methods of representative embodiments described below. The processor 108 may be implemented using a processing device, such as processor/microprocessor, a digital signal processor (DSP), one or more application specific integrated circuits (ASICs), one or more field-programmable gate arrays (FPGAs), or combinations thereof, using software, firmware, hard-wired logic circuits, or combinations thereof. When using a computer processor and/or a DSP, for example, the memory 109 stores executable software/firmware and/or executable code that allows the processor 108 to perform the various functions of the methods of the representative embodiments. The memory 109 may be a non-transitory computer readable medium, and may include any number, type and combination of random access memory (RAM) and non-volatile memory (e.g., read-only memory (ROM)), for example. Finally, it is contemplated that the controller 106 and the processor 108 are a single component associated with memory 109. This single component could be a dedicated processor of the instrument that embodies the mass spectrometer 100. As will become clearer as the description continues, input 107 to the controller 106 can be provided from the processor 108 and memory 109. Accordingly, a single component configured to implement the methods of the representative embodiments is contemplated.
As ion packets travel through the TOF mass spectrometer, their arrival time at the detector varies according to mass. Moreover, the longer the time ions spend in the instrument, the greater the spread in arrival times for ions of the same mass becomes. The measured peak width (defined as full-width at half maximum (FWHM)) for an ion species is given by ΔTi. Generally, a given ion species in a TOF experiment ΔTi can be represented by:
ΔT=(ΔT02+ΔTm2)1/2
where ΔT0 is a fixed peak broadening term, and ΔTm is a peak broadening term that is proportional to the mass of the ion. ΔT0 can be impacted by factors such as the detector impulse response width and jitter in the timing of the high voltage pulse that launches the ion packet, as well as timing errors in the data acquisition system. ΔTm comprises mass dependent aberrations including imperfect alignment of the ion optics, mechanical vibrations, turn-around time arising from the initial velocity spread of the ions, power supply noise, collisions with gas molecules in the high vacuum region, etc. For small mass-to charge ratios, m/z, the pulse width is limited by the impulse response of the detector, ΔT0.
Generally, each mass spectrometer will have a characteristic curve for the peak width versus time of arrival. Once this relationship is determined, it can be used as a calibration curve in accordance with the methods of the representative embodiments described below. According to one aspect of the method the calibration curve for a given instrument can be determined by measurements of ΔT versus time of arrival at the detector by multiple pulse-and-wait measurements. Moreover, once the calibration curve is determined, it can be interpolated and extrapolated to determine ΔT for ion peaks not specifically determined through the calibration.
In accordance with a representative embodiment, the ions are provided by the ion accelerator 102 in a pulsed manner to the mass analyzer 103 at a fixed repetition rate based on the ion of the sample having the greatest time of flight. Notably, the fixed repetition rate is merely illustrative, and as described below, the pulse interval is otherwise set. In the present illustration, the greatest time of flight of an ion in the calibration sample to reach the detector is 469 μs (m/z 2122). It is contemplated that the unknown sample may contain ions with higher m/z species than the calibration sample. In the present illustration, it is desired to measure unknown samples potentially containing ions with m/z≦3150. Consequently, the greatest time of flight in the sample (572 μs) is longer than the greatest time of flight of a calibration ion. Furthermore, for certain samples, the greatest time of flight of an ion in the sample may be shorter than the greatest time of flight of the calibration data. Instruments in accordance with the present teachings typically offer comparatively high and comparatively low mass ranges (e.g. 3200 amu and 1700 amu, respectively) for liquid chromatography, and mass ranges in gas chromatography can be even lower. As such, it can be beneficial to use a single calibrant mixture that covers the widest mass range needed rather than maintain separate calibrants.
The pulser 105 is configured to command the ion accelerator 102 to provide pulses of ions from a stream of ions from the ion source 101 at a rate that is a fraction of the greatest time of flight. Continuing with this illustration, the ion accelerator 102 provides ions to the mass analyzer 103 (and ultimately to the detector 104) at an interval that is approximately one-fourth the greatest time of flight of the unknown sample or every 143 μs. As such, the mass spectrometer 100 is configured to provide ions at a rate that is four times faster than would be allowed for a known pulse-and-wait or non-interleaved mass spectrometer measurement of m/z≦3150.
As described more fully below, ion arrival events arising from several consecutive pulses are assigned to an appropriate flight time based on their measured peak widths after the data from multiple pulses are summed at the processor 108 to provide a spectrum. Beneficially, the present teachings make use of information present in the overlapped mass spectrum when summed over multiple transients. As such, a greater number of ions (in this example four times as many) are provided to the detector 104 than would be provided in the known pulse-and-wait or non-interleaved TOF instrument. It is emphasized that the pulse rate/frame period described above is merely illustrative, and other pulse rates/frame periods are contemplated according to the present teachings.
Returning to
As noted above, the sample used to provide the calibration data can be a sample with known chemical species, or may be an unknown sample, such as a portion of the actual sample to be analyzed. As noted above, calibration will be routinely checked with the standard calibration sample (tuning mix). In the case that calibration fails (e.g., due to the presence of large intact proteins, where ΔTm is larger than it is in the tuning mix data), some of the unknown sample data can be used to self-calibrate in a crude way. Specifically, the measured (aliased) spectrum can be reviewed from shortest time of flight to greatest time of flight (i.e., left to right on the ordinate of
After the calibration data are acquired, a new pulsing interval equal to a fraction of the greatest time of flight is determined, as will be described later. In the present illustration, the fraction is one-fourth of the greatest time of flight, or 143 μs. This value represents the data acquisition time for TOF mass spectrometer 100. As such, the ion accelerator 102 provides a packet of ions every 143 μs, and peak width/flight time data are acquired for an unknown sample. The difference between the true and aliased arrival times (the length of the arrows between calibration data points 201˜208 and data points 213˜219) is exactly a multiple of the 143 μs pulsing interval. First through fourth frames 209˜212 are depicted, and are useful in the explanation of method of representative embodiments.
After the calibration curve 200 is determined, multi-pulsing begins at the selected pulse rate, which in this case is 143 μs. Data points 213˜219 are acquired over the data acquisition time (four frames), which in this example is 572 μs. Notably,
Note that
Data point 213 has a measured flight time of approximately 40 μs and a peak width of approximately 1.1 ns. However, as noted above, no ion from a first pulser firing could have reached the detector 104 in only 40 μs and have a peak width greater than that of calibration data point 201. So, data point 213 must have been a slower moving ion from a pulse prior to the pulse at T=0 μs. As such, one or more frame lengths must be added to data point 213 to determine its actual arrival time for the measured peak width. Notably, the data point 213 has the same peak width as data point 202 and maps to this point (data point 202) on the calibration curve 200. As such, according to the present teachings data point 213 is determined to have a peak width of 1.1 ns and a flight time to the detector of approximately 40 μs plus one frame period (143 μs) or 183 μs. As such, it is determined that the data point 213 is from first frame 209, but arrives in frame 210.
Data point 214 has a measured peak width of approximately 1.4 ns and a flight time of approximately 100 μs. As noted above, the minimum peak width for the sample under measurement is approximately 1 ns (calibration data point 201). As such, the measured flight time of data point 214 cannot be its actual flight time, and data point 214 represents the peak width of an ion from an earlier pulse frame. Mapping data point 214 to the calibration curve 200 reveals its true flight time is approximately 240 μs. So, by adding one frame period to data point 214 the actual arrival time of an ion having a peak width of approximately 1.4 ns (data point 203) is determined. As such, it is determined that the data point 214 is from first frame 209, but arrives in frame 210.
Data point 215 has a peak width of approximately 1.6 ns and measured arrival time of approximately 20 μs. Again, the minimum peak width for the sample under measurement is approximately 1 ns (calibration data point 201), so the measured flight time cannot be the actual flight time and data point 215 represents the peak width of an ion from an earlier pulse frame. However, if one frame period is added, there is no corresponding data point on the calibration curve 200, so another frame period must be added. By adding two frame periods, an ion with peak width of approximately 1.6 ns (data point 204) is found on the calibration curve 200. Mapping data point 215 to the calibration curve 200 reveals its true flight time is approximately 310 μs. So, by adding two frame periods to data point 215 the actual arrival time of an ion having a peak width of approximately 1.6 ns is determined. As such, it is determined that the data point 215 is from first frame 209, but arrives in frame 211.
Data point 216 has a peak width of approximately 1.8 ns and measured arrival time of approximately 70 μs. Again, the minimum peak width for the sample under measurement is approximately 1 ns (calibration data point 201), so the measured flight time cannot be the actual flight time and data point 216 represents the peak width of an ion from an earlier pulse frame. However, if one frame period is added, there is no corresponding data point on the calibration curve 200, so another frame period must be added. By adding two frame periods, an ion with peak width of approximately 1.8 ns (calibration data point 205) is found on the calibration curve 200. Mapping data point 216 to the calibration curve 200 reveals its true flight time is approximately 360 μs. So, by adding two frame periods to data point 216 the actual arrival time of an ion having a peak width of approximately 1.8 ns is determined. As such, it is determined that the data point 216 is from first frame 209, but arrives in frame 211.
Data point 217 has a peak width of approximately 2.1 ns and measured arrival time of approximately 100 μs. Again, the minimum peak width for the sample under measurement is approximately 1 ns (calibration data point 201), so the measured flight time cannot be the actual flight time and data point 217 represents the peak width of an ion from an earlier pulse frame. However, if one frame period is added, there is no corresponding data point on the calibration curve 200, so another frame period must be added. By adding two frame periods, an ion with peak width of approximately 2.1 ns is found (data point 206) on calibration curve 200. Mapping data point 217 to the calibration curve 200 reveals its true flight time is approximately 390 μs. So, by adding two frame periods to data point 217 the actual arrival time of an ion having a peak width of approximately 2.1 ns is determined. As such, it is determined that the data point 217 is from first frame 209, but arrives in frame 211.
Data point 218 has a peak width of approximately 2.2 ns and measured arrival time of approximately 0 μs. Plainly, an ion with a peak width greater than ΔT0 could not arrive at the detector almost instantaneously after firing from the ion accelerator 102. Again, the minimum peak width for the sample under measurement is approximately 1 ns (calibration data point 201), so the measured flight time cannot be the actual flight time and data point 218 represents the peak width of an ion from an earlier pulse frame. However, if one or two frame periods are added, there is no corresponding data point on the calibration curve 200, so another frame period must be added. By adding three frame periods, an ion with peak width of approximately 2.2 ns is found (data point 207) on calibration curve 200. Mapping data point 218 to the calibration curve 200 reveals its true flight time is approximately 430 μs. So, by adding three frame periods to data point 218 the actual arrival time of an ion having a peak width of approximately 2.2 ns is determined. As such, it is determined that the data point 218 is from first frame 209, but arrives in frame 212.
Data point 219 has a peak width of approximately 2.4 ns and measured arrival time of approximately 40 μs. Again, the minimum peak width for the sample under measurement is approximately 1 ns (calibration data point 201), so the measured flight time cannot be the actual flight time and data point 219 represents the peak width of an ion from an earlier pulse frame.
However, if one or two frame periods are added, there is no corresponding data point on the calibration curve 200, so another frame period must be added. By adding three frame periods, an ion with peak width of approximately 2.4 ns is found (data point 208) on calibration curve 200. Mapping data point 219 to the calibration curve 200 reveals its true flight time is approximately 470 μs. So, by adding three frame periods to data point 218 the actual arrival time of an ion having a peak width of approximately 2.2 ns is determined. As such, it is determined that the data point 219 is from first frame 209, but arrives in frame 212.
While the description of the determination of actual flight times of data points 213˜219 of
When low-abundance ion species in the unknown sample give rise to peaks below the height threshold described above, it is still possible in some cases to assign these peaks correctly based on criteria other than peak width. Some of these alternative criteria are described below.
As alluded to previously, data points 201, 213˜219 are acquired over a large number of pulses of ions sent through the mass spectrometer. Beneficially, the data acquired contain many times (in this case four times) the amount of analyte signals that can be acquired using the known pulse-and-wait TOF measurements in the same measurement period. In the case of sparse spectra containing few peaks, the increase in the measured signal may be 10×-20× higher than the pulse-and-wait approach (and frame periods correspondingly lower) without causing the determination of peak widths to fail due to overlapping peaks. The present teachings can thus be beneficially applied in combination with gas or liquid chromatography or other techniques that spread out the arrivals of the ion species over a separation time. The present teachings may also be beneficially applied to high resolution TOF-MS because with higher resolving power it is possible to simultaneously measure a greater number of closely spaced peaks without overlaps.
The method of the representative embodiment described in connection with
Other constraints can be used in the determining of m/z ratios of ions in a sample according to the present teachings. These constraints may be particularly useful in cases where the peak width cannot be correctly determined (for example, peaks comprising only a single ion or very few ions). If the ion source 101 is configured to provide only a limited range of m/z ions, then any flight time corresponding to a m/z ratio outside the transmission window may be rejected. For example, the quadrupole in a QTOF instrument might be configured with a low mass cutoff that rejects ions with m/z<50, thus allowing the determination that an ion with an early measured flight time cannot belong to the packet launched at time T=0 μs. Furthermore, in
An example of using isotopic peaks as an additional constraint is shown in
The method 500 is a method for determining the mass-to-charge ratios of ions in a sample.
At 501, the method comprises providing a calibration sample comprising ions of different mass-to-charge ratios to a time-of-flight mass analyzer and acquiring data until an ion with a greatest time of flight is incident on an ion detector.
At 502, the method comprises determining a peak width for each ion species of the calibration sample after summing several time-of-flight measurements of the calibration sample to provide calibration data of peak width versus flight time.
At 503, the method comprises determining a data acquisition time, the data acquisition time being a predetermined fraction of the greatest time of flight.
At 504, the method comprises providing ions from a continuous beam of a sample to a time-of-flight mass analyzer at pulse intervals having a duration equal to the predetermined fraction of the greatest flight time.
At 505, the method comprises measuring a peak width and a flight time value for each of the ion species in the sample after summing the data acquired during several pulse intervals.
At 506 the method comprises correcting the measured flight time values according to a correlation of measured peak width values with the calibration data of peak width versus flight time.
At 507 the method comprises converting the corrected flight time values into the mass-to-charge ratios.
In view of this disclosure it is noted that the methods and devices can be implemented in keeping with the present teachings. Further, the various components, materials, structures and parameters are included by way of illustration and example only and not in any limiting sense. In view of this disclosure, the present teachings can be implemented in other applications and components, materials, structures and equipment needed to implement these applications can be determined, while remaining within the scope of the appended claims.
