Radiant energy – Ionic separation or analysis – Ion beam pulsing means with detector synchronizing means
Reexamination Certificate
2002-07-11
2003-06-17
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
Ion beam pulsing means with detector synchronizing means
C250S282000
Reexamination Certificate
active
06580071
ABSTRACT:
BACKGROUND OF THE INVENTION
A time-of-flight mass spectrometer is an analytical device that determines the molecular weight of chemical compounds by separating corresponding molecular ions according to their mass-to-charge ratio (m/z value). In time-of-flight mass spectrometry (tofins), ions are formed by inducing the creation of a charge by typically adding or deleting a species such as a proton, electron, or metal. After the ions are formed, they are separated by the time it takes for the ions to arrive at a detector. These detection times are inversely proportional to the square root of their m/z values. Molecular weights are subsequently determined using the m/z values once the nature of the charging species has been elucidated.
FIG. 1
shows a simplified schematic diagram of a laser desorption/ionization time-of-flight mass spectrometer. For simplicity of illustration, some components (e.g., an analog-digital converter) are not shown in FIG.
1
. The mass spectrometer includes a laser
20
(or other ionization source), a sample substrate
26
, and a detector
36
(also known as the analyzer). A number of analytes are at different addressable locations
26
(
a
),
26
(
b
) on the sample substrate
26
. The detector
36
faces the sample substrate
26
so that the detector
36
receives ions of the analytes from the sample substrate
26
. An extractor
28
and one or more ion lenses
32
are between the detector
36
and the sample substrate
26
. The region between the ion lenses
32
and the detector
36
is enclosed in a vacuum tube and is typically maintained at pressures less than 1 microtorr.
In operation, the laser
20
emits a laser beam
21
that is focused by a lens
22
. A mirror
24
then reflects the focused laser beam and directs the focused laser beam to the sample substrate
26
. The laser beam
21
initiates the ionization process of the analytes at a predetermined addressable location
26
(
a
) on the sample substrate
26
. As a result, the analytes at the addressable location
26
(
a
) form analyte ions
34
. The analyte ions
34
subsequently desorb off of the sample substrate
26
.
The sample substrate
26
and the extractor
28
are coupled to a high-voltage supply
30
and are both at high voltage. The last of the ion lenses
32
is at ground. Applied potentials to each of these elements collectively create an ion focusing and accelerating field used to gather formed ions and accelerate them through the analyzer to ultimately strike the detector. The detector
36
then receives and detects the ions
34
.
The time it takes for the ions
34
to pass from the sample substrate
26
to the detector
36
is proportional to the mass of the ions
34
. This is the “time-of-flight” of the ions
34
. As will be explained in detail below, time-of-flight values are used to determine the m/z values for the analyte ions
34
, and consequently the molecular weights of the analytes ionized.
After the analyte at the addressable location
26
(
a
) is analyzed, the sample substrate
26
is repositioned upward so that an analyte on an adjacent addressable location
26
(
b
) can receive the laser beam
21
. This process is repeated until all analytes at all addressable locations on the substrate
26
are ionized and the m/z values for the analyte ions are determined.
Although the above-described mass spectrometer can accurately determine the m/z values of analyte ions, systematic errors are present in the m/z values. One factor that can cause systematic errors is the change in the electrical field strength that accelerates the ions
34
. The change in position of the sample substrate
26
, which is at high voltage, alters the ion extraction electrical field strength. The changing electrical field strength modifies the acceleration of the ions and consequently the time-of-flight values for the ions. Errors in the time-of-flight values for the analyte ions translate into errors in the obtained m/z values.
A user can calibrate the mass spectrometer to correct for the errors. Two calibration strategies are typically employed: external standard calibration and internal standard calibration.
In an external calibration process, a calibration substance is ionized on the sample substrate. The calibration substance is adjacent to the analyte to be analyzed and has a known mass and ions of a known m/z value. The obtained time-of-flight value for the calibration substance may be used to correct the time-of-flight value of the analyte. A more accurate m/z value can be calculated from the corrected time-of-flight value.
While the external calibration process is effective in some instances, a number of improvements could be made. For example, the calibration substance takes up space on the substrate surface that could otherwise be used for an analyte. This decreases the number of analytes per sample substrate that can be analyzed and consequently decreases the throughput of the analytical process. The throughput is also decreased, because time-of-flight measurements are made for a number of calibration substances. Time that could be otherwise used to process analytes is spent processing the calibration substances. Furthermore, forming discrete deposits of calibration substances on each sample substrate takes time and resources. Moreover, in this conventional process, the calibration substance and the analyte are spatially separated from each other. The substrate is still repositioned between the ionization of the analyte and the ionization of the calibration substance. Although error is reduced, a small amount of error is present because the repositioning of the substrate between the ionization of the calibration substance and the adjacent analyte may introduce changes in the accelerating electrical field strength.
Another calibration process is the internal standard calibration process. In an internal standard calibration process, a sample having an analyte is spiked with at least one calibration substance. The calibration substance has a known m/z value and is present at the same addressable location on the sample substrate as the analyte. Both the calibration substance and the analyte ionize and desorb simultaneously. The time-of-flight value for the ionized calibration substance can be used to correct the time-of-flight value for the ionized analyte. The internal calibration approach typically provides about a 10-100 fold improvement in mass accuracy compared to external standard approaches.
However, a number of problems are associated with the use of internal calibration substances. For example, if the calibration substance has a mass that is close to the mass of the unknown analyte, the signal from the calibration substance can “mask” the signal for the ions of the unknown analyte. As a result, the signal for the unknown analyte may not be observed. Also, if the ionization potential of the calibration substance exceeds the ionization potential of the analyte, the formation of analyte ions can be suppressed. Because of the difficulties of applying internal standard calibration approaches, external standard measurements are employed most routinely.
Embodiments of the invention address these and other problems.
SUMMARY OF THE INVENTION
Embodiments of the invention are directed to methods for calibrating mass spectrometers, mass spectrometers, and computer readable media including computer code for calibrating mass spectrometers.
One embodiment of the invention is directed to a method for calibrating a time-of-flight mass spectrometer, the method comprising: a) determining time-of-flight values, or values derived from the time-of-flight values for a calibration substance at each of a plurality of different addressable locations on a sample substrate; b) identifying one of the addressable locations on the substrate as a reference addressable location; and c) calculating a plurality correction factors for the respective addressable locations on the substrate using the time-of-flight value, or a value derived from the time-of-flight value, for the calibration substance on the reference a
Gavin Edward J.
Weinberger Scot R.
Youngquist Michael G.
Ciphergen Biosystems Inc.
Lee John R.
Smith II Johnnie L
Townsend & Townsend & Crew LLP
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