Time-of-flight mass spectrometry
Time-of-flight mass spectrometry is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined by a time of flight measurement. Ions are accelerated by an electric field of known strength. This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the ion to reach a detector at a known distance is measured. This time will depend on the velocity of the ion, and therefore is a measure of its mass-to-charge ratio. From this ratio and known experimental parameters, one can identify the ion.
Theory
The potential energy of a charged particle in an electric field is related to the charge of the particle and to the strength of the electric field:where Ep is potential energy, q is the charge of the particle, and U is the electric potential difference.
When the charged particle is accelerated into time-of-flight tube by the voltage U, its potential energy is converted to kinetic energy. The kinetic energy of any mass is:
In effect, the potential energy is converted to kinetic energy, meaning that equations and are equal
The velocity of the charged particle after acceleration will not change since it moves in a field-free time-of-flight tube. The velocity of the particle can be determined in a time-of-flight tube since the length of the path of the flight of the ion is known and the time of the flight of the ion can be measured using a transient digitizer or time to digital converter.
Thus,
and we substitute the value of v in into.
Rearranging so that the flight time is expressed by everything else:
Taking the square root yields the time,
These factors for the time of flight have been grouped purposely. contains constants that in principle do not change when a set of ions are analyzed in a single pulse of acceleration. can thus be given as:
where k is a proportionality constant representing factors related to the instrument settings and characteristics.
reveals more clearly that the time of flight of the ion varies with the square root of its mass-to-charge ratio.
Consider a real-world example of a MALDI time-of-flight mass spectrometer instrument which is used to produce a mass spectrum of the tryptic peptides of a protein. Suppose the mass of one tryptic peptide is 1000 daltons. The kind of ionization of peptides produced by MALDI is typically +1 ions, so q = e in both cases. Suppose the instrument is set to accelerate the ions in a U = 15,000 volts potential. And suppose the length of the flight tube is 1.5 meters. All the factors necessary to calculate the time of flight of the ions are now known for, which is evaluated first of the ion of mass 1000 Da:
Note that the mass had to be converted from daltons to kilograms to make it possible to evaluate the equation in the proper units. The final value should be in seconds:
which is about 28 microseconds. If there were a singly charged tryptic peptide ion with 4000 Da mass, and it is four times larger than the 1000 Da mass, it would take twice the time, or about 56 microseconds to traverse the flight tube, since time is proportional to the square root of the mass-to-charge ratio.
Delayed extraction
can be improved in axial MALDI-TOF mass spectrometer where ion production takes place in vacuum by allowing the initial burst of ions and neutrals produced by the laser pulse to equilibrate and to let the ions travel some distance perpendicularly to the sample plate before the ions can be accelerated into the flight tube. The ion equilibration in plasma plume produced during the desorption/ionization takes place approximately 100 ns or less, after that most of ions irrespectively of their mass start moving from the surface with some average velocity. To compensate for the spread of this average velocity and to improve mass resolution, it was proposed to delay the extraction of ions from the ion source toward the flight tube by a few hundred nanoseconds to a few microseconds with respect to the start of short laser pulse. This technique is referred to as "time-lag focusing" for ionization of atoms or molecules by resonance enhanced multiphoton ionization or by electron impact ionization in a rarefied gas and "delayed extraction" for ions produced generally by laser desorption/ionization of molecules adsorbed on flat surfaces or microcrystals placed on conductive flat surface.Delayed extraction generally refers to the operation mode of vacuum ion sources when the onset of the electric field responsible for acceleration of the ions into the flight tube is delayed by some short time with respect to the ionization event. This differs from a case of constant extraction field where the ions are accelerated instantaneously upon being formed. Delayed extraction is used with MALDI or laser desorption/ionization ion sources where the ions to be analyzed are produced in an expanding plume moving from the sample plate with a high speed. Since the thickness of the ion packets arriving at the detector is important to mass resolution, on first inspection it can appear counter-intuitive to allow the ion plume to further expand before extraction. Delayed extraction is more of a compensation for the initial momentum of the ions: it provides the same arrival times at the detector for ions with the same mass-to-charge ratios but with different initial velocities.
In delayed extraction of ions produced in vacuum, the ions that have lower momentum in the direction of extraction start to be accelerated at higher potential due to being further from the extraction plate when the extraction field is turned on. Conversely, those ions with greater forward momentum start to be accelerated at lower potential since they are closer to the extraction plate. At the exit from the acceleration region, the slower ions at the back of the plume will be accelerated to greater velocity than the initially faster ions at the front of the plume. So after delayed extraction, a group of ions that leaves the ion source earlier has lower velocity in the direction of the acceleration compared to some other group of ions that leaves the ion source later but with greater velocity. When ion source parameters are properly adjusted, the faster group of ions catches up to the slower one at some distance from the ion source, so the detector plate placed at this distance detects simultaneous arrival of these groups of ions. In its way, the delayed application of the acceleration field acts as a one-dimensional time-of-flight focusing element.
Reflectron TOF
The kinetic energy distribution in the direction of ion flight can be corrected by using a reflectron. The reflectron uses a constant electrostatic field to reflect the ion beam toward the detector. The more energetic ions penetrate deeper into the reflectron, and take a slightly longer path to the detector. Less energetic ions of the same mass-to-charge ratio penetrate a shorter distance into the reflectron and, correspondingly, take a shorter path to the detector. The flat surface of the ion detector is placed at the plane where ions of same m/z but with different energies arrive at the same time counted with respect to the onset of the extraction pulse in the ion source. A point of simultaneous arrival of ions of the same mass-to-charge ratio but with different energies is often referred as time-of-flight focus.An additional advantage to the re-TOF arrangement is that twice the flight path is achieved in a given length of the TOF instrument.
Ion gating
A Bradbury–Nielsen shutter is a type of ion gate used in TOF mass spectrometers and in ion mobility spectrometers, as well as Hadamard transform TOF mass spectrometers. The Bradbury–Nielsen shutter is ideal for fast timed ion selector —a device used for isolating ions over narrow mass range in tandem MALDI mass spectrometers.Orthogonal acceleration time-of-flight
Continuous ion sources are generally interfaced to the TOF mass analyzer by "orthogonal extraction" in which ions introduced into the TOF mass analyzer are accelerated along the axis perpendicular to their initial direction of motion. Orthogonal acceleration combined with collisional ion cooling allows separating the ion production in the ion source and mass analysis. In this technique, very high resolution can be achieved for ions produced in MALDI or ESI sources.Before entering the orthogonal acceleration region or the pulser, the ions produced in continuous or pulsed sources are focused into a beam of 1–2 mm diameter by collisions with a residual gas in RF multipole guides. A system of electrostatic lenses mounted in high-vacuum region before the pulser makes the beam parallel to minimize its divergence in the direction of acceleration. The combination of ion collisional cooling and orthogonal acceleration TOF has provided significant increase in resolution of modern TOF MS from few hundred to several tens of thousand without compromising the sensitivity.