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Time of Flight Mass Spectrometry

From the physical principle time of flight (TOF) may be the simplest way to perform mass spectrometric analysis (Fig. 1.26). TOF is the measure of the time that ions need to cross in a field free tube of about 1 m length [63, 64]. It is a pulsed technique and requires a starting point. The motion of an ion is characterized by its kinetic energy Ec = 0.5m x v2 (m = mass, v = speed). Therefore, the speed of ions or the time to fly through the tube is proportional to their ^Jm/z value. The velocity of the ions formed is generally low and they are accelerated by strong electric fields (2-30 kV) in the direction of the detector. Low mass ions reach the detector more rapidly than high mass ions. Due to the short flight time (50-100 msec) and the good transmission, a spectrum can be generated within 100 ms over an almost unlimited mass range. Detection of the ions is performed with a multichannel plate detector (MCP, see Section 1.5) which has a relatively small dynamic range (generally two to three orders of magnitude).

With soft ionization techniques such as MALDI, ions of m/z 200000 can be routinely detected. The mass range is mainly limited by the fact that with the detector the response decreases with increasing m/z of the ions. The mass resolution of a TOF mass analyzer is relatively poor (unit mass resolution and less) and is affected by factors that create a distribution in the flight time of ions with the same m/z. The simplest way to increase the mass resolution is to increase the length of flight tube or to reduce the kinetic energy spread of the ions leaving the source.

One way to reduce the kinetic energy spread is to introduce a time delay between ion formation and acceleration, referred to as delayed pulsed extraction. After a certain time delay ranging from nanoseconds to microseconds a voltage pulse is applied to accelerate the ions out of the source.

The second way to improve the mass resolution significantly is to use an electrostatic mirror (mass reflectron) placed in the drift region of ions (Fig. 1.27).

Source Detector 1

Source Detector 1

Detector 2 Reflectron

Reflect ron on

Fig. 1.27 Schematic of a time of flight mass spectrometer equipped with a reflectron. The instrument can be operated in the linear mode (reflectron off) or in the reflectron mode (reflectron on).

Detector 2 Reflectron

Reflect ron on

Fig. 1.27 Schematic of a time of flight mass spectrometer equipped with a reflectron. The instrument can be operated in the linear mode (reflectron off) or in the reflectron mode (reflectron on).

Briefly, the ions with high energy penetrate deeper into the ion mirror region than those with the same m/z at a lower energy. Because of the different trajectories, all ions of the same m/z reach the detector at the same time. Thus, all ions of the same m/z have then a much lower energy dispersion. With the reflectron the flight path is increased without changing the physical size of the instrument. In reflectron mode a mass resolving power of 15 000 is standard but the mass range is limited to several thousand m/z units. TOF instruments are non-scanning mass spectrometers resulting in an increased sensitivity compared to quadrupole mass spectrometers.

In general the commercial TOF instruments have two detectors; one for the linear mode and one for the reflectron mode. The combination of MALDI with TOF is ideal because both techniques are pulsed techniques. However, it is also possible to arrange a continuous beam as generated by electrospray ionization. For that purpose orthogonal acceleration was developed [65]. The ion beam is introduced perpendicularly to the TOF and packets are accelerated orthogonally (oa-TOF) at similar frequencies improving the sensitivity. While a packet of ions is analyzed, a new beam is formed in the orthogonal acceleration.

Time of flight instruments are mainly used for qualitative analysis with MALDI or atmospheric pressure ionization. With MALDI ionization one of the main applications is the identification of proteins by analyzing their peptides after trypsin digestion (peptide mass finger print; PMF). Further structural information of the peptides can be obtained from metastable transitions or collision-induced dissociations generated in the drift tube prior to entering the reflectron. This technique is called post-source decay (PSD). A metastable ion is an ion which dissociates in the free field region of the mass spectrometer. For TOF instruments the acquisition rate is in the range 10-20 Hz, making these mass analyzers best suited for the interfacing of fast liquid chromatographic separations or capillary electropho-resis using electrospray ionization.

Due to their fast acquisition rate and high resolution capabilities TOF mass analyzers are often used as the last mass analyzing stage in hybrid tandem mass

Fig. 1.28 Schematic of a quadrupole-time of flight instrument. Quadrupole q0 is used for collisional cooling and ion focusing. Nitrogen or argon is generally used as collision gas. The ion modulator pushes the ions orthogonally to their initial direction into the TOF analyzer.

spectrometers such as quadrupole-time of flight instruments. A quadrupole-time of flight instrument (QqTOF) is the result of the replacement of the last quadrupole section (Q3) of a triple quadrupole instrument by a time of flight analyzer (Fig. 1.28), a powerful combination in regards of mass range (m/z 5 to m/z 40 000), mass resolving power of 10 000 and sensitivity [66, 67]. In single MS mode the quadrupoles (q0, Q1, q2) serve as RF ion guides and the mass analysis is performed in the TOF. To accommodate ion injection a pulsed field is applied in the ion modulator to push the ions orthogonally to their initial direction into the TOF analyzer.

In tandem MS mode, because the product ions are recorded with the same TOF mass analyzers as in full scan mode, the same high resolution and mass accuracy is obtained. Isolation of the precursor ion can be performed either at unit mass resolution or at 2-3 m/z units for multiply charged ions. Accurate mass measurements of the elemental composition of product ions greatly facilitate spectra interpretation and the main applications are peptide analysis and metabolite identification using electrospray ionization [68]. In TOF mass analyzers accurate mass determination can be affected by various parameters such as: (i) ion intensities, (ii) room temperature or (iii) detector dead time. Interestingly, the mass spectrum can be recalibrated post-acquisition using the mass of a known ion (lock mass). The lock mass can be a cluster ion in full scan mode or the residual precursor ion in the product ion mode. For LC-MS analysis a dual spray (LockSpray) source has been described, which allows the continuous introduction of a reference analyte into the mass spectrometer for improved accurate mass measurements [69]. The versatile precursor ion scan, another specific feature of the triple quadrupole, is maintained in the QqTOF instrument. However, in pre cursor scan mode the sensitivity is lower in QqTOF than in QqQ instruments. The lack of good quality product ion spectra on conventional MALDI-TOF instruments made the use of MALDI on QqTOF instruments an interesting alternative for the sequencing of peptides. As in electrospray TOF, in the case of QqTOF the MALDI ion production needs to be decoupled from mass measurements. The technique to interface MALDI with QqTOF is named orthogonal MALDI (o-MALDI) TOF with collisional cooling. With o-MALDI the pulse is almost converted in a continuous beam equivalent to that originated from an electro-spray source.

The TOF mass analyzer has a low duty cycle, and the combination with an ion accumulation device such as an ion trap is therefore very advantageous. It offers also MSn capabilities with accurate mass measurement. In all acquisition modes, the ions are accelerated into the time of flight for mass analysis. Various other hybrid mass spectrometers with TOF have been described, including quadrupole ion trap [70] and linear ion trap [58]. High energy tandem mass spectrometry can be performed on TOF-TOF mass spectrometers [71, 72].

Fourier Transform Mass Spectrometry

1.4.6.1 Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

The main components of a Fourier transform ion cyclotron resonance mass spectrometer are a superconducting magnet and a cubic or cylindrical cell (Fig. 1.29). Typically, the magnet field strengths (B) are in the range 3.0-9.4 Tesla. Ions are stored in the cell according their cyclotronic motion arising from the interaction of an ion with the unidirectional constant homogenous magnetic field. A static magnetic field applied on the z direction confines ions in the x— and — y directions according the cyclotronic motion. To avoid the escape of ions along the z axis, a low electrostatic potential is applied to the end cap electrodes [73].

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