Orbitrap

An Orbitrap mass spectrometer, as a result of its outstanding mass resolving power, high sensitivity, accurate mass measurement ( 5 ppm), full-scan, and/or MS capabilities, is an attractive alternative to a TOF instrument, especially for analysis of small molecules (m/z 1000). The Orbitrap 

The resolving power of a current Orbitrap instrument is defined at either m/z 400 for LIT Orbitrap (LTQ Orbitrap XL or LTQ Orbitrap Velos) or m/z 200 for a stand-alone Orbitrap (Exactive) to achieve various mass resolutions. The LTQ Orbitrap XL has a set of resolutions available at 7,500, 15,000, 30,000, 60,000, or 100,000, whereas the Exactive has a selection at 10,000, 25,000, 50,000, or 

The selection of resolving power in an Orbitrap must be fit for purpose and is associated with both the analyte concentration and the complexity of matrices. A study indicated that a resolving power of 7,000-10,000 could be sufficient for detection of analytes in samples of intermediate complexity at concentrations down to 25 (Tg/kg with accurate mass measurement (mass errors 5 ppm) for lower concentration levels and/or more accurate mass assignment, a higher resolving power (18,000-25,000) was needed and in highly complex extracts, a resolving power of 35,000-50,000 or even 

The orbitrap is the most recently invented mass analyzer. Like with the QIT, ions are trapped and stored in a potential well. However, instead of ejecting the ions for external detection the frequency of the trapped oscillationg ions is measured. This method provides substantially better resolution and mass accuracy in normal operation. 

The orbitrap (Fig. 2.18) was invented by Makarov in 1999 [244, 245]. It can be seen as a modified Knight/Kingdon trap because of its general construction. It can also be seen as a modified quadmpole trap that uses electrostatic fields instead of dynamic. Ions move in stable trajectories both around the central electrode and in harmonic 

Split outer electrode, also used for detection of image current 

Ion detection is carried out using image current detection with subsequent Fourier transform of the time-domain signal in the same way as for the Fourier transform ion cyclotron resonance (FTICR) analyzer (see Section 2.2.6). Because frequency can be measured very precisely, high m/z separation can be attained. Here, the axial frequency is measured, since it is independent to the first order on energy and spatial spread of the ions. Since the orbitrap, contrary to the other mass analyzers described, is a recent invention, not many variations of the instrument exist. Apart from Thermo Fischer Scientific s commercial instrument, there is the earlier setup described in References 245 to 247. 

Performance Parameters. Typical resolving power for the commercial instrument is up to about 130,000 (FWHM) for m/z 400 Th. The mass resolving power is m/z dependent it decreases with fmfz (see the FTICR, described in Section 2.2.6, which decreases linearly with m/z). 

Although, originally, the Orbitrap device was designed for ESI, with an AP ion source, difterent MALDI sources have since been developed and are now commercially available. Contrary to classical MALDI-sources, these operate at atmospheric or intermediate pressure of typically lO mbar. The collisional cooling of such sources limits the PSD of ions to prepare them for longer detection times in comparison with axial TOF analyzers, for example. 

The potential of the DCaux rods is pulsed in such a way as to secure maximal ion transmission into the analyzer. The typical timing is such that the ions of a preselected number of laser exposures are accumulated. Further information on the source design of the intermediate pressure MALDl source and the implementation of Automatic Gain Gontrol in MALDl mode of operation can be found elsewhere [124]. 

One of the main applications of both Orbitrap systems and their MALDl ion sources is for the imaging of biological specimens. The Thermo Fisher Scientific 

The Thermo Scientific ion source can be retrofitted to the LTQ Orbitrap XL line of instruments, while the TransMIT ion source can be retrofitted to the Exactive series instrumentation. The MassTech AP MALDI PDF+ ion source has also been adapted to both hybrid Orbitrap devices, as well as to bench-top Orbitrap systems. 

The AP MALDI PDF+ ion source can be flanged to the atmospheric pressure interface (API) of ion trap-, ion trap-Orbitrap-, or bench-top Orbitrap instruments. The ion source is equipped with a frequency-tripled Nd YAG laser operating at 355 nm with 3 ns pulse duration, while the laser beam is typically coupled into a 400 pm core diameter fiber forming a spot size of 500 x 600 pm on the plate (a smaller-diameter fiber can also be applied). The ease-of-use of this ion source for regular MALDI applications, and the possibility ofa rapid source exchange between MALDI and ESI, have led to this source becoming an excellent tool for today s analytical studies. Details on the coupling of this source with the Exactive series instrumentation are available from the manufacturer. 

The FT-ICR method described in Section 2.3.4 suffers only from one weak point It requires magnetic fields with intensities (or =) 3 Tesla. Consequently, cryomagnets are required, with high costs either for acquirement or for maintenance. The commercial availability of mass spectrometers exhibiting high performances, but low initial cost, modest maintenance cost, and reduced size, is surely of great interest, and the Orbitrap system (Hu et al., 2005) is the answer to this need. 

Makarov invented a new type of mass spectrometer by modifying the Kingdom trap with specially shaped outer and inner electrodes (see Fig. 2.28). Also, in this case a purely electrostatic held is obtained by a dc voltage applied to the inner electrode. Ions injected into the device undergo a periodic motion that can be considered the result of three different periodic motions (1) rotation around the inner electrode (2) radial oscillation and (3) axial oscillations. These three components exhibit well-defined frequencies  

Ions are injected at an angle and offset from the center of the trap. 

The momentum of the ions causes them to orbit around, and oscillate along, a central spindle-like electrode. 

The lateral oscillation of the ions along the inner electrode induces a transient (image) current in the split outer electrode. 

The recorded image current is interpreted using Fourier transform analysis to provide miz values and intensities. 

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With recent instrumental development, such as fast LC, fast GC and two-dimensional gas chromatography (GCxGC) and advanced tandem hybrid MS detection systems (i.e., QqTOF, QqLIT, Orbitrap) the analysis of complex mixtures... 

Sectors(EB 8E EBE Quadnipole (Q, QqQ) Ion trap, Orbitrap Time of flight (TOP) Hybrids (BEqQ QTOF.)... 

Figure 2.7 Mass spectra recorded at different resolutions. Mass spectrum obtained by a two dimensional ion trap at low resolution (a) and by an Orbitrap at resolving power 50000 (b). Mass spectrum of a mixture of three isobaric species [C19H7N]+, [C20H9]+, [C13H19N302]+ obtained at low resolution (black line) and at resolving power 50000 (grey line) (c). It is noteworthy that at low resolution the three peaks are completely unresolved...

Magnetic and electrostatic sectors, quadrupole, and time of flight analyzers belong to the first group, while ion trap, Orbitrap and Fourier transform ion cyclotron resonance analyzers separate ions in time. 

FT-ICR and the Orbitrap belong to this group. In these analyzers the m/z values of the ions are not directly measured, but they are obtained by Fourier transform treatment of the signal (Figure 2.12). 

The Orbitrap. The Orbitrap analyzer, [26] invented by Alexander Makarov, has been defined by the company that commercially produces it as the first totally new mass analyzer to be introduced to the market in more than 20 years . Its name recalls the concept of trapping ions. Indeed, ions are trapped in an electrostatic field produced by two electrodes a central spindle-shaped and an outer barrel-like electrode. Ions are moving in harmonic, complex spiral-like movements around the central electrode while shuttling back and forth over its long axis in harmonic motion with frequencies... 

The Orbitrap allows very high resolution to be achieved (the resolving power in commercial instruments is 100000, rivalling that of FT-ICR instruments) and routine mass measurement accuracies less than 2 ppm. It finds applications in many fields, such as biology, proteomics, food chemistry and cultural heritage. 

Figure 2.13 The Orbitrap analyzer. From the Thermo web site (http //www.thermo.com)...

FIGURE 5.2 Ion path for LTQ-Orbitrap. (Courtesy of Thermo Fisher Scientific, Waltham, Massachusetts.)... 

MS11 capabilities. However, ions may then subsequently be detected at unit resolution using an electron multiplier or, alternatively, focused in a C-Trap (Figure 5.2) and then transferred and detected at high resolution using the Orbitrap. In our experience with the LTQ-Orbitrap, ions may be measured with a resolution of approximately 60,000 with online LC/MS in the full scan mode. 

One recent advance in MS hardware that has been found to be useful for metabolite identification studies is the Orbitrap. This MS has a mass resolution of 30,000 to 100,000 (two models). For many applications, 30,000 mass resolution capability is sufficient. While only a few current literature references cite the Orbitrap MS for metabolite identification, it is safe to predict that the Orbitrap will be the subject of many references in the future. Two references related to its use for metabolite identification are Peterman et al.190 and Lim et al.182 Lim s group related an an impressive example of the use of high mass resolution to differentiate a metabolite from a co-eluting isobaric matrix component, as shown in Figure 7.14. 

Figure 2.18. Schematic of an orbitrap analyzer. The z-direction oscillary motion of the ions induces an image current that is detected by the electrodes.

The orbitrap mass accuracy is better than all quadmpoles and TOF instruments just right after the FTICR and sector instruments, that is, around 2 ppm with internal calibration [248]. 

The acquisition speed is, as for the FTICR, resolution dependent. With Thermo Fischer Scientific s orbitrap the desired mass resolving power can be selected. With the lowest setting (7500 FWHM) the acquisition time for one ion injection is 0.3 s and with the highest setting (100,000 FWHM) it is 1.9 s. 

In principle, it would be possible to perform multistage mass spectrometry like in an ICR analyzer although with no gas CID would of course not be possible, but other dissociation methods could be employed. There might, however, be technical issues. At the time of writing, fragmentation is performed in the linear QIT preceeding the orbitrap in Thermo Fischer Scientific s instrument. Both pulsed and continuous ion sources can be employed. There are several ion sources that can be employed with Thermo Fischer Scientific s orbitrap. 

A mass calibration for FTICR analyzers with superconducting magnets is very stable and is valid for many days for normal applications. Mass accuracy < 1 ppm can be obtained over a fairly wide mass range. Unique elemental composition can be determined for masses over 800 Da [262]. Recently, 0.1 ppm mass accuracy, which required a mass resolving power >300,000, has been achieved for several thousand peaks by a 14.5 T instrument [263] and commercial instruments with mass accuracy <0.2 ppm are available. As with the orbitrap (see Section 2.2.5) the frequency is... 

Since a minimum of about 100 ions is needed to generate a detectable signal under normal circumstances (ion counting is inherently more sensitive than image current detection) and space-charge effects become influential with more than 106 to 107 ions, the dynamic range is relatively poor, about 104. The same applies to the FTICR as to the QIT and orbitrap. The signal depends on other species present in the trap at the same time, which limits quantification quality. 

Image current detection is (currently) the only nondestructive detection method in MS. The two mass analyzers that employ image current detection are the FTICR and the orbi-trap. In the FTICR ions are trapped in a magnetic field and move in a circular motion with a frequency that depends on their m/z. Correspondingly, in the orbitrap ions move in harmonic oscillations in the z-direction with a frequency that is m/z dependent but independent of the energy and spatial spread of the ions. For detection ions are made... 

M. Hardman and A. Makarov. Interfacing the Orbitrap Mass Analyzer to an Electrospray Ion Source. Anal. Chem., 75(2003) 1699-1705. 

J. R. Yates, D. Cociorva, L. Liao, and V. Zabrouskov. Performance of a Linear Ion Trap-Orbitrap Hybrid for Peptide Analysis. Anal. Chem., 78(2006) 493-500. 

A. Makarov, E. Denisov, A. Kholomeev, W. Balschun, O. Lange, K. Strupat, and S. Homing. Performance Evaluation of a Hybrid Linear Ion Trap/Orbitrap Mass Spectrometer. Anal. Chem., 78(2006) 2113-2120. 

M. Scigelova and A. Makarov. Orbitrap Mass Analyzer Overview and Applications in Proteomics. Proteomics, 6(2006) 16-21. 

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