Fourier transform ion cyclotron resonance

The ion cyclotron resonance (ICR) theory was developed in 1932 by Ernest O. Lawrence (Nobel Prize in Physics in 1951) and Stanley M. Livingston.^148,149 In 1951 the principle of ion cyclotron resonance was first incorporated into a mass spectrometer, called the omegatron, by Sommer and co-workers, who successfully applied the concept of cyclotron resonance to determine the charge-to-mass ratio of the proton.^150-153 Since then, FT-ICR-MS has had tremendous growth for the analysis of a wide variety of compounds.

The FT-ICR mass spectrometer has four key components: a superconducting magnet with a strong magnetic field that can vary from 3 T to 20 T, the analyzer cell or ICR cell placed in the center of the strong magnetic field where the ions are stored, analyzed and detected, an ultra-high vacuum system and a sophisticated data system. The analyzer cell can take on different geometries but generally consists of a front and back trapping electrode, two opposite excitation electrodes and two opposite detection electrodes, as indicated in Figure I.12.

The analyzer cell is a low pressure (10^–10 mbar) Penning trap^‡ in which ions can be stored for extended periods of time. The timescale of the experiment is one of the first distinctions of FT-ICR-MS, and is extensively used to study ion/molecule reactions, conformational changes in molecules, the dissociation of very large molecules with a large number of degrees of freedom, and many more processes that require both gas-phase ions and relatively long time to complete.¹⁵⁴

‡The Penning Trap was named after F. M. Penning by Hans Georg Dehmelt who was awarded with the Nobel Prize in Physics in 1989 for developing the first trap.

I.3. Mass spectrometry and gas-phase ion chemistry

33

Figure I.12 ICR ion trap configurations. E=excitation; D=detection; T=end cap (“trapping”). (a) cubic; (b) cylindrical;

(c) end caps segmented to linearize excitation potential (“infinity” trap); (d) and (e) open-ended; (f) dual; and (g)

“matrix-shimmed”.¹⁵⁵

Fundamental aspects of FT-ICR are summarized below:^154,155

 Ion cyclotron frequency, radius, velocity and energy, as a function of ion mass, ion charge and magnetic field strength, follow directly from the motion of an ion in a spatially uniform static magnetic field (eq. 1.3).

𝐹𝑜𝑟𝑐𝑒 = 𝑚𝑎𝑠𝑠 𝑥 𝑎𝑐𝑒𝑙𝑒𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑚^𝑑𝑣

𝑑𝑡 = 𝑞𝒗 𝑥 𝑩 (1.3)

in which m, q, and v are ionic mass, charge, and velocity, and the vector cross product term means that the direction of the magnetic component of the Lorentz force is perpendicular to the plane determined by v and B.

 If the ion maintains constant speed (i.e. no collisions), then the magnetic field bends the ion path into a circle of radius r, the cyclotron motion (Figure I.13).

34

Figure I.13 Ion cyclotron motion; ωc is the angular cyclotron frequency, and r is the ion cyclotron radius. (Adapted from Reference 155).

 Ion cyclotron motion becomes coherent by the application of a spatially uniform RF electric field (excitation) at the same frequency as the ion cyclotron frequency. The ICR signal results from induction of an oscillating “image” charge on two conductive infinitely extended opposed parallel electrodes. A frequency-domain spectrum is obtained by Fourier transformation of the digitized ICR signal (Figure I.14).

Figure I.14 An rf burst accelerates the ions, generating a transient ion image current signal (left). The signal is digitalized, stored in the computer and a Fourier transform is applied to the data to convert the information into a mass spectrum (right).¹⁰⁶

 Confinement of ions by application of a three-dimensional axial quadrupolar dc electric field shifts the ion cyclotron frequency, whereas excitation and detection remain essentially linear, but with a reduced proportionality constant (Figure I.15).

 Collisions broaden the ICR signal in a simple way, and actually make it possible to cool and compress an ion packet for improved detection.

I.3. Mass spectrometry and gas-phase ion chemistry

35

 Although FT-ICR-MS has been coupled to many types of ion sources, most ion sources work best outside the magnet. Thus, several methods have been developed to guide the externally generated ions into the ion trap inside a high-field magnet.

Figure I.15 Schematic representation of the three natural motions of an ion confined in an ICR cell (m-magnetron rotation; c-cyclotron rotation; T-trapping oscillation).¹⁵⁵

 The above features may be combined in various experimental “event sequences” to perform tandem-in-time mass spectrometry (MS/MS or MSⁿ). In most regular mass spectrometers, after ion formation the ions are accelerated forming a focused beam and selected according to their m/z after passing through distinct fields, electric or magnetic.

The ion/molecule collisions take place in a separated division and the ultimate stage is ion detection. In the FT-ICR-MS, all experimental steps occur in the same space, the ICR cell.

An FT-ICR mass spectrometer combines high resolution, high mass-accuracy, non-destructive multichannel detection, long ion-observation times, the possibility of performing gas-phase reactions on trapped ions, and, perhaps most importantly, tools for structural analysis of large biomolecules.

The FT-ICR-MS is a complete ion laboratory by itself.¹⁵⁴ The nature of an FT-ICR-MS experiment implies that the different analytical steps are separated in time. A typical sequence of events for a tandem mass FT-ICR-MS experiment is shown in Figure I.16. A usual ICR experiment consists of four time-spread events: quenching, ion formation/injection, excitation, and detection. All of these functions are performed in the ICR cell, which consists of three pairs of opposing plates;

each pair performs a distinct function: trapping, excitation, or detection of ions (Figure I.17). The total duration of a tandem FT-ICR-MS experiment in which no collision gas is used is approximately 1 s.¹⁵⁴

36

Figure I.16 FT-ICR-MS sequence showing the order of the different time-separated process steps.¹⁵⁴

Figure I.17 Schematic representation of the operation of FT-ICR/MS. The mass analysis involves three steps: (a) ion formation and storage; (b) excitation of the trapped ions by an external broad-frequency range pulse; (c) detection of the ions by measuring their image current.¹⁵⁴

I.3. Mass spectrometry and gas-phase ion chemistry

37 I.3.3 Collision-induced dissociation (CID)

Collision-induced dissociation, or sometimes also mentioned as collision-activated decomposition (CAD) became very important in mass spectrometry, since the beginning of soft ionization techniques such as fast atom bombardment¹⁵⁶, electrospray ionization^157,158, and matrix-assisted laser desorption/ionization¹⁵⁹. The ability to obtain practically intact molecular ions for many classes of compound was enormously useful but the ability to obtain structural information through characteristic fragmentation patterns was therefore lost.

CID occurs when some of the translational energy of an accelerated ion is converted into internal energy upon collision with a residual gas – a process designated as collision endothermicity, q.

CID leads to an increase of the internal energy that can induce decomposition (fragmentation) of the ions and can be employed to increase the number of precursor ions that fragment and also the number of fragmentation pathways.

The CID process is a two-step mechanism, where the excitation of the precursors and their fragmentations are separated in time since the activation time for fast-moving ions is generally orders of magnitude faster than the dissociation time. The first step is very fast (10⁻¹⁴ to 10⁻¹⁶ s) where a collision between an ion and a neutral target results in an increase in the internal energy of the ion. This extra energy is redistributed amongst its vibrational modes (3N - 6 for an ion with N non-linear atoms) – this can be seen as an ergodic ion activation mode. The second step is much slower in which there is unimolecular decomposition of the excited ion to generate product ions and neutral fragments. Because the first step is much faster than the second, large ions are more difficult to fragment as they have more vibrational modes in which to deposit the extra energy.

Therefore, the dissociation products that are observed result from a series of competitive and consecutive reactions and the fragmentation pathways depend on the amount of energy deposited and not on the method of ion activation used. As the energy is distributed with an equal probability among all of the internal modes of the ion, this leads preferentially to cleavage sites at the weakest bonds. For the same reason, molecules with more atoms will need more energy, or more time, to dissociate.

The total available energy for the transfer of kinetic energy to internal energy is called relative energy (Ecom) and depends on the collision partners’ masses (Eq. 1.4).

38

𝐸𝑐𝑜𝑚 = (_𝑚^𝑁

𝑝+𝑁)𝐸𝑙𝑎𝑏 (1.4)

Elab is the ion’s kinetic energy and N and mp represent the masses for the neutral target and precursor ion, respectively. The CID process is highly dependent on the relative masses of the two species. Ecom represents the maximum amount of energy that can be converted into internal energy of the precursor ion. This energy, increases with the target’s mass, allowing more of the ion’s kinetic energy to be converted into internal energy. Also, Ecom decreases as a function of 1/mp, meaning that larger precursor ions have less internal energy available for fragmentation through the collision process.¹⁶⁰

As explained above, activation of the selected ions occurs by collision(s) with neutral gas molecules in a collision cell. This experiment can be done at high collision energies (range of keV), usually using tandem sector and time-of-flight instruments, or at low energies (1- 100 eV), more commonly observed in triple quadrupoles (QqQ) and trapping devices, such as quadrupole ion traps and Fourier-transform ion cyclotron resonance instruments.

One of the main inconveniences of CID is the limitation of the energy transferred to an ion and thus the limitation of its degree of fragmentation. With large ions, the energy is distributed on a greater number of bonds and thus the result is a slower reaction rate of the fragmentation. To minimize those issues, several other ion activation methods that have become progressively useful for specific applications (Table I.5).¹⁶¹

Table I.5 Different ion activation processes.

Method Energy range Type of instrument

Collision-induced dissociation (CID) Low

Electron-capture dissociation (ECD) Low FT-ICR

Infrared multiphoton dissociation (IRMPD) Low IT, FT-ICR

Blackbody infrared radiative dissociation (BIRD) Low IT, FT-ICR

SID is very similar to CID, but allows increased control for energy deposition and can lead to increased fragmentation, especially for large ions with high dissociation thresholds. ECD is a unique technique for the observation of non-ergodic dissociation behavior of multiply charged

I.3. Mass spectrometry and gas-phase ion chemistry

39 cations and exhibits distinct fragmentation mechanisms. IRMPD works with IR radiation, usually by the use of low-power continuous-wave CO2 lasers, in order to cause fragmentation of ions in trapping instruments, and BIRD can thermally activate ions in FT-ICR instruments. It allows the determination of activation energies and Arrhenius constants for dissociation reactions, and can ultimately assist in the elucidation of specific fragmentation mechanisms. These activation techniques can be used separately or in combination to yield very interesting and comprehensive results. Nevertheless, CID still remains the most common ion activation technique employed nowadays.^162-164