Orbitrap mass analyzer The Orbitrap mass analyzer is an orbital ion trap developed by Alexander Makarov in 2000, starting from a modification of the Kingdon trap, dating back to the 1920s. The Kingdon trap consisted in: a thin-wire central electrode, an electrically insulated coaxial outer cylindrical electrode two end-plates Ions could be either generated directly inside the trap through electron ionization or injected from an external source.

A purely electrostatic field was adopted for ion trapping, based on a logarithmic potential: where r is the radial distance from the central electrode. Ions with appropriate velocities could move in stable orbits around the central electrode (for a few seconds). Application of a potential to the end-plates could be used to achieve ion trapping in the axial direction. Stored ions could be eventually transferred outside to a mass analyzer (e.g. to a ToF analyzer).

Representations of ion motion inside a Kingdon trap obtained from different perspectives:

In 1981 the shape of the Kingdon outer electrode was modified by Knight to generate an additional quadrupolar potential, thus having a quadrupolar-logarithmic field: z r In this case ions were subjected to orbital trapping in the radial direction and axial confinement along the z direction (like in a 3D-ion trap), with harmonic oscillation induced by applying an AC potential between the two halves of the split outer electrode. Ions could be either generated inside the trap, by laser etching of a metal target, or injected through the outer electrode (corresponding to a metal mesh). Ion exiting the trap in axial/radial direction were detected using an electron multiplier or a collector plate, respectively.

The Orbitrap can be considered an evolution of the Knight-style Kingdon trap, in which the inner electrode has a spindle-like shape, resulting in a purely harmonic potential in the z direction: z φ r The potential generated inside the Orbitrap, expressed as a function of the radial and axial co-ordinates is: where: k = field curvature Rm = Orbitrap characteristic radius C = constant

Standard: R1  6 mm and R2  15 mm Compact (High Field): The optimal geometry for an Orbitrap is provided by the following relationship between the z and r coordinates: R2 R1 where the 1 and 2 subscripts represent the central and outer electrode, respectively, and R1 and R2 correspond to the largest radii of the two electrodes, i.e. the radii at z=0: R1 and R2 can assume different values, according to the type of Orbitrap analyzer: Standard: R1  6 mm and R2  15 mm Compact (High Field): R1  9 mm and R2  15 mm

Since the applied electric potential does not contain r-z cross terms, the ion motion inside an Orbitrap analyzer is characterized by two independent components: a harmonic oscillation along the z axis; an orbital motion around the central electrode ( motion). Additionally, an oscillation of the rotation radius occurs. Only ions with orbital radii less than Rm will be trapped.

Characteristic frequencies Three peculiar frequencies can be determined for the Orbitrap motion. The first is related to the axial oscillation, resulting from the applied potential and described by the following differential equation: whose solution is: where: Ez is the initial kinetic energy of an ion in the axial direction and the axial oscillation frequency in an Orbitrap depends on the m/z (indicated as m/q in this case) ratio and on the field curvature

The other two frequencies arise from the solution of complex differential equations and are both directly proportional to the axial oscillation frequency: radial oscillation: rotation (around the spindle-electrode): Note that both frequencies depend on the radial distance and that the Rm/R ratio should be at least greater than 21/2.

Ion injection into the Orbitrap Ions can be injected into the Orbitrap at a z-position offset from the equator (z=0). The approach is known as electrodynamic squeezing: 0 V The monotonic increase of electric field strength with time has the effect of contracting the radius of the ion cloud, as well as pulling the ion packet closer to the z-axis, thereby preventing collisions with the outer electrode as the packets begin their axial oscillations. After all the ions of interest have entered the trap and moved far enough from the outer electrodes, the voltage on the central electrode is stabilized and stable z-oscillation starts.

Detection of image current By analogy with FT-ICR-MS, ion detection with an Orbitrap is based on broadband excitation, image current detection and conversion to mass spectrum through Fast Fourier Transform (FFT): Rings accounting for the radial oscillation of ions 30 mm 12 - 18 mm 10-10 mbar Note that the image current is measured between the two halves of the outer electrode by a differential amplifier.

Example of image current transient obtained with a Orbitrap for bovine insulin ions.

Ion rotational motion in the Orbitrap Although not used for mass analysis, the rotational (r, ) motion of ions in the Orbitrap is important for their trapping. By analogy with an electrostatic analyzer, in which a matching between ion kinetic energy and electric field will lead to a stable ion trajectory (independently from m/z ratios), a stable orbital motion in the Orbitrap will be obtained only if the ion kinetic energy before injection is suitably matched to the radial component of the electric field. The matching can be explained using SIMION simulations of ion (m/z 609) motion at z =0. The trajectories are observed from one of the ends of the Orbitrap. Incoming ion kinetic energy poorly matched The ion trajectory, shown for a few rotation periods, is highly eccentric, non-circular.

Incoming ion kinetic energy poorly matched After hundreds of rotation periods, the trajectories describe a ring with a poorly defined border. Ion kinetic energy well matched The orbit is nearly circular and the locus of orbits appears as a thin ring even if thousands of periods are shown. Two ion kinetic energies fairly matched The loci of orbits for the two energy correspond to concentric thin rings

Representations of ion motion inside an Orbitrap analyzer obtained from different perspectives:

Figures of merit for Orbitrap analyzers As the axial oscillation frequency is independent on energy and spatial spread of ions, high mass accuracies (1-2 ppm) and resolving powers (up to 240000) can be achieved. Compared to ICR or 3D-ion traps, the main advantages provided by Orbitrap are: very accurately defined electric field; increased space charge capacity at higher masses (due to independence of trapping potential on the m/z ratio); larger trapping volume; concomitant achievement of high mass accuracy, large dynamic range and high m/z range.

It is interesting to note that the resolving power provided by the Orbitrap becomes better than that obtained by a ICR-MS spectrometer at high m/z ratios. The improvement of resolution is even more evident when the Compact HF (High Field) Orbitrap is used.

The high resolving power available with Orbitrap-MS can be appreciated in the separation of two isotopomers of the M+2 isotopologue related to the singly charged ion of the MRFA peptide: The m/z ratios relevant to MRFA isotopomers including either a 34S or two 13C atoms are 526.2607 and 526.2716, respectively.

A powerful hybrid system: Linear Ion Trap-Orbitrap A hybrid mass spectrometer combining a Linear Ion Trap (LTQ technology) and a Orbitrap has been recently introduced on the market and represents one of the most powerful instruments currently existing in MS: API Ion source Linear Ion Trap C-Trap Orbitrap

Ions generated in the API ion source are first trapped in the linear ion trap and can be then analyzed using the MS and MSn modes typical of the LTQ. Afterwards, precursor or product ions generated in the linear ion trap can be axially ejected from it and collected in a C-shaped ion trap (C-trap), from which they are subsequently passed into the Orbitrap mass analyzer:

C-trap The C-trap consists in the arrangement of four curved electrodes having a rectangular cross-section around a central bent axis: The inside rod (right) includes a slit, used to eject ions towards the Orbitrap analyzer. top bottom left right RF-waveforms out of phase by 180 degrees are applied to the top/bottom rods and to the right/left rods, respectively. top bottom left right

Ions with different m/z ratios are trapped and confined along the centerline of the trap; collisions with filling N2 gas enable ion cooling. Afterwards, the RF potentials are rapidly (ca. 200 ns) ramped down and DC potentials are applied to the rods to eject ions through the slot of the inside electrode. The curved shape of the C-trap electrodes enables the focusing of ejected ions with different m/z ratios:

Performances of LTQ-Orbitrap Accuracy > 4 ppm rms (external calibration) > 2 ppm rms (internal calibration) Resolution 60000 at m/z 400 with a scan repetition rate of 1 Hz Maximum Resolution >100000 Mass Range 50-2000; 200-4000 Sub-fmol sensitivity (LC/MS) MS/MS and MSn capabilities Wide dynamic range > 2500

The high accuracy of LTQ-Orbitrap can be clearly seen in the following spectrum, relevant to a peptide mixture:

Isotope distribution can be obtained even for highly charged proteins, as shown for the +10 ion of apo-myoglobin:

Possible operating modes for LTQ-Orbitrap Maximum productivity Cycle time: 1 second Maximum accuracy Cycle time: 2 seconds SE1 Full Scan MS SE1 Full Scan MS SE2 MS/MS SE2 MS/MS SE3 MS/MS SE4 MS/MS SE3 MS/MS SE4 MS/MS 1 LTQ Orbitrap high resolution full scan 3 parallel low resolution ion trap MS/MS scans 1 LTQ Orbitrap high resolution full scan 3 LTQ Orbitrap high resolution MS/MS scans

Maximum productivity mode: drugs mix Ketitofen S O N High resolution full scan 310.12653 R=81567 C19H20O1N1S1 = 1.67 ppm 96.08 RT: 2.87 MS2 of m/z 310 Scan # 214 50 100 50 100 Buspirone Propanolol Ketitofen 386.25583 R=72923 C21H32O2N5 = 2.01 ppm 292.11 260.16489 R=90556 C16H22O2N1 = 1.48 ppm 249.07 198.12 220 240 260 280 300 320 340 360 380 400 155.08 282.13 m/z 80 100 120 140 160 180 200 220 240 260 280 300 320 100 183.08 RT: 2.88 MS2 of m/z 260 Scan # 216 116.10 O N H O H 50 157.06 98.09 129.06 218.11 242.15 60 80 100 120 140 160 180 200 220 240 260 m/z 122.07 100 N N N O RT: 2.88 MS2 of m/z 386 Scan # 215 N N O 50 150.10 222.14 265.19 168.10 291.20 343.21 100 150 200 250 300 350 400

Maximum accuracy mode: drugs mix N O 50 100 150 200 250 300 350 400 122.07127 R=16524 C6H8N3 = -0.1 ppm 222.14905 R=12090 C13H20N1O2 = 0.9 ppm 150.10257 R=14682 C8H12N3 = -0.1 ppm 265.19122 R=11082 C15H25N2O2 = 0.6 ppm 343.21298 R=9670 C19H27N4O2 = 0.4 ppm 291.20752 R=10097 C17H27N2O2 = 2.8 ppm 168.10161 R=13750 C9H14N1O2 = -1.7 ppm Ketitofen High resolution full scan RT: 2.88 MS2 of m/z 386 Scan # 215 220 240 260 280 300 320 340 360 380 400 m/z 310.12653 R=81567 C19H20O1N1S1 = 1.67 ppm 386.25583 R=72923 C21H32O2N5 = 2.01 ppm 260.16489 R=90556 C16H22O2N1 = 1.48 ppm 50 100 Propanolol Buspirone 80 100 120 140 160 180 200 220 240 260 280 300 320 50 96.08073 292.11569 249.07368 198.12767 155.08583 282.13168 R=18184 C6H10N1 = -0.5 ppm R=10650 C19H18N1S1 = 0.8 ppm R=11447 C17H13S1 = 1.7 ppm R=13090 C14H16N1 = 0.3 ppm R=14022 C12H11 = 1.9 ppm R=10075 C18H20N1S1 = 2.1 ppm O N H 183.08052 116.10705 157.06490 218.11758 98.09639 242.15410 129.06998 R=13448 C13H11O1 = 0.4 ppm R=16833 C6H14N1O1 = 0.5 ppm R=14358 C11H9O1 = 0.7 ppm R=12101 C13H16N1O2 = 0.1 ppm R=18093 C6H12N1 = -0.4 ppm R=11542 C16H20N1O1 = 0.7 ppm R=14538 C10H9 = 0.8 ppm 60 80 100 120 140 160 180 200 220 240 260 m/z 50 RT: 2.87 MS2 of m/z 310 Scan # 214 RT: 2.88 MS2 of m/z 260 Scan # 216 S O N Note that accuracies are always better than 5 ppm.

Resolving Isobaric Compounds with LTQ-Orbitrap Lys-des-Arg-Bradykinin An example of the power of LTQ-Orbitrap is the resolution of two almost isobaric peptides, whose exact masses differ by less than 0.04 u: Lys-des-Arg-Bradykinin KRPPGFSPF 1031.5552 Val5-Angiotensin II DRVYVHPF 1031.5188

Even using a relatively high resolving power, 60000, the discrimination between the two peptides is only slight: [M+2H]2+ [M+H]+

Lys-des-Arg-Bradykinin When the resolving power is raised to 100000 a clear separation between peaks for the two peptides is achieved: Val5-Angiotensin II DRVYVHPF Lys-des-Arg-Bradykinin KRPPGFSPF

The evolution of mass peak separation with RP is apparent from this graph:

Comparison between the most common MS analyzers pros cons applications Quadrupole (Q) energy and spatial distribution of ions not critical; simple scanning method; low cost easy coupling to other quadrupoles or other MS analyzers • low-resolution (1000) low mass accuracy (100-1000 ppm) • limited mass range (approximately m/z 4000) • general low-resolution MS instruments • molecular weight determination 3D-ion trap (IT) energy and spatial distribution of ions not critical • low cost • small device • inherent MS/MS and MSn capabilities • low-resolution (1000-2000), unless special methods (like ZoomScan) are used • general low resolution MS instruments • molecular structure elucidation

pros cons applications Analyzer pros cons applications Linear Ion trap (LIT) • high-resolution achievable at low acquisition rate (30000 in Ultra-zoom scan mode) high sensitivity; high scan speed (> 15000 u/s) MSn capabilities up to n = 15 • limited mass range (approximately m/z 4000) • higher price than 3D- ion traps • high sensitivity measurements • molecular structure refined elucidation Time Of Flight (TOF) • high scan rate (up to 20000 scans/s) • high resolution (up to 20000) when reflectron(s) and high-speed electronics are used • virtually no limit on mass range (actual limit 106 u) • high sensitivity (femtomoles) • good mass accuracy (10-100 ppm) • strict demands on initial energy and spatial distribution of ions • high-performance electronics needed • low resolution (1000-2000) in linear mode. • high-resolution measurement using reflectron(s) • high-sensitivity measurements

pros cons applications Analyzer pros cons applications Orbitrap high mass accuracies (1-2 ppm) and resolution (150000) sub-femtomole sensitivity concomitant high accuracy and large dynamic and m/z ranges • high price • high vacuum requirements • high resolution measurements • high sensitivity measurements Ion Cyclotron Resonance (ICR) cell • unsurpassed resolution (up to 106) • high mass accuracy (1-2 ppm) • high detection efficiency • high mass range • detection not destructive (ion stability experiments possible) • inherent tandem MS capabilities. • high price (106 euros) • expensive mantainance (magnet) • highly demanding on computing facilities (FT calculations) • extremely high resolution measurements (low m/z ratios) • fundamental ion chemistry studies