Ion traps Quadrupole (3D) traps Linear traps
3D-Ion traps A 3D-ion trap can be considered the tridimensional analogue of the linear quadrupole mass analyzer: B A C D The central ring electrode of the 3D-ion trap can be imagined as generated by rods A and C, connected after being deformed. Rods B and D are ideally modified to generate the end caps.
The ring electrode and the end caps are assembled through insulating spacers, avoiding any possible electrical contact between them: The assembled 3D trap is actually less than 10 cm long and less than 5 cm large: End cap Ring electrode End cap
Geometry of 3D-ion traps A different co-ordinate system (with respect to quadrupole) needs to be defined to describe ion motion within the 3D-ion trap: The internal diameter of the ring electrode defines the r axis, whereas the axis of the two end caps corresponds to the z axis. The maximum displacements from the trap center permitted along the two axes are r 0 and z 0, respectively. An rf voltage is applied between the ring and end-cap generate an electric quadrupole field. electrodes to
The potential at any point (r,z) in the quadrupolar field is given by: where 0 is the potential applied to the ring electrode: As for the quadrupole, U and V are the dc potential and the rf potential amplitude, respectively, is the rf potential angular frequency. Ions of a given m/z value may undergo stable or unstable motion in the trap. The quantitative solution to stability conditions is described by second order differential equations of the Mathieu form. The solutions are readily summarised in the form of a stability diagram.
Mathieu stability diagram for a 3D-ion trap The Mathieu diagram for a 3Dion trap is still drawn within a (q,a) plane, but the co-ordinate definitions are slightly different than those for quadrupoles: The stability regions are now labelled as z-stable and r-stable and are not symmetrical. Once again, different z-r stable regions, like those marked as A and B, can be found in the diagram.
Region A is the one commonly exploited for 3D-ion trap operation: In the figure, lines corresponding to the same z and r parameters, in turn correlated to the oscillation frequency of ions in the z and r directions (by analogy with x and y oscillations of ions within the quadrupole), are also reported. It is worth noting that the stability region crosses the q axis, in this case.
After U, V, (i.e. 2 f, with f = 1.1 Mhz) have been fixed, along with r 0 and z 0, a and q coordinates can be calculated for any m/z ratio at any time and the motion for the relevant ion can be simulated: r z r direction A stable trajectory within the 3D-ion trap implies a continuous oscillation between the end caps and inside the ring electrode aperture. The oscillations describe a three-dimensional 8, which is slightly bended. Small oscillations are super-imposed on the main motion.
The ion motion can be inferred also by considering the potential surface in a quadrupole ion trap: During positive excursions of the potential applied to the ring electrode, the ion is pushed towards the end caps (i.e. towards z 0 or z 0 ) but far from the ring surface (r 0 0). During negative excursions, the surface shape changes so that the ion returns to the center (z 0 0) along the z axis but is pushed towards the inner surface of the ring (i.e. towards r 0 or r 0 ).
Starting from the a and q parameters for a specific ion under fixed experimental conditions (U, V, ), its oscillation frequencies along the z and r direction, known as secular frequencies, can be calculated: where u is equal to z or r, according to the cases, and u obtained from the Mathieu diagram. can be The fundamental secular frequencies (i.e. those corresponding to n = 0) are usually considered, since higher order frequencies are of little practical significance.
Ion trap operation The process leading a 3D-ion trap to a mass scan can be divided into four stages: 1. Ion trapping and ion cooling 2. Ion selection 3. Ion excitation 4. Mass scan
During stages 1-4 no further ion can be admitted within the trap, thus a electrostatic ion gate, i.e. a system of electron lenses controlling ion transfer into the trap, has to be present before the trap. Ions can be loaded within the trap only if the ion gate is open. For example, in the case of negative ions, this means applying a positive potential to electron lenses: When ion loading is complete the gate potential is switched to negative values, so that no further ions can enter the trap and the scan procedure can begin. After inverting the potentials the same scheme can be applied to positive ion mode.
Ion trapping and cooling Ion entering the ion trap can be trapped, i.e. stored within the device without hitting any of the electrodes or exiting through the end cap apertures, only if their (q, a) co-ordinates are included in a z-r stability region of the Mathieu diagram. Simulations of motion for ions with a specific m/z ratio, under certain U, V and conditions will show if trapping will actually occur.
Actually, optimum trap operation requires that the ions have favorable initial conditions, in particular, a proper distribution of kinetic energies. This adjustment is achieved by using low pressure helium gas to remove excess kinetic energy from the ions and cause them to occupy the central region of the trap (ion cooling):
The importance of ion cooling by helium buffer gas is clearly evidenced by these examples: MRFA
Ion trap loading: Automatic Gain Control (AGC) Typically the ion trap can hold up to 10 5 10 6 ions before mutual coulombic repulsions significantly affect their trajectories (space-charge effects) and greatly reduce mass resolution. In most modern 3D-ion trap instruments an automatic control of ion trap loading (Automatic Gain Control) is available to minimize such effects. The main stages of a AGC process are: 1) Loading of the ion trap for a definite time (10 ms) 2) Pre-scan of trapped ions 3) Evaluation of the signal obtained at the detector (AGC prescan signal) 4) Comparison between the obtained signal and a user-defined target value (depending on the MS experiment: full scan, SIM, SRM, CRM, etc.) 5) Adjustment of ion injection time to get compliance with target values.
The AGC Prescan Signal can be expressed by the relationship: where the number of ions is related to the loading time. The injection time (ion time) can be calculated, as a multiple of the presccan loading time, by the following simple equation:
Effect on mass resolution of the ion population loaded inside a 3D-trap; MRFA peptide:
Ion scanning based on mass-selective instability Differently from the quadrupole, in a 3D-ion trap a ion is required to become unstable, along the z-axis, to be transferred to the detector and thus provide a mass signal. The main approach to ion scanning with a 3D-trap is thus based on the mass-selective instability: In this case, the dc component of the 0 potential is equal to 0 and only a RF voltage is applied to the ring electrode: The rf voltage amplitude is raised to a certain value during ion trapping, then slightly lowered, to promote ion cooling. Afterwards, a linear increase is effected, to promote destabilization of ions with increasing m/z ratios.
According to the Mathieu co-ordinates for a 3D-ion trap: working with U = 0 means that the mass scan line is the q z axis: As the rf voltage amplitude, V, is increased, ions move to higher q values. Eventually, ions with the lowest m/z ratios reach the stabiliity boundary (q = 0.908).
Simulations show that a ion reaching the condition q > 0.908 becomes unstable in the z direction: 264 502 As the rf voltage V is increased, ions with increasing mass will reach the critical co-ordinate q z and will be ejected from the ion trap.
Stability diagram and mass range for a 3D-ion trap The m/z ratio of the highest mass ejected ion, for a given rf voltage V, can be easily calculated using the equation: In this case q z has to be replaced with the ejection value, 0.908. For typical ion trap geometries, like the one based on the equation r 02 = 2 z 02, with r 0 = 1 cm, a maximum rf voltage amplitude, V max, equal to 7500 V at = 1.1. MHz will determine a maximum ejectable m/z ratio equal to 650. The mass range can be extended: by increasing V max reducing the trap dimensions (r 0 and /or z 0 ) decreasing reducing the q z ejection value
The initial (and also minimum) amplitude of the rf voltage, V min, has a similar influence on the low mass limit, usually called exclusion limit or lowmass cut off (LMCO) of the ion trap. Variation of the low mass limit with the rf voltage can be easily seen by comparing the relative positions of ions with three different mass-to-charge ratios along the mass-selective instability line (a z = 0):
q z, Low Mass Cut Off (LMCO) and secular frequency calculation Let us consider a butylbenzene ion (m/z 134) in an ion trap with r 0 =1.00 cm and with z 0 = 0.783 cm and under the following conditions: The corresponding q z value can be easily calculated: As 0.450 < 0.908 the ion will follow stable trajectories inside the ion trap.
As far as the LCMO value is concerned, it is worth noting that the m q z product is constant at constant V: As a consequence, the follow relationship can be written: With a rf potential of 757 V (0-P) applied to the ring electrode, only ions with m/z > 66.4 will be stored in the ion trap.
The general equation for q z can be re-written in a further form: Consequently, also the (m/e)/v ratio is constant, for a given q z. The potential V to be applied to the ring electrode to get a given LMCO can be calculated by the following equation:
Calculation of secular frequencies can be made starting from the already shown equation: and considering that z (and, equivalently, r ) is given approximately by the relation: For the butylbenzene ion (m/z 134) at a rf voltage of 757 V, q z = 0.450, thus z = 0.318 and the fundamental secular frequency for the axial direction, z,0, corresponds to 1.049 10 6 rad s -1 or, equivalently, 167 khz. Similarly, a fundamental secular frequency for the radial direction, r,0, can be estimated.
Resonance Excitation As the motion of ions confined in a quadrupole 3D-ion trap is characterized by two secular frequencies, axial and radial, ion motion can be excited upon resonant excitation at either or both of these frequencies. Excitation along the the z axis can be effected, after ion cooling, by applying a small (a few hundred mv) supplementary potential, oscillating at the secular frequency, across the end-cap electrodes, thus working in dipolar mode. Under the effect of the exciting potential ions are forced to move away from the trap center and acquire higher kinetic energies (up to tens of ev).
A simulation of ion motion shows the destabilization occuring along the axial direction:
Resonant excitation can be used for several purposes: (i) to remove unwanted ions generated during ionization, thus isolating a narrow range of m/z ratios; in this case, wavebands of frequencies are applied to the end-cap electrodes to excite and eject many ion species simultaneously, thus leaving a single ion species (or a small range of m/z ratios) isolated within the ion trap; (ii) to increase ion kinetic energy, thus promoting endothermic ionmolecule reactions; (iii) to deposit internal energy in ions through momentum-exchange collisions with helium atoms, a fundamental process to promote fragmentation; (iv) to eject ions while the amplitude V of the main r.f. potential is being ramped up, a procedure known as axial modulation.
Space-charge effects and axial modulation One of the inherent features of ion trap is that while ions with lower m/z ratio (red) are being scanned out of the trap, those with higher m/z ratios (orange, blue) are still inside the trap. a q lim = 0.908 q The motion of higher m/z ratio ions generates an interference (spacecharge effects) on the trapping potential, causing a broadening of peaks related to lighter ions. The phenomenon, deleterious for mass resolution, can be reduced significantly by the so-called axial modulation, consisting in applying, between the end caps, a supplementary oscillating field of 6 V (p-p) at a frequency about one half that of the rf voltage applied to the ring electrode.
Under these conditions the secular motion of ions enters into resonance with the supplementary field just before they are ejected from the trap. The ions are energized and come into step, i.e. the energy broadening due to space-charge effects is recovered. Interestingly, axial modulation can be exploited also to extend the m/z range over the limit imposed by the maximum value attainable for the RF voltage on the ring electrode. As shown by the following calculations, supplemental RF voltages in the khz range result in extension of the higher limit of the ion trap m/z range from 650 units (attainable with a 7500 V rf voltage amplitude, at 1.1 MHz, in a ion trap with r 02 = 2 z 02, with r 0 = 1 cm) to 6500 units: q lim q lim
Resonance ejection A supplemental RF voltage applied to the end-caps can be exploited for a systematic ejection of unwanted ions from the ion trap. In this case also the supplemental RF voltage can be scanned, like the fundamental RF voltage:
In a figurative sense the application of a supplemental voltage at a specific frequency results in opening a hole (actually a resonance or instability point) in the stability diagram: The q z value at which the hole is located can be easily calculated from the z value corresponding to the secular frequency:
Ion isolation using resonance points A careful use of resonant ejection and RF voltage scan at the ring electrode can lead to isolation of ions having a narrow m/z range in the ion trap. At 1000 V none of the ions has a q z value approaching that of the resonance point so the ions remain inside the trap. Resonance (or instability) point q = 0.227 At 3000 V the ion with m/z 500 has been ejected and the one with m/z 1,000 is about to be ejected. At 6000 V the q z values for all the ions are greater than 0.227, the value of the resonance point.
A combination of forward and reverse resonance ejection ramps may be used to isolate ions in the trap: (i) reverse scanning resonantly ejects ions from high to low m/z (ii) forward scanning resonantly ejects ions from low to high m/z (iii) isolation of ions with a single m/z value is achieved
Further approaches to ion isolation Upper apex isolation Supposing that the orange ion, located in the A point of the stability diagram, is to be isolated, the following steps are followed: a the ion is moved to point B by increasing the RF voltage of the ring electrode: several (lighter) ions along the q axis will be ejected; q lim = 0.908 a dc voltage U is also applied to the ring electrode: the orange ion is lifted to the C point, the upper apex of the diagram. All the other ions are istantaneously placed outside the stability diagram. q the orange ion is brought back to the q axis.
Two-stage isolation The orange ion is moved from the A to the B point by reducing the RF voltage at the ring electrode; a it is then moved to the C point by applying a negative dc voltage to the ring; all the heavier ions (like the light blue ion) will be outside the stability diagram; q lim = 0.908 the dc voltage is put to 0 (C B), the RF voltage is increased (B D), then a positive dc voltage is applied to the ring: the orange ion is lifted to the E point. At this stage, all the other ions (lighter) are destabilised; the orange ion is taken back to the q axis (E D) q
A MS/MS experiment in a 3D-ion trap After isolation in the 3D-trap, an ion can be dissociated through collisions with helium atoms. Before proceeding with fragmentation, a stabilization is performed by lowering the RF voltage applied to the ring electrode: The procedure leads to a decrease of the q co-ordinate for the isolated ion:
Collisional Induced Dissociation (CID) is started by applying a Supplementary AC Voltage pulse between the end caps: Product ions, with lower m/z ratios, are generated inside the trap. Due to preliminary stabilization of the precursor ion (green), most new ions are also stable, as their q co-ordinates are lower than the critical value:
During the collisional stage the excitation voltage amplitude (usually referred to as Collisional Energy, CE) plays a key role in determining the relative intensity of the precursor and product ions in the MS/MS spectrum that will be subsequently acquired: Precursor ion (Product ions) It is worth noting that high excitation voltages result in loss of ions and thus of total intensity. This is due to ejection of the precursor and parent ions through the end-caps apertures before actual MS/MS scanning.
Product ions and residual precursor ions are progressively ejected outside the trap by raising the Main RF voltage applied to the ring electrode. Axial modulation is usually applied in this stage: a supplementary AC voltage is applied between the endcaps, at a frequency corresponding to a q value slightly lower than the critical one. A hole is then opened in the stability diagram. In this snap-shot of the Mathieu stability diagram, red ions are just falling into the hole : Conversion dynode Electron multiplier
While proceeding with Main RF and Supplementary AC voltages scan also yellow ions are ejected. In this snap-shot of the ion trapdetector assembly, the last electrons generated on the conversion dinode by red ions are visible while yellow ions are just being ejected from the trap: Conversion dynode At this stage, blue product ions and residual green precursor ions are still inside the trap: Electron multiplier
When ions hit the conversion dynode electrons are generated and subsequently accelerated towards the electron multiplier. The current signal is then registered as a function of the m/z ratio: a MS/MS spectrum is obtained. Conversion dynode Electron multiplier
A 3D-ion trap mass spectrometer: Thermo LCQ Skimmer 3D-ion trap Heated Capillary Interoctapole Lens Electrospray Source Tube Lens First Octapole Second Octapole Detector
Pros and Cons of 3D-Ion Trap Mass Analysers Compactness and mechanical simplicity Enhanced specificity and sensitivity for MS/MS experiments MS n (n up to 9-10) capability (simply by the use of additional ion isolations/fragmentations, performed sequentially in time) Poor dynamic range, due to space-charge effects (reduced by procedures like AGC) Relatively low resolution (about 1000-2000 at normal scan rates) and mass accuracy (usually higher than 100 ppm) Poor trapping efficiency (1-10% for externally generated ions)
Linear ion traps Since 1988 linear ion traps have been studied by the researchers at Finnigan as a possible alternative to 3D-ion traps. In 1995 Mark Bier and John Syka patented several types of ion traps with increased ion capacity, including the linear ion trap, which could be operated in the mass selective instability mode, the same method used in all commercial 3D traps for producing mass spectra.
Among traps patented in 1995 one of the most successful (and then applied to commercial instrumentation) is based on a quadrupole with hyperbolic rods cut into three sections, insulated electrically by quartz spacers: 37 mm 12 mm 12 mm Y Z X Front Section Center Section Back Section Slot: 30 mm 0.25 mm The length of front and back sections, identical, is 1/3 of that of the center section, whose peculiarity is having slots cut in the x rods.
In order to correct non-linearities in the quadrupole fields generated by the slots in the center section, x rods are slightly more distant than y ones from the quadrupole center: Y 0 = 4 mm X 0 = 4.76 mm The slots cut into the x rods are exploited for ion radial ejection towards two detectors, placed at the sides of the linear trap, as shown in the upper view of the device: Detector 1 Center Section Front Lens Front Section Detector 2 Back Section Back Lens
The division of quadrupole into three sections has a remarkable effect on the electric field operating within the center section. Field simulations obtained with the SIMION software Three-section trap show that fringe fields, clearly observed in a single quadrupole, are absent in the center section, thus enabling ion radial ejection to occur along the entire length of the section: One-section trap
Ions generated in the source are transferred into the linear ion trap by complex ion optics, subject to differential pumping. In the case of the Thermo Electron LTQ XL spectrometer, having a ESI interface with orthogonal geometry, the general scheme is the following: Electrospray Source Ion Transfer Ion Transfer Tube Tube Skimmer Square Quadrupole Skimmer Inter-multipole Lens 1 Split Gate Lens Detector 1 3 10-3 Torr He Center Section Tube Lens Square Quadrupole Octopole Tube Lens Front Lens Detector 2 Back Lens Front Section Back Section 8.3 L/s 25 L/s 300 L/s 400 L/s
A snapshot of ion transfer from the ESI source to the linear ion trap of the LTQ XL spectrometer: The picture shows ions with different m/z ratios (different colors) while being transferred through the Octopole preceding the linear ion trap. Helium atoms (white particles) are present inside the trap.
The introduction of octapoles (like in the LCQ instrument) and square quadrupoles (like in LTQ XL), extends ion transmission efficiency over wider mass ranges:
Linear ion trap voltages During linear ion trap operation three types of voltage can be applied to its sections. DC 1 DC 2 DC 3 y The LTQ uses three dc axial trapping voltages, one for each rod section, to establish axial (Z-axis) trapping. DC 1 DC 2 DC 3 z For positively charged ions the operating potentials for the three sections during ion storage and mass analysis are: Section Front (DC 1) Center (DC 2) Rear (DC 3) Ion Storage - 9 V - 14 V - 12 V Mass analysis + 20 V - 14 V + 20 V
Ion storage is effected by applying only an AC voltage (V) between the rod opposite couples. This means working on the q axis ot the Mathieu stability diagram for quadrupole: q y Once ions have been stored, a AC voltage (at a 1.2 MHz frequency), with amplitudes up to 10 kv peakto-peak, is applied between couples of opposite rods: RF - RF + RF + RF - y x
A comparison of potential surfaces obtained with a 3D and a linear ion trap shows that the trapping space is about 20 times larger in the case of the linear trap: 3D ion trap linear ion trap
Mass scan is performed by increasing linearly the AC voltage (Main RF Voltage): The limit q-coordinate is progressively reached by higher m/z ratios, which are then ejected through the slots cut in the x rods: q y
Supplementary voltages in the linear ion trap Ion isolation, excitation and ejection, useful for MS/MS and MS n operation, can be performed by applying an AC voltage only to two the exit rods (x), while the y rods are grounded (GND): AC+ GND GND AC - y x Three voltage types are usually adopted: ion isolation waveform voltage, i.e. an AC voltage with a distribution of frequencies between 5 and 500 khz, containing all resonance frequencies except those corresponding to the ions to be trapped; resonance excitation RF voltage, applied to excite a previously isolated ion and promote its dissociation induced by collisions with helium atoms; resonance ejection RF voltage, useful to promote resonance in ions that are about to be ejected, to improve mass resolution.
A combination between the main RF voltage and a supplementary AC voltage, applied between the x rods, can provide isolation of ions with a single m/z ratio: In this case, once grey and green-colored ions have been ejected using the Main RF voltage, a square pulse in the waveform voltage opens a hole in the stability diagram, where blue ions fall:
A MS/MS experiment with the linear ion trap Once isolated in the linear ion trap, red ions, representing precursor ions for a subsequent MS/MS experiment, can be stabilized by lowering the Main RF Voltage, so that their q co-ordinate is decreased: q y As a result, the oscillations of red ions within the trap are minimized:
Excitation of red ions is then promoted by a pulse of Supplementary AC voltage at the same frequency as their secular frequency. q y Product ions (blue, green and grey) are generated by collisions with helium atoms (Collision Induced/Activated Dissociation, CID/CAD):
In order to obtain a MS/MS spectrum, fast, contemporary scanning of the Main RF Voltage and of the Supplementary AC Voltage is effected: Product ions (and residual parent ion) are thus progressively destabilised, ejected through the x-rod slots and finally detected by one of the detectors placed outside the trap. In the snap-shot on the right green ions are being ejected from the trap and transferred towards the detectors, while red ions are still stable inside the trap:
Ion ejection towards the two side detectors occurs along most of the trap length, through the slot in the x-rods: A significant increase in sensitivity is achieved through this approach. Actually, due to high sensitivity, linear ion trap instruments can be operated in MS n mode with n up to 15!
Comparison between LTQ Linear Ion Trap and 3D Ion traps Trapping efficiency: 10 Detection efficiency: 2 Global efficiency: 10 Ionic capacity 20 Scan speed (u/s): 4 Zoom Scan scan speed: 4 Ultra Zoom scan speed: 27 u/s
Comparisons between LTQ Linear Ion Trap and 3D Ion traps Single scan 0.5 s Myoglobin spectrum and deconvolution Deconvolution 3D- trap Single scan 0.41 s Deconvolution Linear trap
Isotope distributions for caffeine (195), MRFA (524) and Ultramark (1522) in a LTQ MS spectrum obtained with a scan rate of 16700 u/s: A similar resolution can be obtained by LCQ only at a quite lower scan rate (5500 u/s).
Zoom scan spectra obtained by LTQ at 1100 u/s for the Melittin peptide (GIGAVLKVLTTGLPALISWIKRKRQQ, the principal active component of bee venom) at different charge states: A similar Zoom Scan resolution can be obtained by LCQ only at a 280 u/s scan rate.
Ultra-zoom scan is a peculiar, very slow mass scan (27 u/s) performed by the linear ion trap, providing very high resolutions, up to 30000:
Multiple-event MS experiments with linear ion traps In the MS jargon, an event is a specific MS, MS/MS or MS n acquisition, performed sequentially with others during a MS experiment. High scan rate MS instruments, like those based on ion traps, can perform several MS events in very short times. In the case of linear ion trap instruments this characteristic is pushed to astonishing results: without with Sequence of MS events acquired by LTQ within 4 s!
RP-LC-ESI-MS chromatogram, in Base Peak mode, for a mixture of 7 drugs, obtained by LTQ: ketoconazole 100 95 90 85 buspirone 4.49 317.29 6.04 531.45 6.28 480.36 80 75 Relative Abundance 70 65 60 55 50 45 40 35 timolol 4.01 350.15 5.31 386.45 6.80 243.23 bifonazole nicardipine 30 25 20 15 10 5 ampicillin 7.78 494.05 glyburide 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (min)
Multiple-event scan for ketoconazole: MS and MS/MS [M+H] + [M+2H] 2+ Wide band activation is a special MS/MS experiment in which both the parent ion and potential product ions arising from H 2 O or NH 3 losses are fragmented contemporarily.
Multiple-event scan for ketoconazole: MS 3 and MS 4 The high sensitivity of the linear ion trap provides high S/N ratio spectra even in MS 4 mode.
It is interesting to point out that noise is decreased more than signal when subsequent MS stages are considered, thus the S/N ratio will be higher for MS n events than for MS or MS/MS ones:
The fragmentation pattern rapidly obtained for each analyte using MS, MS/MS and MS n acquisitions can be used for structural elucidations: MS 2 (event 3) + N O O O N N Cl MS 3 (event 13) O + N N Cl MS 4 (event 14) + O Cl Cl m/z 446 Cl Cl m/z 281 m/z 173 N O N N O Ketoconazole [M+H] + m/z 531 N O O Cl Cl MS 2 (event 3) -CH 2 =CO (H migration) + N N O O O N N Cl MS 3 (event 4) O + N N Cl MS 4 (event 5) O + Cl m/z 489 Cl Cl m/z 255 Cl m/z 187