Tendenze nell innovazione della strumentazione in spettrometria di massa: Trappola lineare, orbitrap, ion mobility e nuova strumentazione René Magritte La Condition Humaine 1
Ion Trap Mass Spectrometry Ion trap has been developped by the Bonn group in the early sixties. It represents one of the most simple devices (from the hardware point of view!!) with a series of capabilities which make it unique. Analizzatori quadrupolari Alcuni dispositivi basati su campi quadrupolari studiati dal gruppo di Paul (Univ. Di Bonn) negli anni sessanta. 2
Quadrupole ion trap q = -4zV/mr²ω² Quadrupole Mass Analyzers Differential Equations of Motion 2 2 z ( 4e/ m( r 0 2z 0 ))( U Vcos t) z 0 2 2 r ( 2 e/ m( r 0 2z 0 ))( U Vcos t) r 0 2 du [a2q cos ( )] u 2 0, where = t/2 d khz Mathieu Equation u( ) AC cos( 2n ) BC sin( 2n ) u 2n 2n n= n= u = ½ * where ion frequency of motion 3
The ion motion inside an ion trap will depend by: -the m/z ratio of the trapped ions; -The ion trap dimension (r); -The dc voltage applied to the intermediated electrode U; -The rf voltage applied to the intermediate electrode V; -The rf frequency. It can be convenient to express the stability conditions of an ion inside the trap by introducing two quantities which consider all the above parameters a=-16zu/mr 2 2 q=8zv/mr 2 2 Apply Waveform to Isolate Parent Ion 4
Apply Waveform: Excite and Fragment Mass Summary: Conceptual Ion Trap Operation API Source Ion Gate Ion Injection Scanned RF Helium Bath Gas Waveforms Ion Current Data System Detector 5
Ion trap limits: They are mainly due to space charge effects; An optimum number of ions must be present inside the trap for its effective operative conditions; The limitation of ion number inside the trap reflects on sensitivity limitation Possible solution: Linear ion traps (LIT) Shematic diagram of the Thermo Finnigan LIT mass spectrometer. The ion beam travels from left to right. Typical operating pressures are given. 6
Angled view of three sections of the two-dimensional LIT. The detector faces the center section. Ion Trap Space Charge limit 9.00E+10 6.00E+10 3.00E+10 Storage Space Charge Limit TIC = 7.3E10 TIC 1.00E+10 5.00E+9 Spectral Space Charge Limit TIC = 7E9 1.00E+9 1.00E+8 1.00E+7 Spectral Space Charge Limit TIC = 2E7 0.00E+00 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 7
Ion Ejection 2D vs 3D Ion Transfer Tube Skimmer Tube Lens Myoglobin Tryptic Digest 1fmol LCMS/MS 8
By LIT an effective increase of sensitivity has been achieved. To increase selectivity a further step is required, based on the development of systems operating in high resolution. Simulation Peptide mixture: [Val 5 ]-Angiotensin II Lys-des-Arg 9 -Bradykinin Sequence: DRVYVHPF KRPPGFSPF Formula: C 49 H 69 N 13 O 12 C 50 H 73 N 13 O 11 Exact mass: [M+2H] 2+ = 516.76671 [M+2H] 2+ = 516.78490 m (mmu): 18.2 mmu RP = 18,000 RP = 56,700 100 516.77581 (observed) 100 516.76671 516.78490 80 80 Intensity (%) 60 40 516.76671 (correct) 516.78490 (correct) Intensity (%) 60 40 20 20 0 516.65 516.70 516.75 516.80 516.85 516.90 m/z 0 516.65 516.70 516.75 516.80 516.85 516.90 m/z 9
Finnigan LTQ FT - FTICR MS Means: Fourier Transform Ion Cyclotron Resonance Mass Spectrometry The mathematical method applied to convert the acquired transient signal into a frequency spectrum The way masses are separated different masses have different cyclotron resonance frequencies in a magnetic field The analytical technique 10
1.a Risposta: FTICR La trasformata di Fourier 11
La serie di Fourier 12
Attention must be paid with the Fourier transform! 13
Linear Ion Trap FTICR Hybrid 2D Ion Trap Differentially pumped by turbo pumps ICR Cell 7 T Actively Shielded Superconducting Magnet The Orbitrap: a new mass spectrometer Qizhi Hu, a Robert J. Noll, a Hongyan Li, a Alexander Makarov, b Mark Hardman c and R. Graham Cooks a a Purdue University, Chemistry Department, West Lafayette, IN 47907, USA b Thermo Electron (Bremen), Hanna-Kunath-Str. 11, Bremen 28199, Germany c Thermo Electron (San Jose) 355 River Oaks Parkway, San Jose, CA 95134 USA JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2005; 40: 430 443 14
Electrostatic Traps...Static charges can not be stable in electrostatic fields... Linear traps Segmented-ring traps Orbital traps 15
Trajectories in the orbitrap Characteristic frequencies: Frequency of rotation ω φ Frequency of radial oscillations ω r Frequency of axial oscillations ω z r z z 2 R m R 2 1 φ k U ( r, z) z 2 2 2 r / 2 R ln( r / 2 m R m ) r z z R m R k m / q 2 2 Lord of the ion rings: Forging of the ring Electrodynamic Squeezing A.A. Makarov, Anal. Chem., v.72 (2000), No.6, p.1156-1162. A.A. Makarov, US Pat. 5,886,346, 1999. Fast Injection 16
Lord of the ion rings: Retainment of the rings k m / z 1. Frequencies are determined using a Fourier Transformation 2. For higher sensitivity AND resolution, transients should not decay too fast Ultra-high vacuum Ultra-high precision 17
LTQ-Orbitrap: 2 nd generation of the fast injection (orthogonal) 1. Ions are stored in the linear trap 2. are axially ejected 3. and trapped in the C-trap and squeezed into a smaller cloud 4. then a voltage pulse across C-trap ejects ions towards the Orbitrap 5. where they are trapped and detected V Gas <1 mtorr Orthobi (2004)- Thermo Bremen; Prototypes/Series x V -2 k V Central Electrode Voltage y -3.5 k V 18
Introduction to ion mobility methods 19
Ion mobility To perform an ion mobility experiment, ions are introduced into an atmospheric pressureregion(called drift tube ) across which an electric field is uniformely applied. The uniform field is generated by connecting a series of evenly spaced rings with equal value resistors. The time required for the ions to reach the detector depends upon the ion s - Collision cross section (averaged over all possible orientation of the ion); - charge state; - mass; - drift tube operating parameters (electric field strength, drift tube length, buffer gas pressure, temperature) 20
Some definitions: Reduced ion mobility K 0 = L*P*273 / t D *E*760*T Where t D is the drift time, L the length of the drift tube, P the pressure, E the electric field strength and T the temperature Collision cross section 18 16 1 2 ze k T 1 2 mi mb L P 273 N b 1 1 1 2 t D E 760 T 1 Conventional ion mobility spectrometers From Ion Source To Detector GATE Electric Field High Mobility Ion Low Mobility Ion 21
Protein conformation GATE Measuring the ion mobility of an ion can yield information about its structure as small, compact, ions drift quicker than large extended ions 22
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Flexibility Fragmentation can be induced in both TRAP and TRANSFER T-WAVES The system can operate in both Mobility- Tof and Tof only mode 24
Passato prossimo - presente Synapt HDMS system ESI Mass Spectrum of -Lactalbumin 8+ 100 7+ 9+ 10+ % 11+ 13+ 12+ 0 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 m/z 25
PAP_10.raw : 1 PAP_10.raw:1 PAP_10.raw : 1 2000 7+ 8+ m/z 1500 1000 11+ 12+ 13+ 10+ 9+ Trap Injection Voltage 5 V PAP_10.raw:1 7+ Drift Time High efficiency ion mobility Excitation in Trap T-Wave results in a more open conformation 26
PAP_10.raw : 1 PAP_10.raw:1 PAP_10.raw : 1 2000 7+ 8+ m/z 1500 1000 11+ 12+ 13+ 10+ 9+ PAP_10.raw:1 Trap Injection Voltage 25 V 7+ Drift Time Something new! Direct analyses on living bodies* *The lecturer advise against to try to perform these experiments by yourselves 27
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Paul Delvaux (1956) - Loneliness 29
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