GRADUATE COURSE IN MASS SPECTROMETRY: LECTURE 2
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1 DEPARTMENTS OF CHEMISTRY AND BIOCHEMISTRY GRADUATE COURSE IN MASS SPECTROMETRY: LECTURE 2 Mass Analysers Shabaz Mohammed October 20, 2015
2 High Vacuum System Turbo pumps Diffusion pumps Rough pumps Rotary pumps Inlet Ion Source Mass Filter Detector Data System Sample Plate Target HPLC GC Solids probe MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap FTMS Microch plate Electron Mult. Hybrid Detec. PC s UNIX Mac
3 Mass Analyzers Quadrupole Analyzer (Q) Ion Trap Mass Analyzer (QIT) Time-of-Flight Analyzer (TOF) Orbitraps(Orbi)
4 Electromagnetism Force or F = q 2 x q 1 4πεr 2 or q 2 x V 1 -V 2 d q 2 Lens at Voltage V 1 r q 2 d q 1 charge Lens at Voltage V 2
5 Electromagnetism In mass spectrometry we work with charges and lenses 0V +200V + Same situation as +1000V +1200V -200V 0V It s the difference that matters
6 Electromagnetism In mass spectrometry we work with charges and lenses 0V -200V + Same situation as +1000V +800V -200V -400V It s the difference that matters
7 Electromagnetism Work or energy W= q 2 x V 1 -V 2 d x d Lens at Voltage V 1 q 2 d Lens at Voltage V 2 q is often expressed in multiples of e (charge on an electron) i.e energy is expressed in ev (electron volts)
8 Electromagnetism V Work or energy W= 1 -V q 2 2 x x d d 0V -200V + Potential energy 300 ev If q is 1 charge then: Loses 200 ev of potential energy Gains 200 ev of kinetic energy 0 ev
9 Time-of-flight Mass Analyzer Analyser performs the equivalent of the 100m sprint for ions Start line Known Distance (L) Finish line Ions are given the same amount of energy through a pulse (Energy is proportional to charge and the applied potential. E=zeV, z is number of charges, e is the amount of charge on an electron, V is volts) Ions then move at the speed determined by their mass (E=0.5mv 2, v for velocity (L/t)) i.e. velocity goes down as mass goes up Distance to finish line is established and stopwatch (t) is accurate to 1 nanosecond or better So. zev 2 mv 2 which can be turned into m z t 2eV L 2
10 ToF: Problems Starting position is not always the same! A) Temporal: Ions of the same mass can form at different times but have the same KE. B) Spatial: Ions of the same mass form having the same KE but in different parts of the source; C) Kinetic Energy: Ions of the same mass can form with different KEs. D) Direction: There is also the problem that when an ion forms, it may do so with an initial velocity in the opposite direction.
11 Ion formation problem solution: Time Lag Focussing/Delayed extraction 0 V Ions of same mass, different velocities Step 1: No applied electric field. Ions spread out. 0 V. 20 kv V. Step 2: Field applied. Slow ions accelerated more than fast ones kv. + Step 3: Slow ions catch up with faster ones. 0 V.
12 Improving resolution: The reflectron Reflectron is an electrostatic hill for the ions to try and overcome kv In reality they fail and are turned around Ions that have a higher velocity will have more kinetic energy and will climb the hill further before being turned around.
13 Improvement of resolution Linear Spec #1 MC=>SM3=>MC=>MC[BP = , 2191] Continuous extraction 70 % Intensity Resolution = 500 Delayed extraction Mass (m/z) Reflector Linear Spec #1 MC=>SM3=>MC=>MC[BP = , 1617] Linear mode % Intensity Resolution = 3,500 + Delayed extraction % Intensity % Intensity Mass (m/z) 4700 Reflector Spec #1 MC=>MC[BP = , 2642] Resolution = 10, Mass (m/z) Reflector Spec #1 MC=>MC=>MC[BP = , 1751] m/z = Resolution = 20, Reflector Reflector mode Delayed extraction + Reflector Reflector mode + Delayed extraction + Reflector Mass (m/z)
14 Quadrupole Mass Analyser: Description -[U+Vcos( t)] -(U+ 2r y y z x x +[U+Vcos( t)] +(U+Vcos t Consists of Four Metal Rods Opposite Rods have same voltage and adjacent rods have opposite voltage The voltage is a combination of a constant potential (U) and an alternating potential (V)
15 Quadrupole Mass Analyser: Description Voltage cos( t) t 3 0V t 2 t 4 U V t 1 t t 1 2 t t 3 4 Taken from Roman Korner: quadrupole mass analyzers. BM58/BMP58 Course 1998.
16 Quadrupole Mass Analyser: Description Ions are given a small amount of kinetic energy (1-2 ev) to allow them to traverse the quadrupole to the detector (assuming they have stable trajectories)
17 Quadrupole Mass Analyser: Scanning voltage Complete mass scan time
18 Quadrupole Mass Analyser: Scanning products of fib a m+2h collsion gas =6.8e-4, ce=40 SM (3.073) Daughters of 769ES+ 4.72e % m/z
19 Quadrupole Mass Analyser Used to be one of the cheapest mass spectrometers on the market Very good for precursor ion scanning and selective reaction monitoring
20 -[U+Vcos( t)] -(U+ Vcos t 2r y +[U+Vcos( t)] y z x x +(U+Vcos t
21 Quadrupole Ion Traps: Description
22 Quadrupole Ion Traps: Description Taken from: Web Site of Chemistry Department Purdue University,Indiana, USA RF voltage is applied to ring electrode to produce a three-dimensional quadrupole electric field for trapping ions.
23 Quadrupole Ion Traps Taken from: Web Site of Chemistry Department. Purdue University, Indiana, USA
24 2D Ion Trap Mass Analyzer LTQ
25 A Little Bit About Orthogonal ToFs DETECTOR Important Factors in on-axis MALDI-ToF: TOF Resolution and mass accuracy depend on knowing exact distance, time and energies given to the ions SOURCE Ions form in the same direction as ToF analyser and so the source conditions are important factors in mass accuracy and resolution e.g. Starting position of ions Starting kinetic energy and direction of ions
26 A Little Bit About Orthogonal ToFs Collisional Cooling of ions (usually in a quadrupole) before they enter the ToF region helps further improves ToF sensitivity and Resolution. Cooling allows better positioning of ion packet into pusher region of the ToF
27 A Little Bit About Orthogonal ToFs DETECTOR TOF Reducing the ions velocity here Allows better focusing of ions here SOURCE Collisional cooling of ions increases mass accuracy and resolution
28 The Orbitrap Positive ion moving very fast + Positive ion attracted and has its path bent - + Attraction of rod is high, Velocity of ion is high.. Ion makes an orbit around rod Very high negative potential Similar to a satellite around the earth
29 LTQ Orbitrap Hybrid Mass Spectrometer Finnigan LTQ Linear Ion Trap API Ion source Linear Ion Trap C-Trap Differential pumping Orbitrap Differential pumping
30 LTQ Orbitrap Operation Principle 1. Ions are stored in the Linear Trap 2.. are axially ejected 3.. and trapped in the C-trap 4.. they are squeezed into a small cloud and injected into the Orbitrap 5.. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier Ions of only one mass generate a sine wave signal
31 Detection:Fourier Transform Samples are typically not just 1 ion but several and on top there will be ions with several different m/z values. Typical image current for a simplish mixture looks like this: Time How do we deduce the individual frequencies?
32 Detection:Fourier Transform Luckily waves do not affect each other and so within the messy image current, the waves are present intact. Fourier transform is a mathmatical technique which can deduce the frquencies present.
33 Resolution The longer you measure, the better the spectrum.more datapoints for final deduction
34 Frequencies and Masses The axial oscillation frequency follows the formula Where w = oscillation frequency k = instrumental constant m/z =. well, we have seen this before k m / z Many ions in the Orbitrap generate a complex signal whose frequencies are determined using a Fourier Transformation
35 Collision induced dissociation Mixture MS 1 Collision cell MS 2 Accelerate ions into collision cell Potential energy -10 ev -50 ev Delta Kinetic energy 40 ev 0 ev
36 m/z Collision induced dissociation Mixture MS 1 Collision cell MS 2 sm (0.950) Cm (11:20) 100 TOF MS ES+ 7.02e3 sm (0.781) Cm (6:19) 100 TOF MSMS ES+ 1.64e3 sm (0.043) Cm (1:23) 100 TOF MSMS ES+ 708 % % % 0 m/z m/z A mass spectrum of a mixture Isolation of an ion In our case a protonated peptide A mass spectrum of the fragments Produced by the ion
37 Q Exactive Beam CID fragmentation mass selection Remove neutrals D30 high resolution MS and MS/MS
38 Q- ToFs
39 Orbitrap Fusion CID Ion trap CID fragmentation mass selection Beam CID fragmentation Redesigned instrument capable of: 15 MSMS HCD (orbitrap) per second 20 MSMS CID (ion trap) per second
40 Mass Analyzers Quadrupole Analyzer (Q) Low (1 amu) resolution, fast, (relatively) cheap Ion Trap Mass Analyzer (QIT) Fair resolution, all-in-one mass analyzer Time-of-Flight Analyzer (TOF) Good resolution, exact mass, fast, no upper m/z limit, costly Orbitraps(Orbi) High resolution, exact mass, costly
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