Modern Mass Spectrometry and Coupling Techniques
Rob Nieckarz
Office: HCI D325
nieckarz@org.chem.ethz.ch
Special Thanks to Dr. Richard Smith at the University of Waterloo
Gas Chromatography Liquid Chromatography Capillary Electrophoresis Spectroscopy
MS
MS + =
Added informaOon and greater
confidence in our analysis
Mass Separation and the Lorentz Force
NOTE:
• All mass analyzers function on the basis of the Lorentz Force equation which describes the force exerted on a charged particle in an
electromagnetic field. The particle will experience a force due to the
electric field (qE), and due to the magnetic field (qv x B). Combined they give the Lorentz force equation:
F = q(E + v x B)
– F is the force (in newtons)
– q is the electric charge of the particle (in coulombs) = ze – E is the electric field (in volts per meter)
– B is the magnetic field (in webers per square meter, or equivalently, teslas)
– v is the instantaneous velocity of the particle (in m/s)
Mass Spectrometry September 2004 4
Mass resolution: Definition
Valley
Peak width at half height
By courtesy of Spektrum Akademischer Verlag
Mass Resolution
• The FWHM definition is easier to apply (only need one peak), but gives a resolution about twice that of the
10% valley definition
• Resolution for sector instruments is usually given as the 10% valley figure.
• High resolution has some obvious advantages:
-It allows one to resolve ions that are isobaric
-The narrower a peak, the easier it is to measure its position accurately
Mass Spectrometry September 2004 5
Why do we need high resolution?
• To resolve single, adjacent peaks of high molecular weight compounds
• To enhance specificity
• To determine the elemental composition of a compound:
Example: Three different molecules may have the nominal mass 28:
CO = 27.9949
ΔM = 0.0112 M/ΔM = 2800 N2 = 28.0061
ΔM = 0.0252 M/ΔM = 1120 C2H4 = 28.0313
Mass resolution: peak shape
By courtesy of Spektrum Akademischer Verlag
16
Mass Resolution
• Low resolution: <2,000. Suitable only for nominal mass measurement.
• Medium resolution: 2,000-20,000. Suitable for accurate mass measurement. Resolve isotope clusters of high charge states.
• High resolution: >20,000. Better than medium resolution. You can never have too much resolution!
• In practice, there is a trade-off between resolution and sensitivity.
The ions are not coming from a point source: they exit the
source through a slit of finite dimensions, and cannot be perfectly focussed. Slits and lens help to compensate for this by cutting out ions from the centre of the beam and focussing. To get very high resolution, the slits have to be narrowed, which means that a lot of ions are lost.
Types of Mass Analyzers:
• Time of flight mass spectrometers (Tof)
• Fourier transform ion cyclotron resonance (FTICR)
• Orbitrap
• Linear quadrupoles (Q -‐mass filters)
• Three dimensional quadrupoles (ion traps -‐ IT)
• Linear iontraps (2D)
• Sector instruments
• Tandem instruments
Time of Flight Mass Analyzer
Fast, simple, high mass accuracy and resoluOon
Time of Flight (Tof)
Principle:
Ions of different mass (accelerated by the same field, V) have different velocities and thus flight times. The larger the mass the slower the ion:
K.E. = zeV = mv²/2 Ion formation:
Ions are introduced to the Tof in pulses (e.g. MALDI or orthogonal extraction from a continuous beam such as ESI)
Ion detected by analogue or time to digital converter (GHz ADC or TDC)
• Linear Tof (high mass range but low mass resolution)
• Reflectron Tof (lower mass range but high mass resolution)
54
Mass Separation: Time of Flight (Tof) MS
acceleration region
(drift region)
Basic Principles
Since the initial kinetic energy of the ions is given by:
zeV = mv²/2 (i)
velocity: v = (2zeV/m)1/2 (ii) time of flight: t = L/v = L[m/(2zeV)]1/2 (iii)
m/z = 2eVt2/L2 (iv)
Example:
For C6H5+. and C7H7+., (m/z 77 and 91), accelerated at 10kV, what are the
velocities of these 2 ions and how long would it take them to traverse a 2m flight tube?
using eqn (ii) v77 = (2x1x1.6022x10-19x10,000/m)1/2 m(kg) = 0.077/6.022x1023 = 1.279x10-25
v77 = 158,306m/s = 9498 km/h or 12.63μs similarly for v91 v91 = 145,621m/s = 8737 km/h or 13.73μs
V is the extraction pulse potential (V) L is the length of field free drift zone (m) t is the measured time-of-flight of the ion (s)
56
Example cont
• From eq (iii), difference in flight time:
tA/tB = (mA/mB)1/2
• Consequently, this square root relationship causes Δt for a given Δm/z to decrease with increasing m/z
• For example:
Δt/amu is calculated to be 114ns at m/z 20 to be 36ns at m/z 200 to be 11ns at m/z 2000
• Tof mass analyzer depends on the ability to accurately measure these short time intervals to make it a useful MS
57
Linear Tof
• Transmittance as high as 90%
• Ions introduced into the flight tube have a temporal and kinetic energy distribution which yields relatively poor mass resolution.
• Kinetic energy spread can be reduced by employing Delayed Ion Extraction
Principle of Delayed Ion Extraction:
• Ions are formed during a short pulse of a few nanoseconds
• The acceleration (extraction) field is only applied after a delay of some hundreds of nanoseconds:
• At the beginning of the extraction ions with high initial velocities have traveled further than slower ones. Therefore after the second extraction pulse they do not experience the full acceleration
potential.
• Thus the initially faster ions will be accelerated less than the initially
58
Reflectron Tof
Same m/z but different kinetic energy
• In a reflectron Tof, the ions traverse the drift tube and penetrate into an electric field (ion mirror) where their direction is reversed.
• Faster ions (with higher kinetic energy) penetrate farther into the electric field than slower ions (with lower kinetic energy).
• Thus faster ions have a longer flight path and therefore need
approximately the same flight time as the slower ions which have a shorter flight path.
59
Tof: Advantages and Disadvantages
• Good mass accuracy – reflectron ~ 5-10ppm
– limited with quadrupole MS, poor with ion traps and linear Tof
• High mass resolution
– reflectron ~5,000 to 20,000
– Quadrupole MS, ion traps and linear Tof operate closer to unit mass resolution at m/z ~ 103
• High mass range
– linear >105 Da, reflectron <104 Da
– Ion traps and quadrupoles are limited to ~6,000 Da
• Acceptable linearity for linear and reflectron Tof
– not as good as quadrupole MS, but similar to ion traps
• Very good scan-to-scan reproducibility for linear and reflectron Tof – as good as quadrupole MS
Fourier Transform Ion Cyclotron Resonance Mass Analyzer
UlOmate in mass accuracy and resoluOon
Expensive, difficult to operate
FTICR MS
• Basic Construction:
– a cell where ions are trapped by intense, constant magnetic field and applied voltage
– The cell accepts ions in a “pulsed” mode from the continuous ion beam – Detection of the ions is based on the FT deconvolution of the image
current the circulating ions induce in a pair of detector plates after excitation with a resonant Rf pulse.
63
Ion Trapping and FTICR MS
• ions enter the cell (or are created internally) and they begin their cyclotron motion, orbiting around the centre of the magnetic field
• since the magnetic field is quite high (typical minimum of 4.7T, but this is increasing) the ions are trapped in the radial (x,y) direction.
• Resolving power and scan speed increase linearly with B
• by applying small, equal potentials to the two end or “trapping”
electrodes, the ions are confined in the z or axial direction.
• ions can be confined for very long periods of time such that ion/molecule reactions or even slow unimolecular dissociation processes can be observed and monitored.
Ion Trapping and FTICR MS
m T
Cyclotron frequency Trapping oscillaKon frequency Magnetron frequency
FTICR MS Detection
• In FT detection, all ions, regardless of their mass are detected at the same time.
• Once ions are trapped inside the ICR cell they are excited by a fast sweep of all the Rf frequencies, exciting the ions to cyclotron motion with a larger radius.
Ions before excitation.
They have their natural cyclotron radius within the magnetic field.
67
FTICR MS - Detection
When a packet of ions (+ve)
approaches an electrode, electrons are attracted from ground and accumulate in that electrode causing a temporary current.
As the ions continue to orbit, the electrons accumulate in the other
electrode. The flow of electrons in the external circuit represents an image current. The amplitude of the current is proportional to the number of ions in the packet.
FTICR - Detection
• the frequency of the image current oscillation is the same as the frequency of the ion’s cyclotron motion which is related to mass. A small AC voltage is created across a resistor and is amplified and detected.
• using FT techniques all ion packets, each containing ions of the same mass, are detected. The decay of the image current (as the excited cyclotron orbit radius decays) is detected in time and transformed into a frequency domain signal by a
Rf Excitation
Detected time domain image current
Resulting mass domain Spectrum
Fourier Transform
Time
Time
m/z
How fast are the ions moving?
Frequency = 1 x 106 s-‐1
d = 0.05 m
Distance travelled = πd = 0.157 m
Speed = 0.157 m / 1x 10-‐6 s = 157 000 m/s
= 9 424 km/hr Time for one revoluOon = 1 x 10-‐6 s
FTICRMS
• Very high resolution is possible. The current record is 8x108, and routine values are 100,000 or so.
• Long trapping times are possible, allowing for ion-molecule reactions.
• Good sensitivity.
• Like the ion trap, the FTICR cell works well with pulsed sources.
• MSn capability
• However, expensive because of the cost of superconducting magnets and the very high vacuum requirements.
• Difficult to operate
Orbitrap Mass Analyzer
The only new mass spectrometer concept to be developed in the last 30 years
The only commercial instrument that can come close to the
performance of an FTICR
Orbitrap Summary
-‐ High performance mass analyzer -‐ Resolu:on up to 200,000
-‐ Mass Range up to 50,000
-‐ High mass accuracy (1-‐2 ppm) -‐ Non-‐destruc:ve ion analyzer -‐ MSn possible
-‐ CID und H/D exchange possible
Quadrupole Mass Analyzer
Cheapest instrument Fast acquisiOon Omes
20
Linear Quadrupoles (2D - mass filters)
Linear Quadrupoles (2D - mass filters)
• Four hyperbolic rods (cheap version: circular rods) – compromise!
• Opposite pairs of rods are connected electrically but are of opposite polarity
• Each pair of rods has a DC (U) + AC (V0 cosωt) Rf voltage applied:
1 pair of rods: -(U + V0 cosωt) and the opposite pair: +(U + V0 cosωt) where, ω = radial frequency = 2πf
• During a mass scan, the DC and AC voltages are ramped but the ratio of DC/AC (ie U/V0)is kept constant
• For a given DC and AC amplitude, only ions with a given m/z (or m/z range) have stable oscillations and are transmitted and can be detected
22
Quadrupole (end view)
Hyperbolic Round
Equipotential Field Lines
Linear Q: Equations of Motion
From the electrical part of the Lorentz equation, we can derive the equation of motion (x and y directions) for a particle in a combination of DC and AC Rf fields the Mathieu equation:
– u represents the x or y transverse displacement. We do not
consider displacement in the z direction because the electric field is 0 along the asymptotes of the hyperbolic rods.
– The 2 parameters characteristc of the field (a and q) are given by:
d u
d a
uq
uu
2
2
2 2 0
ξ + ( − cos ξ ) =
2 2
8
ϖ
mr a zeU
a
x= −
y= 4
2 2ϖ mr q zeV
q
x= −
y= −
and
26
• Where the variable ξ is the time in radians of the applied field = ωt/2
• U is the DC voltage and V is the AC Rf voltage of frequency ω
• r0 is the radius of the instrument aperture
• Plotting a against q gives the Mathieu stability diagram of the linear quadrupole field - a/q = 2U/V
• Typical values are:
- U = DC voltage (~200 - 1000V)
- V = AC voltage (~1000 - 6000V, 1-2MHz),
- m = mass of ion, e = electonic charge, z = # of charges on ion - 2r0 = distance between the rods - 1-2 cm
Linear Q: Equations of Motion
Stability Diagram
28
aq Space
• Note:
– Both +ve and –ve abscissa with a values ranging up to 10 and q values ranging up to 20
– In practice we only operate in the +ve area of region I Why?
– Because in order to have a and q values >1 we would require VERY high DC and AC voltages which is not practical
Stability Diagram
X unstable
L1, only 1 ion has a stable trajectory all others ions are lost therefore adjacent ions are resolved from each other
L2, 3 ions have a stable trajectory at the same time therefore these 3 ions would not be resolved from each other
In practice, the ratio of a/q is changed by changing the DC voltage
0.1 0.3
0.2
a
Y unstable
X and Y Stable
L1
L2 L1 = L2 Operating lines
a/q constant
. .
. . . . . . .
.
What would happen if no DC voltage is applied?
31
Conceptualizing a Q scan
0.1
0.8 0.4
0.3
0.2
a
q
m1 < m2 < m3
stable region of m1 stable region of m2
stable region of m3
Operating or scan line
Mass Range and Resolution
• Depends on 5 parameters:
• Rod length (L) – 50 to 250mm
• Rod diameter (r) – 6 to 15mm aligned to μm accuracy
• Maximum supply voltage (Vm)
• AC (Rf) fequency (f)
• Ion injection energy (Vz) - ~5 volts
• From the theory of quadrupole operation the following relationship can be derived:
Mmax = 7x106Vm/f2r2
Consequently, as r and f increase, Mmax decreases and as r and f decrease, Mmax increases
33
• The resolution limit of a quadrupole is governed by the number of cycles of the Rf field to which the ions are exposed:
M/ΔM = 0.05 fL m/2eVz
Mass Range and Resolution
2
• Consequently, as both f and L increase so does resolution.
If L in increased then f can be decreased and vice versa
• Scanning speeds as high as 6,000 amu/sec and mass resolution of 10,000 is attainable
Linear Q
Advantages:
• Small and light weight ~20 cm long
• Inexpensive
• Simple to operate – complete computer control
• Low accelerating voltage – handles high source pressures better
• Full scan mass spectra and selected ion monitoring (SIM) for quantitation
Disadvantages:
• Unit mass resolution only and limited mass range
• High mass discrimination
• Rod contamination causes further imperfections in the quadrupole field – compromises resolution and sensitivity
35
Linear Q
Other applications:
• QQQ for MS/MS
• Hybrid instruments eg BEQQ and QqTof
• Ion lenses (hexapoles and octapoles)
• Collision chambers for MS/MS ie QQQ and BEQQ etc
• Prefilter – before mass resolving rods to reduce contamination
Quadrupole 3D Ion Trap (QIT)
Ion trap consists of three electrodes:
• ring electrode (hyperbolic shape)
• 2 hyperbolic electrodes - end caps
• Orifice for ion injection
• Orifice for ion ejection
• Pulsed introduction of ions
Cap
Cap Ring Cap
Cap Ring Cap
Cap Ring Cap
Cap
Ring r0
38
QIT (properties)
• Ion trap volume very small (7mm i.d.)
• High sensitivity (10-18 mol) (scan mode)
• High mass range : 6,000
• Higher mass resolution than Q ~x2-3
• High dynamic range: 106 depending on space charging
• MSn capabilities
• Low mass cut-off is a disadvantage
• Helium is introduced intentionally into the ion trap (10–3 mbar)
– Needed as a buffer to absorb kinetic energy of incoming ions without chemical interaction so they can feel the effect of the trapping field - dampening
(cooling) of oscillations
– collision partner for MS/MS and MSn
• Ions are concentrated in center of ion trap
• Better resolution and better sensitivity than Q
QIT (ion motion)
• Between the three electrode a quadrupole field exists, which forces the ions to the center of the trap
• The farther the ion is removed from center of trap the stronger is the exerted electric force
• The ions oscillate within the trap, but with a rather complex sinusoidal motion
• The ion motion can be described by Mathieu’s differential equations
40
Quadrupole 3D Ion Trap (QIT)
• For the QIT, the electric field has to be considered in 3 dimensions. The electric field can be descibed by the expression:
Φx,y,z = Φ0(r2 - 2z2) r02
• The Mathieu equation still applies:
• The equations of ion motion in the r and z direction are:
d²z/dt² - (4e/mr0²) [(U - V cos2ωt)z = 0 d²r/dt² + (2e/mr0²) [(U - V cos2ωt)r = 0
d u
d a
uq
uu
2
2
2 2 0
ξ + ( − cos ξ ) =
Quadrupole 3D Ion Trap (QIT)
•Solving these Mathieu type differential equations yields the parameters a
zand q
za
z= -2a
r= 16zeU
m(r
0² + 2z
02) ω ² and q
z= -2q
r= -8zeV
m(r
0² + 2z
02) ω ²
Where ω = 2 π f, f = fundamental R
ffrequency of the
trap (~1MHz)
42
QIT (Ion stability diagram)
courtesy of Spektrum Akademischer Verlag
Ring Electrode
Ring Electrode Endcap
Endcap
q = 0.908
q < 0.908
QIT (stability diagram)
• Ions are only stable both in r and z direction for certain defined values of a and q
• Ions oscillate with so called “secular frequency”, f, which differs from the frequency of applied Rf field because of inertia (in addition oscillations of higher order)
• Ions of different m/z are simultaneously trapped, V
determines low mass cut-off at qz = 0.908, which increases with V
44
QIT (mass selective ion stability scan)
• Mass scan is possible by increasing the amplitude of the voltage on the ring electrode (U = 0, az = 0 ie no DC
voltage)
• Scan line: While scanning along this line (a=0) ions become increasingly non stable and exit the stability diagram at qz = 0.908.
• Trajectory of these ions in z- direction.
• Ions exit from trap through holes in end cap.
• Linear scan function
Space Charging
m/z 530 0
20 40 60 80 100
Relative Abundance
524.3
525.3
526.3
530
0
20 40 60 80
100 524.4
525.4
526.3 527.5
530 0
20 40 60 80
100 524.5
525.5
526.5 527.5
530 0
20 40 60 80
100 524.8
525.7
526.7
522 522 522 522
~ 300 Ions ~ 1500 Ions ~ 3000 Ions ~ 6000 Ions
Good resolution and mass accuracy
Poor resolution
and mass accuracy
48
QIT (space charge)
• With increasing number of ions trapped the space charge increases
• Space charge distorts the electric field
• Deterioration of resolution, sensitivity and mass accuracy Solution:
Pre-scan or measure in real time to control the number of ions (or more correctly, the number of charges) in the trap (a maximum of ~103 - 104)
Linear (2D) traps
• Similar idea to 3D traps with a “new” 2D geometry
• Rf only quads with DC voltage end electrodes
• Larger size than 3D IT – higher ion capacity (~x50) therefore fewer space charge problems
• More than one design for this type of trapping instrument
• Hybrids such as QQQ where Q3 can also be used as a linear trap and LT-FTICR
50 Axial Trapping
Exit Lens Radial Trapping RF Voltage
Radial Trapping RF Voltage
Axial Trapping
DC Voltage
Resonance Excitation
Trapping Forces in a Linear Ion Trap
Courtesy of Sciex
Linear Ion Trap vs 3D Trap
No low mass cut-off
Trapping Efficiency: >10
Detection Efficiency: doubled Overall Efficiency: >10
Ion Capacity (Spectral): >20 Scan Rate (amu/sec): 4x
Highly Efficient MSn: 5x over 3D IT
Electric and MagneOc Sector Mass Analyzer
The ‘original’ commercial MS systems High resoluOon, but comes at the cost of sensiOvity
detector!
m/z = eB
2r
2/2V
• Therefore specific values of V and B allow ions unique in m/z to pass to the detector. Variations in V or B will cause
ions to collide with the walls of the flight tube therefore at any unique value of V or B only one specific ion will be passed to the detector. In practice only B scans are preferrred when generating full scan data over a large (>50Da) mass range
• One exception to this is when high resolution, accurate mass measurements are made where Vacc scanning is
preferred as voltages can be controlled and measured much
Mass Separation: Magnetic Fields
7
Deflection of ions of different masses in a constant magnetic field
•This is how Aston’s original mass spectrograph operated!
• In modern instruments, the magnetic field is scanned to bring ions of different m/z ratios successively to the detector
Directional (angular) focusing of a magnetic field
Divergent ions of the same m/z will be brought into focus by a magnetic field
9
Mass Separation: Magnetic Fields
• One significant drawback with employing B scans is that the initially accelerated ions have a kinetic energy spread which exhibits itself as increased peak width ie low
resolution.
• To overcome this problem an electric sector (ESA) is combined with the magnetic sector to produce what is called a double focusing instrument.
“New” Developments in Magnetic Sector Instruments
• Large, high field magnets
– Mass range up to 10,000 Da at full accelerating potential (10 kV) for analysis of large biopolymers – Example: bovine insulin (MW 5734)
• Laminated magnets
– To reduce magnetic hysteresis
– Total cycle time < 1 sec, fast scanning
8 C15
C9 C21
Analysis of diesel fuel sample 1:1000 in DCM, 60m DB5 column
Petroleum Hydrocarbons
Analysis of diesel fuel:
• typically by GC or 2D GC-‐(TOF)MS
• what other compounds can be detected with ultra high resoluKon mass spectrometry?
Zoom
Diesel 1:1000 in MeOH + 0.2% formic acid
10
Zoom
Analysis of diesel fuel sample
Zoom
Analysis of diesel fuel sample
12 C17H34ONa+1 (0.03 ppm) C16H30O2Na+1 (0.00 ppm)
C15H26O3Na+1 (0.28 ppm) C14H22O4Na+1 (0.11 ppm)
C13H18O5Na+1 (0.18 ppm) C17H18O2Na+1 (0.00 ppm)
C13H18O4K+1 (0.11 ppm)
C15H26O2K+1 (0.14 ppm) C18H22ONa+1 (0.40 ppm) C18H22K+1 (0.32 ppm)
Analysis of diesel fuel sample
110,000 FWHM 29,000 FWHM 7,000 FWHM 1,500 FWHM C17H18O2Na+1 (0.00 ppm)
Analysis of diesel fuel sample
14 110,000 FWHM
2048K data points
29,000 FWHM 512K data points
7,000 FWHM 128K data points
1,500 FWHM 32K data points
Analysis of diesel fuel sample
110,000 FWHM 29,000 FWHM 7,000 FWHM 1,500 FWHM
Analysis of diesel fuel sample
16 110,000 FWHM
2048K data points
29,000 FWHM 512K data points
7,000 FWHM 128K data points
1,500 FWHM 32K data points