Download Modern Mass Spectrometry and Coupling Techniques

Modern Mass Spectrometry and Coupling Techniques Rob Nieckarz Office: HCI D325 [email protected] Special Thanks to Dr. Richard Smith at the University of Waterloo MS + Gas Chromatography Liquid Chromatography Capillary Electrophoresis Spectroscopy 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)
q = ze therefore, F
= zeE+zevB
2
Mass resolution: Definition
Valley Peak width at half height By courtesy of Spektrum Akademischer Verlag Mass Spectrometry September 2004 4 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
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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 Spectrometry September 2004 5 Mass resolution: peak shape
By courtesy of Spektrum Akademischer Verlag Mass Spectrometry September 2004 6 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.
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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)
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Mass Separation: Time of Flight (Tof) MS
acceleration region
(drift region)
54
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
v91 = 145,621m/s = 8737 km/h or 13.73μs
similarly for v91
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)
e = 1.6022x10-19C
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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
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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
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slower ions.
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.
58
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
•
Very fast aquisition time (up to kHz)
59
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.
61
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
63
Ion Trapping and FTICR MS
•
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.
64
Cyclotron frequency Trapping oscillaKon frequency Magnetron frequency m T FTICR MS Detection
•
In FT detection, all ions, regardless of their mass are detected at the
same time.
Ions before excitation.
They have their natural
cyclotron radius within the
magnetic field.
•
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.
65
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.
67
FTICR - Detection
Rf Excitation
Time
Detected time domain
image current
Time
Fourier Transform
Resulting mass domain
Spectrum
•
•
m/z
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
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Fourier transform.
How fast are the ions moving? d = 0.05 m Frequency = 1 x 106 s-­‐1 Time for one revoluOon = 1 x 10-­‐6 s Distance travelled = πd = 0.157 m Speed = 0.157 m / 1x 10-­‐6 s = 157 000 m/s = 9 424 km/hr 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
69
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 Linear Quadrupoles (2D - mass filters)
20
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
21
Quadrupole (end view)
Hyperbolic
Round
Equipotential Field Lines
22
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:
2
d u
+ ( au − 2qu cos 2ξ )u = 0
2
dξ
– 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:
8 zeU
a x = −a y =
mr02ϖ 2
and
− 4 zeV
q x = −q y =
mr02ϖ 2
25
Linear Q: Equations of Motion
•
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
26
Stability Diagram
27
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
28
Stability Diagram
L1, only 1 ion has a stable trajectory all
others ions are lost therefore adjacent
ions are resolved from each other
a
0.3
Y unstable
0.2
Operating lines
a/q constant
.
.
.
.
. . ..
..
0.1
L1
L1 = L2
L2
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
X unstable
X and Y Stable
0.4
0.8
q
What would happen if no
DC voltage is applied?
29
Conceptualizing a Q scan
a
0.3
Operating or scan line
stable region of m1
0.2
stable region of m2
m1 < m2 < m3
stable region of m3
0.1
0.4
0.8
q
31
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 R
( f) 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
32
Mass Range and Resolution
• The resolution limit of a quadrupole is governed by the
number of cycles of the Rf field to which the ions are
exposed:
2
M/ΔM = 0.05 fL m/2eVz
• 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
33
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
34
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
35
Quadrupole 3D Ion Trap (QIT)
Ion trap consists of three electrodes:
Cap
Cap
Ring
r0
Cap
• ring electrode (hyperbolic shape)
• 2 hyperbolic electrodes - end caps
• Orifice for ion injection
• Orifice for ion ejection
• Pulsed introduction of ions
36
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
38
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
39
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 = Φ (r2 - 2z2)
r02
0
• The Mathieu equation still applies:
d 2u
+ ( au − 2qu cos 2ξ )u = 0
2
dξ
• 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
40
Quadrupole 3D Ion Trap (QIT)
•Solving these Mathieu type differential equations
yields the parameters az and qz
az = -2a r = 16zeU
m(r0² + 2z02)ω²
and qz = -2qr =
-8zeV
m(r0² + 2z02)ω²
Where ω = 2πf, f = fundamental Rf frequency of the
trap (~1MHz)
41
QIT (Ion stability diagram)
Endcap
Ring Electrode
q = 0.908
q < 0.908
Ring Electrode
Endcap
42
courtesy of Spektrum Akademischer Verlag
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
43
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
44
Space Charging
~ 300 Ions
100
524.3
~ 1500 Ions
100
524.4
~ 3000 Ions
100
524.5
~ 6000 Ions
100
80
80
80
60
60
60
60
40
40
Relative Abundance
80
40
40
525.3
20
525.4
20
526.3
0
522
m/z
530
Good resolution
and mass accuracy
0
522
20
526.3
527.5
530
0
522
524.8
525.7
525.5
526.5
527.5
530
526.7
20
0
522
530
Poor resolution
and mass accuracy
47
Courtesy of Agilent
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)
48
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
49
Trapping Forces in a Linear Ion Trap
Radial Trapping RF Voltage
Axial
Trapping
DC
Voltage
Axial
Trapping
Exit Lens
Radial Trapping RF Voltage
Courtesy of Sciex
Resonance Excitation 50
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
52
Electric and MagneOc Sector Mass Analyzer The ‘original’ commercial MS systems High resoluOon, but comes at the cost of sensiOvity detector!
Mass Separation: Magnetic Fields
m/z = eB2r2/2V
• 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
more accuartely than can B
5
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
7
Directional (angular) focusing of a magnetic
field
Divergent ions of the same m/z will be brought into focus by a magnetic field
8
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.
9
“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
19
Petroleum Hydrocarbons Analysis of diesel fuel: •  typically by GC or 2D GC-­‐(TOF)MS C15
Analysis of diesel fuel sample 1:1000 in DCM, 60m DB5 column C9
•  what other compounds can be detected with ultra high resoluKon mass spectrometry? C21
8 Diesel 1:1000 in MeOH + 0.2% formic acid Zoom 9 Analysis of diesel fuel sample Zoom 10 Analysis of diesel fuel sample Zoom 11 Analysis of diesel fuel sample C18H22K+1 (0.32 ppm) C17H18O2Na+1 (0.00 ppm) C14H22O4Na+1 (0.11 ppm) C15H26O2K+1 (0.14 ppm) C18H22ONa+1 (0.40 ppm) C15H26O3Na+1 (0.28 ppm) C13H18O5Na+1 (0.18 ppm) C13H18O4K+1 (0.11 ppm) C16H30O2Na+1 (0.00 ppm) C17H34ONa+1 (0.03 ppm) 12 Analysis of diesel fuel sample C17H18O2Na+1 (0.00 ppm) 110,000 FWHM 2048K data points
29,000 FWHM 512K data points
7,000 FWHM 128K data points
1,500 FWHM 32K data points
13 Analysis of diesel fuel sample 110,000 FWHM 2048K data points
29,000 FWHM 512K data points
7,000 FWHM 128K data points
1,500 FWHM 32K data points
14 Analysis of diesel fuel sample 110,000 FWHM 2048K data points
29,000 FWHM 512K data points
7,000 FWHM 128K data points
1,500 FWHM 32K data points
15 Analysis of diesel fuel sample 110,000 FWHM 2048K data points
29,000 FWHM 512K data points
7,000 FWHM 128K data points
1,500 FWHM 32K data points
16