Transcript Mass Filters in Mass Spectrometry

Mass Filters in Mass
Spectrometry
Separations of ions based on properties of
mass and charge.
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Mass Spec is “A Universal Technique”
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Analysis by MS does not require:
 Chemical modification of the analyte
 Any unique or specific chemical properties
 In theory, MS is capable of measuring any gasphase molecule that carries a charge
Analyzed molecules range in size from H+ to megaDalton DNA and intact viruses
As a result, the technique has found widespread use
Organic, Elemental, Environmental, Forensic,
Biological, Reaction dynamics
All experiments have this basic backbone,
but range of applications implies a
diversity of experimental approaches.
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What does the mass filter do?
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A mass spectrometer determines the mass-tocharge ratio (m/z) of gas-phase ions by
subjecting them to known electric or magnetic
fields and analyzing their resultant motion.
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Sectors – magnetic or electric
Quadrupole
Ion Trap
Time of flight (TOF)
Ion Cyclotron Resonance (FT-MS)
Tandem system (MS-MS, MS-MS-MS, etc)
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Vacuum Requirement
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Mean Free Path: the average distance a molecule travels
between collisions.
For typical MS conditions, can be estimated as:
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L in cm, p in mTorr
Suggests pressures on the order of 10-5 torr to move a
molecule across a meter without collision.
Requires moderately sophisticated, and moderately
expensive systems of vacuum pumps
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Vacuum Systems
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All mass spectrometers operate at very low pressure (high
vacuum) to reduce the chance of ions colliding with other
molecules in the mass analyzer
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Experiments are conducted under high vacuum conditions
(10-2 to 10-5 Pa or 10-4 to 10-7 torr)
Requires two pumping stages:
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collisions cause the ions to react, neutralize, scatter, or fragment.
these processes will interfere with the mass spectrum
mechanical pump - provides rough vacuum ~0.1 Pa (10-3 torr)
second stage uses diffusion pumps or turbomolecular pumps to
provide high vacuum
ICR instruments have even higher vacuum requirements
(often includes a cryogenic pump for a third pumping
stage)
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The Mass Spectrum
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A mass spectrum is a plot of signal intensity vs. m/z
To compare different MS techniques we need to provide
numerical indications of how good the data is . . .
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Information from a Mass Spectrum
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Identification of molecular mass
Determination of structure
Determination of elemental composition
Determination of isotopic composition
Quantification
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Not inherent – requires consideration of ionization
efficiencies, ion transmission, detector response …
Qualitative information is much easier to extract!
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m/z
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The mass-to-charge ratio is often referred to as m/z and
is typically considered to be unitless:
m: mass number = atomic mass in u
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z: charge number = Q in e
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with 1 u = 1/NA g
with 1 e = 1.6022×10-19 C
the Thompson has been proposed as a unit for m/z, but
is only sometimes used
Historically, most ions in MS had z = 1
 with new ionization techniques, this is no longer true
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Signal Intensity
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Depending on the type of mass spectrometer, ions
may be detected by direct impact with a detector
or by monitoring of an induced current image.
Recorded signal can be measured in:
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Counts per unit time (Digital)
Voltage per unit time (Analog)
Power (Frequency domain)
To a first approximation, relative signal intensity
reflects relative ion abundance
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Figures of Merit for the Mass Spec
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Selection of appropriate MS instrumentation and
conditions depends on analysis sought and key
figures of merit.
Sensitivity
Ion Transmission
Duty Cycle
m/z Range
Mass Resolving Power
Mass Accuracy
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Resolving Power
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Mass peak width ( Dm 50%)
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Mass resolution / Resolving Power (m / Dm 50%)
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Full width of mass spectral peak at half-maximum peak height
Quantifies ability to isolated single mass spectral peak
Mass accuracy
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Mass accuracy is the ability to measure or calibrate the instrument response
against a known entity. Difference between measured and actual mass
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“The history of spectroscopy is the history of resolution …”
- A. G. Marshall, et al, A. Chem., 74(9), 252A, 2002.
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Different charge
but the same mass
Differing in
nominal closestinteger mass
Ions of the same
chemical formula
but different
isotopic
composition
Ions of the same
nominal mass but
different elemental
composition
Note m/z
dependence of
necessary
resolving power
Mass Accuracy
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Mass accuracy is
linked to resolution.
A low resolution
instrument cannot
provide a high mass
accuracy
High resolution and
high mass accuracy
enables
determination of
elemental
composition based
on exact mass
Possible because of
elements’ mass
defects
Requirements
increase as m/z
increases
Exact masses and corresponding formula for various
ions of m/z 180 containing only C, H, N, and O atoms.
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Calculate resolution and accuracy
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Different types of mass filters
Analyzer
System Highlights
TOF
Theoretically, “no limitation” for
maximum m/z, high throughput
Quadrupole
Unit mass resolution, fast, low cost
Ion Trap
Unit mass resolution, fast, low cost
Sector (Magnetic and/or
Electrostatic)
High resolution, exact mass
ICR (FT-MS)
Very high resolution, exact mass,
perform ion chemistry
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Time-of-Flight MS
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To determine m/z values
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A packet of ions is accelerated by a
known potential and the flight times
of the ions are measured over a
known distance.
Key Performance Notes
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Based on dispersion in time
Measures all m/z simultaneously,
implying potentially high duty cycle
“Unlimited” mass range
DC electric fields
Small footprint
Relatively inexpensive
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Time-of-Flight MS
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Time-of-Flight MS
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Ions accelerated by strong
field, E, within short source
region, S.
Drift times recorded across
long, field-free drift region, D
vD depends on starting position
of ion – ideally all ions start
from same plane.
but there are complicating factors . . .
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TOF = total recorded flight time of an ion
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to = Ion formation time after T0 of TOF measurement
ta = Time in acceleration region, which depends on initial position and
initial energy
tD = Time in drift region, which depends on initial position and initial
energy
td = Response time of detector
For any m/z in a time-of-flight mass spectrum, the recorded peak
will be the sum of signals corresponding to multiple, independent,
ion arrival events
Each ion arrival will be recorded at a unique TOF, (see eqn above)
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Resolution in TOF
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TOF’, which is the center of the peak in the mass
spectrum, will be an average of all individual ion
arrival TOFs
The width of TOF’, Dt, will depend on the
distribution of the individual ion arrival TOFs
(and other factors …)
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Improving Resolution in TOF MS
At ionization: U = U0 (initial ion energy)
At exit of extraction:
U = U0 + Eextxq
At beginning of drift:
U = U0 + Eextxq + (V1-V2)q
Tune source voltages and/or delay to
compensate for DU0 and create
space focus at detector. Mass
dependent.
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Improving Resolution in TOF MS Reflectrons
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Reflectron consists of a series of
electrodes, forming a linear field in
direction opposite of initial acceleration.
Ions are slowed by this field, eventually
turning around and accelerating back in
direction of detector.
Penetration depth depends on Us, which
is function of U0 and acceleration field, E.
Reflectron voltages are tuned to create a
space focus at the plane of the detector.
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Two means to the same end
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In Delayed Extraction, we give ions different U to
achieve same TOF.
In Reflectron, ions possess different U. We force
them to travel different D to achieve same same
TOF
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In both cases resolution is enhanced!
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TOF is inherently a pulsed detector
TOFMS is an ideal detector for pulsed
ionization methods
 If ionization event is synchronized with
time zero, high duty cycle is achieve
 But not all sources are “pulsed”
(ex. Electrospray, or stream ions from EI
source, etc.)
 Because of pulsing, ions are wasted when
TOFMS is applied to a continuous source and . . . .
 Increased efficiency comes at the expense of mass range and mass
resolution
 Still, figures of merit and cost make thetechnique desirable
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Use “orthogonal” extraction
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Ions are extraction in a
direction orthogonal to
original analyte stream
trajectory
Extraction event is still
rapid (Dt), but extraction
volume is determined by
length of gate region.
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orthogonalTOFMS
(oTOFMS)
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Able to reduce average
initial energy in ToF
direction to ~0 (resolution
and accuracy)
Independent control of
beam energy and drift
energy, allows maximum
duty cycle.
Want tightly collimated
beam in extraction region
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A review question . . . .
Suppose you are attempting
to separate these two compounds
by LC-MS. The first compound
to appear in your chromatogram
has an intense peak at
m/z = 344.1421.
You know that your mass spectrometer
has mass accuracy greater than 7 ppm.
 What conclusion can you make?
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2.
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Compound A is the first to come off of the column
Compound B is the first to come off of the column
You are measuring an average of the two compounds that contains
mostly A
Nothing -- You do not have sufficient mass accuracy to determine
which compound(s) you are measuring
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1. Compound A is the first to come off of the column
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Other detectors . . .
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TOFMS
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Pulse packet of ions introduced into analyzer
All m/z in packet reach detector (“simultaneous detection”)
m/z determination based on dispersion
Based on static, DC fields
Quadrupole MS
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Continuous introduction of ions into analyzer
Transmit only specific m/z value to detector
m/z determination based on band-pass filtering
Based on time-vary, RF fields
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Quadrupole Geometry / operation
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Quadrupole consists of
four parallel rods
Typical length might be
10’s of cm
Precise dimensions and
spacing
Rods connected
diagonally in pairs
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Quadrupole Geometry / operation
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Voltage of all rods have a DC
component, U.
All rods have RF component
of voltage with MHz
frequency = ? /2p and
amplitude Vo.
Potentials on the two sets are
out of phase .
Quadrupole fields cause no
acceleration along z axis.
V1 = V3= -Fo = -U – Vocos ? t
V2 = V4= Fo = U + Vocos ? t
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Quadrupole stability diagrams
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Stability diagram for fixed Rf
frequency, fixed m/z.
An ion will have stable trajectory
through quadrupole if x and y are
always less than radius of quadrupole.
(Sim A) With no RF and positive U,
positive ion is stable along X (repelled
to center), attracted to negative Y rod
causes instability
(Sim C) RF field has stabilized Y
trajectory.
Note that with increased U, need greater
Vo to achieve this stability.
(Sim E) Instable along x-axis.
Note that as U increases, lower Vo will
induce this instability.
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Forces – Generalizing for all m/z
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Stability Diagram for a Quadrupole
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Mass Selectivity
Many conditions (U, V, m) fall within stability region – there is more than one
way for ion to pass through
 For selectivity, must also consider
stability of other mass values
 Apex of generalized stability
diagram is at a = 0.237, q = 0.706
 To select transmit narrow mass
window, adjust U and Vo such that
a = 0.237, q = 0.706 (e.g., Ion B)
 For any value m we find
a/q = 2U/Vo
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To scan values of m through narrow
transmission window, hold other
parameters constant and scan U and
Vo with constant ratio
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U/Vo = ½(0.233 / 0.706)
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Mass Selectivity
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For ANY value m
a/q = 2U/Vo
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For, example:
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Reduce U, Hold
Still stable, slope of
“scan line” is reduced
What effect does this
have on resolution?
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Mass Scanning
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Scan line shows
U/Vo = ½(0.233 / 0.706)
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Increase in mass
requires proportional
increases in U and Vo
to maintain this ratio
and these a and q
values.
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Quadrupole Performance
Typical Quadrupoles
 Maximum m/z ~ 4,000
 Resolution ~ 3,000
 Quadrupoles are low resolution instruments
 Usually operated at ‘Unit Mass Resolution’
 Small, lightweight
 Easy to couple with chromatography
Rf-Only quadrupoles
 Operated with U = 0, quadrupole becomes a broad bandpass
filter
 Such “rf-only” quads are an important tool for transferring
ions between regions of mass spectrometers.
 Often denoted with small “q”
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Collisional Cooling
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A common application of rfonly multipoles involves
collisional cooling.
In an ESI source, the
expansion into vacuum
produces a ion beam with
broad energy distribution
Ion optics and TOFMS
experiments rely on precise
control of ion energies
Desire strategies to dampen
energy from external processes
Rf-induced trajectory in high
pressure region yield
collisions, and reduction in
energy
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Collisional Cooling
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Triple Quadrupole Mass Spectrometer
Q1 selects parent; q2 CID fragmentation inside RF-only quad;
Q3 fragment analysis; Detector
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Fragment Ion Scan: Park Q1 on specific parent m/z; scan Q3
through all fragment m/z to determine make-up of Q1
Parent Ion Scan: Park Q3 on specific fragment m/z; scan Q1
through all parent m/z to determine source of fragment
Neutral Loss Scan: Scan Q1 and Q3 simultaneously, with constant
difference, a, between transmitted m/z values (a = MQ1 – MQ3).
Signal recorded if ion of m/z= MQ1 has undergone fragmentation
producing a neutral of m = a.
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Quadrupole Ion Trap
Quadrupole ion storage trap mass
spectrometer (QUISTOR) - recently developed
 traps and analyzes all the ions produced in the source
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the S/N is high.
consists of a doughnut shaped ring electrode and two endcap electrodes
A combination of RF and DC voltages is applied to the electrodes to
create a quadrupole electric field (similar to the electric field for
quadrupole)
electric field traps ions in a potential energy well
 scan the RF and DC fields
 the fields are scanned so that
ions of increasing m/z value are ejected
from the cell and detected
 The trap is then refilled with a new
batch of ions to acquire the next
mass spectrum
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Quadrupole Ion Trap
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Several commercial instruments are available
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this analyzer is becoming more popular.
QUISTORs are very sensitive, relatively
inexpensive, and scan fast enough for GC/MS
experiments
The mass resolution of the ion trap is increased by
adding a small amount 0.1 Pa (10-3 torr) of
Helium as a bath gas.
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Collisions between the analyte ions and the inert bath
gas dampen the motion of the ions and increases the
trapping efficiency of the analyzer
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Sector Instruments (Mag or Elec)
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Magnetic Sector: the first mass spectrometer, built by J.J.
Thompson in 1897, used a magnet to measure the m/z value
of an electron
Magnetic sector instruments have evolved from this concept
Sector instruments have higher resolution and greater mass
range than quadrupole instruments, but they require larger
vacuum pumps and often scan more slowly
The typical mass range is to m/z 5000, but this may be
extended to m/z 30,000.
Magnetic sector instruments are often used in series with an
electric sector, described below, for high resolution and
tandem mass spectrometry experiments.
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Magnetic Sector explained
Magnetic sector instruments separate ions
in a magnetic field according
to momentum and charge
 Ions are accelerated from the source into
the magnetic sector by a 1 to 10 kV electric field
 the radius of the arc (r) traveled depends
upon the momentum of the ion, the charge
of the ion (C) and the magnetic field strength (B).
 Ions with greater momentum follow a larger radius
 Ion velocity - determined by the acceleration voltage (V) and
mass to charge ratio (m/z)
 the m/z transmitted for a given radius,
magnetic field, and acceleration voltage:
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Contribution by Electric Sectors
An electric sector consists of two concentric curved plates.
 A voltage is applied across these plates
 Ion beam bends as it travels through the analyzer
 voltage is set so the beam follows the curve of the analyzer
 The radius of the ion trajectory (r) depends upon the kinetic
energy of the ion (V) and the potential field (E) applied
across the plates
 an electric sector will not separate ions accelerated to a
uniform kinetic energy
 radius of the ion beam is independent
of the ion's mass to charge ratio
 electric sector is not useful as a standalone
mass analyzer
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Electric Sector/Double Focusing Mass Spectrometers
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The mass resolution of a magnetic sector is
limited by the kinetic energy distribution of the
ion beam
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kinetic energy distribution results from variations in the
acceleration of ions produced at different locations in
the source . . . . and
from the initial KE distribution of the molecules
an electric sector significantly improves the
resolution of the magnetic sector by reducing the
kinetic energy distribution of the ions
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Reverse Geometry Double Focusing Mass Spectrometer
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FT-Ion Cyclotron Resonance MS
(FT-ICR MS)
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FT-ICR mass spectrometry exploits the cyclotron
frequency of the ions in a fixed magnetic field
The ions are trapped in a Penning trap (a device for the
storage of charged particles using a constant magnetic
field and a constant electric field) where they are excited
to a larger cyclotron radius by an oscillating electric field
perpendicular to the magnetic field.
The signal is detected as an image current as a function
of time.
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After a Fourier transform, which converts a time-domain signal
(the image currents) to a frequency-domain spectrum (the mass
spectrum), we can get a “traditional” mass spectrum
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The ICR trap explained
m/ = B/
z
2pf
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Advantages of FT-ICR MS
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High Mass Resolution
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enhances sensitivity by making it possible to distinguish
between analyte and background species at or near the
detection limit
narrow peak width allows the signals of two ions of similar
mass to charge (m/z) to be detected as distinct ions. A peak at
mass 800.000 Da can be distinguished from a peak at mass
800.001 Da
Has “almost unlimited” resolution
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/ DM > 10,000,000 is possible,
M/
DM is in the range from 100,000 to 1,000,000 for most
experiments
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Advantages of FT-ICR MS
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ultrahigh mass accuracy (1ppm)
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offers an alternative to tandem mass spectrometry
(MS/MS) for identification, an advantage if the amount
of sample is limited.
The mass accuracy can be less than that of a single
electron, so that chemical compounds with the same
nominal molecular weight but different elemental
compositions can be distinguished by ICRMS
wide mass range
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spectra are collected in a single scan over a wide mass
range without loss of sensitivity
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Advantages of FT-ICR MS
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detect different ions simultaneously, instead of one at a
time (scanning sectors)
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Multiple pulse / collection cycles can be used
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Thus, high speed
Signal averaging
Time dependent studies of ion stability / ion reactions
The other particularity of the FTICR mass spectrometer is
that new fragmentation techniques can be used, such as
infrared laser activation, or electron capture dissociation.
These techniques can be used in combination to fragment
very large molecules such as whole proteins
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Limitations of FT-ICR MS
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The background pressure of an FTICR should be very
low to minimize ion-molecule reactions and ion-neutral
collisions that damp the coherent ion motion. Strict lowpressure requirements mandate an external ion source for
most analytical applications.
Need high magnetic field.
A limit in the sensitivity of FTICR is caused by
broadband image current detection, requires
approximately 100 charges to generate a measurable
signal at a given m/z ratio.
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“Ion-counting” MS require fewer molecules to generate “signal”
Large and Expensive
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Applications of FT-ICR MS
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Macromolecules
Multi-residue analysis
Metabolomics
Proteomics
 by extracting proteins from cells or tissue, fragmenting
them into shorter peptide segments, and then determining
the masses of all fragments
Biomarkers
Complex mixture, e.g. crude oil
Fast and specific analyses of toxins
Elemental composition
Isotopes
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Ion detection - based on charge or momentum
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large signals - faraday cup is used to collect ions and
measure current
most modern detectors amplify the ion signal using a
collector similar to a photomultiplier tube, for example:
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gain controlled by changing HV applied to the detector
detectors are selected for:
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electron multipliers,
channeltrons
and multichannel plates.
speed,
dynamic range,
gain, and
geometry
some detectors are sensitive enough to detect single ions
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