1. Mass Filters in Mass Spectrometry
Separations of ions based on properties of mass and charge.
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2. Mass Spec is “A Universal Technique”
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 gas-phase molecule that carries a charge
Analyzed molecules range in size from H+ to mega-Dalton 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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3. What does the mass filter do?
A mass spectrometer determines the mass-to-charge ratio (m/z) of gas-phase ions by subjecting them to known electric or magnetic fields and analyzing their resultant motion.
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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4. Vacuum Requirement
Mean Free Path: the average distance a molecule travels between collisions.
For typical MS conditions, can be estimated as:
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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5. Vacuum Systems
All mass spectrometers operate at very low pressure (high vacuum) to reduce the chance of ions colliding with other molecules in the mass analyzer
collisions cause the ions to react, neutralize, scatter, or fragment.
these processes will interfere with the mass spectrum
Experiments are conducted under high vacuum conditions (10-2 to 10-5 Pa or 10-4 to 10-7 torr)
Requires two pumping stages:
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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6. The Mass Spectrum
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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7. Information from a Mass Spectrum
Identification of molecular mass
Determination of structure
Determination of elemental composition
Determination of isotopic composition
Quantification
Not inherent – requires consideration of ionization efficiencies, ion transmission, detector response …
Qualitative information is much easier to extract!
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8. m/z
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
with 1 u = 1/NA g
z: charge number = Q in e
with 1 e = ×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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9. Signal Intensity
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:
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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10. Figures of Merit for the Mass Spec
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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11. Resolving Power Mass peak width ( Dm 50%)
Full width of mass spectral peak at half-maximum peak height
Mass resolution / Resolving Power (m / Dm 50%)
Quantifies ability to isolated single mass spectral peak
Mass accuracy
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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12. “The history of spectroscopy is the history of resolution …” - A. G
“The history of spectroscopy is the history of resolution …” - A. G. Marshall, et al, A. Chem., 74(9), 252A, 2002.
Different charge but the same mass
Differing in nominal closest-integer 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
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13. Mass Accuracy 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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14. Calculate resolution and accuracy
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15. 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
Sector (Magnetic and/or Electrostatic)
High resolution, exact mass
ICR (FT-MS)
Very high resolution, exact mass, perform ion chemistry
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16. Time-of-Flight MS To determine m/z values Key Performance Notes
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
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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17. Time-of-Flight MS
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18. Time-of-Flight MS
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.
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19. but there are complicating factors . . .
TOF = total recorded flight time of an ion
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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20. Resolution in TOF
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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21. 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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22. Improving Resolution in TOF MS - Reflectrons
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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23. Two means to the same end
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
In both cases resolution is enhanced!
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24. 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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25. Use “orthogonal” extraction
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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26. orthogonalTOFMS (oTOFMS)
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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27. 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 =
You know that your mass spectrometer
has mass accuracy greater than 7 ppm.
What conclusion can you make?
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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28. 1. Compound A is the first to come off of the column
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29. Other detectors . . . TOFMS Quadrupole MS
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
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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30. Quadrupole Geometry / operation
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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31. Quadrupole Geometry / operation
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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32. Quadrupole stability diagrams
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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33. Forces – Generalizing for all m/z
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34. Stability Diagram for a Quadrupole
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35. 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 = (e.g., Ion B)
For any value m we find
a/q = 2U/Vo
To scan values of m through narrow
transmission window, hold other
parameters constant and scan U and
Vo with constant ratio
U/Vo = ½(0.233 / 0.706)
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36. Mass Selectivity For ANY value m a/q = 2U/Vo For, example:
Reduce U, Hold
Still stable, slope of “scan line” is reduced
What effect does this have on resolution?
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37. Mass Scanning Scan line shows U/Vo = ½(0.233 / 0.706)
Increase in mass requires proportional increases in U and Vo to maintain this ratio and these a and q values.
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38. 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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39. Collisional Cooling
A common application of rf-only 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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40. Collisional Cooling
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41. Triple Quadrupole Mass Spectrometer
Q1 selects parent; q2 CID fragmentation inside RF-only quad;
Q3 fragment analysis; Detector
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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42. Quadrupole Ion Trap Quadrupole ion storage trap mass
spectrometer (QUISTOR) - recently developed
traps and analyzes all the ions produced in the source
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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43. Quadrupole Ion Trap Several commercial instruments are available
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.
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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44. Sector Instruments (Mag or Elec)
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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45. 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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46. 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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47. Electric Sector/Double Focusing Mass Spectrometers
The mass resolution of a magnetic sector is limited by the kinetic energy distribution of the ion beam
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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48. Reverse Geometry Double Focusing Mass Spectrometer
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49. FT-Ion Cyclotron Resonance MS (FT-ICR MS)
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.
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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50. The ICR trap explained
m/z = B/2pf
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51. Advantages of FT-ICR MS
High Mass Resolution
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 Da can be distinguished from a peak at mass Da
Has “almost unlimited” resolution
M / 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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52. Advantages of FT-ICR MS
ultrahigh mass accuracy (1ppm)
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
spectra are collected in a single scan over a wide mass range without loss of sensitivity
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53. Advantages of FT-ICR MS
detect different ions simultaneously, instead of one at a time (scanning sectors)
Thus, high speed
Multiple pulse / collection cycles can be used
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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54. Limitations of FT-ICR MS
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 low-pressure 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.
“Ion-counting” MS require fewer molecules to generate “signal”
Large and Expensive
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55. Applications of FT-ICR MS
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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56. Ion detection - based on charge or momentum
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:
electron multipliers,
channeltrons
and multichannel plates.
gain controlled by changing HV applied to the detector
detectors are selected for:
speed,
dynamic range,
gain, and
geometry
some detectors are sensitive enough to detect single ions
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