how mass spectrometers work
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How Mass Spectrometers Work. Principles New Technologies Application areas for specific technologies. Name of this game is technology, physics and engineering design. J.J. Thompson Discoverer of electron 1912, showed 2 neon isotopes deflected differently by magnet - PowerPoint PPT PresentationTRANSCRIPT
How Mass Spectrometers Work
•Principles
•New Technologies
•Application areas for specific technologies
Name of this game is technology, physics and engineering design.
J.J. Thompson
Discoverer of electron
1912, showed 2 neon isotopes deflected differently by magnet
His student Aston, (1918) designed more elaborate instruments.
Diverse Interfaces/Sources Let us Cope with a variety of Molecular Types
Need to get the molecule into the vapor phase before it can be ionized…. Unfortunately many more molecules are non-volatile than are volatile. The Interface problem
Then we have the problem that different structure types make ions with a variety of mechanisms and degrees of difficulty. The Source problem
We have a range of solutions to the Interface problem. These generally fall into:
Aerosolizing
Blasting off a surface
Heating
A Generic PictureAs liquid, in solution, flow from GC, eluent from LC, solid on probe or moving belt
Interface
Sample is volatilized and ionized either with electrons or charged gas molecules. Fragments break off the starting molecule
Different technologies to achieve this. E.g. Magnets, RF quadrupoles, “ion trap” (cyclotron) time-of-flight tube. All these, in different ways determine specific curves or paths through the separator based on molecular weight/charge. Some parameter e.g. Voltage is scanned, so that at some Vm only masses of m have proper curve and pass to detector
Source
To Vacuum
Typically this is a photomultiplier tube
Charged plate
collimator
Ion Separator
To Vacuum
Slit
Detector
To Vacuum
Sources
As liquid, in solution, flow from GC, eluent from LC, solid on probe or moving belt
Interface
Source
To Vacuum
Sources we will cover
Electron Impact
Chemical Ionization
Fast Atom Bombardment
Matrix Assisted Laser Desorption Ionization
Electrospray Ionization
Atmospheric Pressure Chemical Ionization
APPCI
Secondary Ionization (SIMS)
Different Classes of compounds may need different Ionization
techniquesMany compounds have adequate volatility on heated probe.
Often careful tracking of ion current vs. time will reveal mixtures of compounds selectively “boiling off” a heated probe.
Use by placing about a microgram in solution on probe tip, allow to dry, insert to source.
An important variation on this is Desorptive heating (very fast, like a “thermal shock”
Electron Impact Sources
Incandescent Rhenium filament, electron source, 70ev, can be lowered
To Vacuum, Maintains ca. 10-5 torr
Sample inlet, heated probe, etc. To mass
spectrometer
e-e-e-
Chemical Ionization (CI)-Why?
Variety of chemistry, tailor to the sample
One gas doesn’t work, try another
Dial in more fragmentation (hard, soft CI reagent gases)
Can be universal or compound selective
Positive or negative ion chemistry can be easily achieved
Most organics with mass < 800 Daltons have sufficient volatility for CI.
Chemical Ionization Sources
Inlet, choice of gasses (RG), ca. 1torr.(maintained by valve, pumping system)
Incandescent Rhenium filament, electron source
To Vacuum
Sample inlet, heated probe, etc.
RG+
RG+ RG+ RG+
RG+
RG+
RG+RG+RG+
RG
RG+
To mass spectrometer
RGRG
RG
Ion Chemistry and Chemical Ionization
Proton transfer (proton addition or abstraction)
Charge transfer
Electron capture
Addition of Reagent ion
Higher adduct
Cluster formation
Governed by the heat of formation of the various products vs the reactants
Example for CIOverdose case, gastric contents examined for drugs.
Here, the soft ionization potential of Isobutane as CI reagent gas could be counted on to provide molecular ions of all the compounds.
Milne, et al. Anal. Chem. 1970 (42) 1815-1820.
Different Reagent Gases give somewhat different Mass Specs
We can see why CI data don’t make it into “fingerprint” databases
R.G.Cooks, et al. Org Mass Spec. 1976, 11, 975-983
Better Molecular Ions for fragile compounds
The Ion-molecule chemistry can be diagnostic for stereoisomers
You might say we rationalize the proclivities based on ease of H atom abstraction from para positionHarrison and Lin, Org. Mass Spec. 1984, 19, 67-71,
See also, Keogh, et.al. Anal Chem 1984, 56, 1849-1852.
CI in C6F6
Chemical Ionization-Diversity of Chemistry possible
Different reagent gases
Take advantage of chemical affinities to tailor the ionization
“Hard Ionization” gasses (big-∆H) produce high energy MH+, leading to more fragmentation. Example, Hydrogen
Energy scale in reagent gasses
H2>>CH4>iC4H10>NH3>CH3-ONO>NF3>N2O(last three are proton abstraction reagent gases for negative ion MS. N2O is also pos. CI gas)
Compare proton affinities (PA) of conjugate acid of reagent gas to that of substrate. e.g., C4H9
+ is a strong enough acid to give a H+ to any nitrogenous base.
Contrary example, NH4+is not acidic enough to protonate ethers
Other Reagent Gases
Ar
CS2
N2
Electrospray Ionization (ESI)
Liquid Mobile phase from LC or direct injection of solution
Coulombic explosion after desolvation in vacuum
(M+nH)n+
(M+nH)n+
(M+nH)n+
(M+nH)n+
(M+nH)n+
(M+nH)n+
Lower vacuum
MM
M
H+
H+
H+ H+H+
H+
H+
skimmers
To
mas
s sp
ec
Capilliary Potential at 3-8 kVolts
Limitations on E-Spray
Compound must be polar enough to spray in a substantially aqueous mobile phase
Relies on electrostatic charging of aerosol droplets
Non-volatile salts in mobile phase can foul the ion optics. (Buffers)
Ion optics in a source after using phosphate buffer. Can work for a while but salts on highly charged surfaces can arc etc.
Atmospheric Pressure Ionization(API) Mass Spec
Source works in near to atmospheric pressure
Uses heated nebulizer
Corona discharge uses solvent CI
Useful in Normal Phase HPLC
Logical choice for very non-polar molecules.
Atmospheric Pressure Ionization
Heater
High-velocity nebulizer gas
Liquid sample (LC)
High voltage needle “corona” discharge, ionizes, breaks down, the gas close to it
Vacuum, to mass spec
SolventH+
Sufficient concentration to act as Chemical Ionization Reagent gas
SH+
M
SH+SH+
SH+
SH+
M
M M
M
MH+
MH+
MH+
MH+
And fragments!
Atmospheric Pressure Photoionization Chemical
Ionization (APPCI)
Cutting edge source technology
Uses UV photon flux to transit chromophores to excited states, able to ionize other molecules.
When there is no chromophore, a dopant that has a chromophore, like acetone is used.
Atmospheric Pressure Photoionizaton (APPI)
Atmospheric Pressure Ionization
Heater
High-velocity nebulizer gas
Liquid sample (LC)
MH+
MH+
MH+
MH+ Vacuum, to mass spec
SH+SH+
SH+ SH+
SH+
M
M MM
M
And fragments!
UV light source
h
hh
h
hh
Ion Separation
Ion Separator
To Vacuum
Various Ion Separation Technologies
•Different for different applications
•Big variable in cost
•Different in resolving power
•Differ in the accessible mass range
•We will cover:
Magnetic sector
Quadrupole
Time-of-flight
Ion Trap cyclotron
Combinations, triple quad, Q-TOF, double sector
Magnetic Sector Ion Separation
magnet
Ions from source
Curvature of pathway varies as function of mass/charge
As the magnetic field is scanned, only one mass at a time has the right curvature to make it through the slit
Detector
€
zV =mv2
2
HzV =mv2
R
R =mvHz
mz
=H 2 R 2
2V
Quadrupole mass analysis
Mass range 10–4000 daltons (amu)
Resolution, typically 1000
Scan rate 5000 daltons/min
Accuracy .1–.2 daltons
Not for high resolution mass spec
Great for most organic chemistry applications
How do Quadrupoles separate ions of different m/z?
Accelerated beam of all the ions
pos
Neg
DC voltage
And AC (Radio Frequency)
Ions with wrong spiral, crash into sides and don’t get through
Ions with proper spiral make it to the detector
The Quadrupole Orbits
Where RF and DC are the dc and ac voltages m is the mass and e is the charge, is the radiofrequency and r is the space between the quad rods
€
Q =4eRF
mr02ω 2
A =8eDC
mr02ω 2
Q
AConstant A/Q ratio: steeper slope=lower resolution
Regions of orbit stability Lifetimes are 50-
100 sec
A “Mathieu” diagram
Ion TrapsUse RF fields to bring the ions into orbits (like Quadrupole, but made into a ring.
Scanned RF can sequentially make different masses have stable orbits
Ion chamber is swept clean thousands of times per second, then the RF voltage changed for a new orbit.
Ideal for MS-MS. In this technique we can keep a selected mass in orbit for a long time, then introduce a collision gas for secondary Chemical Ionization.
Either get new fragment, or use to track the origin of smaller fragments (Did that fragment come from the molecular ion, or from fragmentation of a product ion?)
Ion Traps
Ring Electrode
End Cap Electrodes
DC voltage
Electron Multiplier
RF, frequency can be swept.
Ion Source
Ions held in orbits
Ion Trap Equation of motion
€
qz=
4zVmr
0
2ωRF
2
From the stability digram, qz
gives instability at 0.98
V is the peak voltage between ring and endcap electrodes.
Is the RF frequency, ca. MHz for a 1 cm gap(radius)
Resolution and Mass Spec
C20H9+
C19H7N+
C13H19O2N3+
??? All same nominal mass
249
Done with TOF, FT mass spec or double sector magnetic instrument
Exact mass calibration compounds used.
5ppm precision is sufficient
C19H7N+` C20H9+ C13H19O2N3
+
249.058 249.070 249.1479
Exact Mass MS
Curves back, focusses
4-sector instruments;2 electric field, 2 magnet sectors
Highly refocussed, accuracy is is ca 10ppm
detector
Three sector Quad Mass SpecsMS-MS
A second mass separation sector
Can pass single ion m/z for selective Ion Monitoring.
We can exploit this as way to eliminate chemical noise from a dirty matrix
Can have collision cell for further CI on a specific, selected ion or fragment ion.
Collision cell, hexapole acceleration region without selectivity accd to mass
A normal quadrupole for mass separation
Ions from source
Time-of-Flight Mass Spectrometry
Totally different concept. Have a fixed electric field down a “flight tube”
A fixed distance from start to detector.
Accelerate ions to the field, start timing. The time to the detector is inversely proportional to mass (smaller mass gets there first)
Time therefore is calibrated to mass
Great precision, accuracy--can do exact Mass determination
Time-of-Flight mass spectrometry
Simple principle: Drift time over a fixed distance is related to momentum. “time” of transiting over this distance is proportional to
€
mz
Best technique for macromolecules. (have done over 500kDa). Has very high mass resolution, fortunately.
Since biomolecules like this suffer from bad volatility issues, TOF is happily paired with MALDI ionization.
How Flight Tubes work
Electric field “reflectron” gives longer path for resolution
Ion path
Accelerate ions (“pusher”) From source
Detector
MatrixAssistedLaserDesorptiveIonization
Or… What to Do, when your sample has no volatility whatsoever
How do you get a protein to oblige you by vaporizing nicely and going down the mass spec?
laser
Dilute biomolecule in e.g. cinnamic acid for proteins, picolinic acids for nucleotides
Molecular ions
Exquisite sensitivity
Great marriage with TOF for high mass range, resolution
MALDI-TOF
1048.9
1537.6
1296.7
1060.6
A mixture of peptides ca. 10 amino acid in length
Injected to mass spectrometer 20 femtomoles (10-15)each, of angiotensin I, angiotensin II, bradykinin, and fibrinopeptideA
Mass Spec Detectors
Ions+ Voltage to Detector
Conversion Dynode
+ +
+++
+ +
+
Phosphor plate
h
hh
h
hhe-
e-e-
e-
e-e-
Ph
otoMu
ltiplier
Tu
be
Specific Detectors
Cascade of Faraday Cups
The important point is the 106-fold amplification by these stages (e- per ion)