Transcript A typical Quadrupole Assembly

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 Picture
As liquid, in solution, flow
from GC, eluent from LC,
solid on probe or moving belt
Charged plate
collimator
Interface
Source
Sample is
volatilized and
ionized either with
electrons or
charged gas
molecules.
Fragments break
off the starting
molecule
To
Vacuum
Slit
Ion
Separator
Detector
To
Vacuum
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
To
Vacuum
Typically this is a
photomultiplier
tube
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
techniques
Many 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
To Vacuum, Maintains ca. 10-5 torr
Sample inlet, heated
probe, etc.
M+
MMM e- - M+M+M+
e e-
To mass
spectrometer
Incandescent Rhenium
filament, electron
source, 70ev, can be
lowered
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
To Vacuum
Sample inlet, heated
probe, etc.
RG+
RG+
+
RG
RG+ RG+ RG+
RG+ RG+ RG+
MM
M
RG+
RG
RG RG
RG
MM+ +
M+
Inlet, choice of
gasses (RG), ca.
1torr.
(maintained by
valve, pumping
system)
To mass
spectrometer
Incandescent Rhenium
filament, electron source
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 CI
Overdose 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
CI in C6F6
You might say we rationalize the proclivities based on ease
of H atom abstraction from para position
Harrison and Lin, Org. Mass Spec. 1984, 19, 67-71,
See also, Keogh, et.al. Anal Chem 1984, 56, 1849-1852.
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
Lower
vacuum
(M+nH)n+
H+
H+
(M+nH)n+
(M+nH)n+
HH+ +
M
MM
H
+
H+
(M+nH)n+
To mass spec
Capilliary
Potential
at 3-8
kVolts
H+
(M+nH)n+
(M+nH)n+
Coulombic explosion
after desolvation in
vacuum
skimmers
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
Liquid sample (LC)
Heater
High-velocity
nebulizer gas
SH+ SH+
M
SH+ M
+ SH+
M SHM M
MH+ MH+
MH+ MH+
SolventH+
Sufficient
concentration to
act as Chemical
Ionization
Reagent gas
And
fragments!
High voltage
needle “corona”
discharge, ionizes,
breaks down, the
gas close to it
Vacuum,
to mass
spec
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 Ionization
Atmospheric Pressure
Photoionizaton (APPI)
SH+ SH+
Liquid sample (LC)
+ SH+
M SHM M
Heater
High-velocity
nebulizer gas
M
SH+ M
And
fragments!
h
h
h
MH+ MH+
MH+ MH+
h
h
h
UV light
source
Vacuum,
to mass
spec
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
mv 2
zV 
2
mv 2
HzV 
R
mv
R
Ions from
Hz
source
m H 2R2

z
2V
magnet
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
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?
Ions with
proper spiral
make it to the
detector
Ions with wrong spiral, crash
into sides and don’t get
through
DC voltage
And AC (Radio
Frequency)
Accelerated
beam of all
the ions
The Quadrupole Orbits
4eRF
Q
mr 0 2  2
8eDC
A
mr 0 2  2
A
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
Constant A/Q
ratio: steeper
slope=lower
resolution
A “Mathieu”
diagram
Q
Regions of orbit
stability
Lifetimes are 50100 sec
Ion Traps
Use 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
End Cap
Electrodes
DC voltage
Ion Source
Ring
Electrode
Ions held in
orbits
RF, frequency
can be swept.
Electron Multiplier
Ion Trap Equation of motion
4zV
qz  2 2
mr0  RF
V is the peak voltage
between ring and endcap
electrodes.
 Is the RF frequency, ca.
MHz
for a 1 cm gap(radius)
From the
stability
digram, qz
gives
instability at
0.98
Resolution and Mass Spec
C20H9+
Done with TOF, FT mass spec or
double sector magnetic
instrument
C19H7N+
Exact mass calibration
compounds used.
C13H19O2N3+
5ppm precision is sufficient
??? All same
nominal mass
249
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
Three sector Quad Mass Specs
MS-MS
A second mass
separation sector
detector
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
m
z
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
Detector
Ion path
Accelerate ions
(“pusher”)
From source
Electric field
“reflectron” gives
longer path for
resolution
Matrix
Assisted
Laser
Desorptive
Ionization
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?
Molecular ions
Exquisite sensitivity
Great marriage with
TOF for high mass
range, resolution
Dilute biomolecule in
e.g. cinnamic acid for
proteins, picolinic acids
for nucleotides
MALDI-TOF
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
1296.7
1060.6
1048.9
1537.6
Mass Spec Detectors
Voltage to
Detector
Ions+
+ ++
+ + +
+
e-
e-
e-
e-
e-
h
h
h
h
h
h
PhotoMultiplier
Tube
Conversion Dynode
e-
Phosphor plate
+
Specific Detectors
Cascade of Faraday
Cups
The important
point is the
106-fold
amplification
by these
stages (e- per
ion)