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Lecture 7: Mass Spectrometry, Instrumental
In mass spectrometry, components of a mixture are transferred
to the gas phase (if they’re not already there) and separated by
electric (and sometimes magnetic) fields.
The first MS data was collected by this guy:
Wilhelm Carl Werner Otto Fritz Franz Wien,
1864-1928
Wilhelm showed that ‘canal rays’ of positively
charged particles could be deflected by strong
Electric and Magnetic fields
From this evidence, he concluded that
the positively charged species responded
to the EM fields based on their e/m
(charge-to-mass) ratio
Mass Spectrometry: The Beginning
But it was J. J. Thomson who really got things going:
He improved Wilhelm’s apparatus by creating the positive
rays at low pressure. As a result, we was able to separate
various positive ions of gasses, and even Ne20 from Ne22
One of Thomson’s students,
Francis Aston, greatly
improved Thomson’s
instrument
Francis Aston
(1877-1945)
The main difference was that
Aston’s instrument focused
ions with different speeds into
a single line
Mass Spectrometry: The Beginning
At first, it looked like MS would be good mostly for
analyzing isotopes. Prior to WWII, the stable isotopes of
virtually every element were analyzed.
After WWII, the general picture of a mass spectrometer
emerged. It must have the following parts:
An Ion Source – we have to get
stuff into the gas phase
A Vacuum System – we can’t have our
ion beam colliding with neutrals
A Mass Analyzer – we need to
separate or select the desired ions
A Detector – we need to
detect our selected ions
Vacuum Systems: Roughing pumps
In order to keep our ion beams focused, we can’t have them
banging into neutral gasses at atmospheric pressure.
In general, that means we want pressures in the 10-6 – 10-8 Torr
range
How do we know that? We calculate the mean free path .
This is the total distance traveled in time t divided by the
number of collisions:
Path length (vt)

N
Particle density
V
vt
1


vt 
Radii of colliding particles
   (r1  r2 )
Collision cross section
For small molecule gasses at ~10-7 Torr,  = ~ 1000 m
Vacuum Systems: Roughing pumps
Getting to 10-8 Torr ain’t easy. We need to do it in stages.
The first stage uses a roughing or rotary vane pump ~10-3 Torr
Vacuum Systems: Turbo Pumps
From there, we can use a turbomolecular pump:
The mechanism of action is simple: The
fan blades simply ‘hit’ gas molecules out
of the chamber.
This’ll get us down to ~10-7 Torr
But what if we need to get lower?
Vacuum systems: Cryopumps
Cryopumps work by trapping small amounts of gas molecules
by condensing them on to a surface
They have very slow pumping
speeds, but can get down to
~10-12 Torr under the right
conditions
Vacuum Gauges
Once we have our vacuum (and while we’re making it) we
need to monitor the pressure
For the first pumping stage (~10-4 Torr) we can use a ‘Pirani
Gauge’
Pirani gauges work by measuring the
resistance through a wire exposed the
vacuum. Current through the wire heats
it up, exposure to surrounding gas cools
it down. Conductivity is dependent on
temperature.
For lower stages, we usually use a Bayard-Alpert or Penning
hot/cold cathode gauge .
Bayard-Alpert gauges ionize residual gasses in the
vacuum using a stream of electrons. The resulting
positive gas ions are drawn to a negatively charged
collector producing a current (1 mA/.01 Torr)
Detectors
There are two major types of detectors. One, the ‘chanel
electron multiplier’ (CEM) is going to look awfully familiar:
The other is called a microchannel plate (MCP). And it’s
basically an array of tiny CEMs.
http://hea-www.harvard.edu/HRC/mcp/mcp.html
Detectors: Comparison
CEM
Extreme Sensitivity
Can easily measure a single ion
MCP
Lowish Sensitivity
Microchannels are not as
efficient as dynode arrays
Extreme Sensitivity
Threshold
Detection threshold must be
carefully set or data gets noisy!
Still have to set detection
threshold quite carefully
Low Duty Cycle
Very High Duty Cycle
Relatively long (~500 s)
recovery time
Channels can return signal
while others are recovering
Easily Saturated
Easily Saturated
Cannot tell the difference between
lots of ions and tons of ions
Cannot tell the difference between
lots of ions and tons of ions
The Core: Mass Analyzers, Sector
The Mass Analyzer is what makes a mass spectrometer. It’s
how you select or separate ions according to their mass.
The first Mass Spectrometers used sector mass analyzers, so
named because ions are separated first in electric and then
magnetic field sectors.
mv2
2 zV
zV 
,v 
2
m
Fcent
mv2

r
To make a ‘perfect’ arc
sub for v
mv2
 zvH
r
m  2 zV   2 zV

  z 
r m   m
Fmag  zvH
rearrange
m r2H 2

z
2V

H


Variations on Sector Mass Analyzers
Sector instruments aren’t all that popular anymore – they’re
big and clunky due to the need for a powerful magnet. But
some people still use ‘em…
There are a number of
variations on the sector
theme. One of the main
advantages is the ability
to focus ions in the
electric (D) or magnetic
sectors (E) to limit
dispersion
Mass Analyzers: Quadrupoles
Like sector mass analyzers, quadrupoles are mass filters: They
work by letting only the desired mass through.
A quadrupole mass analyzer is a set of four parallel rods:
Quadrupole Mass Analyzers
The unifying aspect of chromatographic methods is that the
separation is not with an applied electric field
x
 0  (U  V cos(t ))
 0  (U  V cos(t ))
Total applied DC
potential
potential
+
+
-
+
Alternating (RF)
potential
-
r0
+
Upon entering the quadrupole, particle will experience the
following accelerations:
d 2x

Fx  m 2   ze
dt
x
d2y

Fy  m 2   ze
dt
y
   0 ( x 2  y 2 )(U  V cos(t )) / r02
y
Quadrupole Mass Analyzers
To describe the motions these forces induce, we need to sub
in our equation for  and differentiate with respect to x or y
d 2 x 2 ze
 2 (U  V cos(t )) x  0
2
dt
mr0
d 2 y 2 ze
 2 (U  V cos(t )) y  0
2
dt
mr0
The trajectory of an ion is stable as long as the neither the x
nor the y values reach or exceed ±r0 which corresponds to
the position of one of the rods.
To get the positions x and y over a timespan (residence time in
the quadrupole), we would have to integrate these equations
(called the Paul equations). But that would be very hard.
The Mathieu Equation
So we use a trick devised by Emile Mathieu. For an oscillator,
we can replace the time term t with 
t

2
But now we have to describe our applied potentials U and V
with respect to .
2 2
8 zeU
au  a x  a y 
m r
2 2
0
4 zeV
qu  q x  q y 
m 2 r02
U  au
m  r0
( ze) 8
m  2 r02
V  qu
( ze) 4
We can now describe the motions of our ions in the form of
the Mathieu Equation:
d 2u
 (au  2qu cos(2 ))u  0
2
d
Stability Diagrams
In the Mathieu equation, r0 is constant and  can be held
constant. If we are interested in a particular ion (m/ze), the
only variables are our applied potentials U and V, which are
proportional to au and qu respectively.
If we plot the stability of an ion with respect to all values of
au and qu we get:
We need a region that is
stable with respect to U and
V and x and y.
The most convenient region
for us is ‘A’.
Stability and Mass Selection
Region ‘A’ represents a stable ion trajectory. For a given m/z,
any au or qu values outside that region will cause the ion to
smack into the quadrupole rods.
So this is how we select
ions. If we ramp up U and
V proportionally so that
our applied voltages pass
through the smallest
possible ‘tip’ of the
stability area for each m/z.
We can change the resolution of our mass filter by
increasing our U/V slope, but we have to be careful not to
miss the peak entirely!
Quadrupole Limits
Resolution: Depends on residence time in quadrupole.
Quadrupoles can get up to a resolution of about 5000 – but
remember, in a quadrupole, higher resolution = lower
sensitivity.
Sensitivity: Inversely proportional to resolution
Mass Accuracy: Partly depends on the precision with which
you can control U and V… which is highly accurate. Around
.1 Da on new instruments
m/z limit: Depends on the maximum RF potential that can
be applied to the quadrupole rods. Normally goes to about
7000 V. The practical limit is about 10,000 – 15,000 V for
electronic and arcing reasons. 7000 V translates into a limit
of about 5000 m/z.
Tandem Quadrupole MS
Many mass spectrometers have quadrupoles set up in tandem
for the purpose of carrying out MS/MS
The most common setup is a ‘tripple quadrupole’ arrangement:
Q1
Collision (Q2)
+
-
+
+
-
+
Q3
-
+
-
+
High Pressure,
RF only
We can characterize molecules via a number of MS/MS modes:
Fragment Ion Scan: Q1 Fixed, Q2 Collide, Q3 Scan
Precursor Ion Scan: Q1 Scan, Q2 Collide, Q3 Fixed
Neutral Loss Scan: Q1 Scan, Q2 Collide, Q3 Scan - offset
Quadrupole (Paul) Ion Traps
In a Quadrupole Ion Trap, we collect ions in a 3D
quadrupolar field with ‘end caps’ to control stability in the z
direction
End Cap
Ring
The normal way to get a mass spectrum from an ion trap is
to ramp the end cap RF which causes steadily larger ions to
be ejected in the z direction.
You can also select the ion of interest by ramping Uz (down)
and Vz (up), called a mass selective stability scan
Ion Trap Limits/Advantages
Ion traps are capable of MSn experiments:
1. Select Ion of Interest
2. Tickle ion of interest (gently) using it’s ‘secular frequency’
3. Allow destructive collisions with damping gas molecules
4. Return to Step 1. Repeat ad nauseum.
Resolution: Depends on the number of RF cycles in the
trapping field. Typically around 8,000 for new instruments.
Up to 30,000 has been reported.
Mass Accuracy: Like resolution, depends on the ramp-up
speed of the RF ‘escape’ voltage. Around .2 Da
Space/Charge Effects: Trapped ions, all like-charged, repel
each other reducing trapping efficiency and, effectively, the
dynamic range of the instrument.
Time-of-Flight (TOF) Mass Analyzers
In TOF mass analyzers, ions are accelerated down a long
flight tube via a brief ‘pulse’ electric field
Ions enter the flight tube close to
a square electric pulse generator
called the pusher.
This imparts a potential energy zU to
a charged particle which we assume
is all converted to kinetic energy:
zU  1 mv2
2
2
Since: v  d t
d 
t
d
2U
m
z
zU  1 m 
2 t
Rearrange
TOF Limits/Advantages
The primary advantage of TOF analyzers is their max m/z,
which is theoretically unlimited. Practically speaking,
though, they go up to about 15,000 – a huge advantage for
studying large proteins
TOFs must use MCP detectors because ions of different
masses hit the detector in rapid succession. This results in a
very high duty cycle (mass spectra from 100 – 10,000 can be
acquired in a second or less) but low sensitivity.
Resolution: Dependent on the length of the TOF tube. Many
TOF instruments permit a ‘W mode’ scan which effectively
doubles the length, but at a price of sensitivity. TOFs can go
up to 17,000 in W mode and about 11,000 in V mode.
Mass Accuracy: Depends ability to generate a very square
pusher pulse and measure accurate ‘arrival times’. Around .2 Da
Fourier Transform Ion Cyclotron Resonance
Fourier Transform Ion Cyclotron Resonance (FT-ICR) is the
highest resolution mass analyzer available.
FT-ICR instruments use a strong magnetic field to cause
ions to go into a circular orbit inside a set of four charged
plates
Trapping plate (+ve)
X = H (into page)
Trapping plate (+ve)
The size of the orbit is defined by the same factors that
mv
made the ‘arc’ in sector instruments: mv2
r
r
 zvH
zH
FT-ICR
We can measure m/z this way because the angular velocity
of the ions  is dependent on their mass:
mv2
 zvH
r
Since v = r…
c 
zH
m
How do we measure c? We do it by detecting the tiny
current induced by the ions as they pass close to the plates
receiver plate
receiver plate
Our signal…
Putting the ‘FT’ in FT-ICR
So our data is in the ‘time domain’, meaning that we are
looking at our signal as a function of time. But we’re
interested in frequency. We’ve seen this before and we
know what we have to do.
Over time, our cloud of ions will loose coherence due to
collisions with gas, ion/ion repulsion etc. This will make our
time domain signal decay exponentially.
Frequency Domain
Time Domain
FT
Tickling Ions in FT-ICR
When ions enter the FT-ICR cell, they will have very small
orbital radii – so they’re not near the receiver plates – and
their motion isn’t coherent – particles with the same m/z are
not ‘bunched together’.
The solution is similar to a quadrupole ion trap: We ‘tickle’
ions at their orbital frequency c by applying an additional
RF potential to the trapping plates. This will solve both of
the above problems at once.
We can either select ions by using a specific RF frequency
(this is used to excite ions for fragmentation or eject
undesired ions) or…
We can use a reverse FT to figure out what RF signal we
need to excite a range of frequencies (this is used to collect
mass spectra)…
Getting Mass Spectra with FT-ICR
Lets say we want to excite ions oscillating at c of 1-5. We
need an excitation profile something like this:
Now we need to figure out what
excitation signal will give us this
excitation profile:
And now we apply that RF profile to the
plates. This is called Stored Waveform
Inverse Fourier Transform (SWIFT)
FT-ICR = The Best Mass Analyzer!
Apart from being very expensive, FT-ICRs are the best kind
of Mass Analyzer. Here’s why:
Resolution: Frequencies can be measured very accurately.
And in FT-ICR those frequencies can be measured over and
over with multiple excitation pulses. FT-ICRs regularly get
resolutions in the 106 range.
Sensitivity: Currents induced by the cycling ions may be
weak, but they can be amplified. FT-ICR cells can detect as
little as one ion! and can store up to ~105!
Mass Accuracy: Can be very good (.01 Da) with careful
calibration
Duty cycle: Pretty quick. Mass Spectra are collected in
milliseconds to seconds.
Ion Sources
The mass analyzer may make the mass spectrometer, but it is
advances in ion generation methods that have made MS
suitable for biological applications.
Since we’re concerned with analytical biochemistry, we’re
going to skip over the following ways of making ions:
Chemical Ionization (CI) – ions from collisions with
reagent gas
Fast Atom Bombardment (FAB) – sample is hit with a
stream of H+, producing secondary ions
Inductively Coupled Plasma (ICP) – sample is passed
through a plasma ‘flame’
Electron Impact/Ionization (EI) – ions from collisions
with electrons
MS for Biomolecules
There is one main ionization process requirement for the
analysis of biological molecules by MS:
It needs to be gentle. We want to see intact biological
macromolecules.
½ of the 2002 Nobel prize in chemistry was for the invention of
soft ionization techniques:
Koichi Tanaka
Matrix
Assisted Laser
Desorption
Ionization
(MALDI)
John Fenn
Electrospray
Ionization
(ESI)
Matrix Assisted Laser Desorption Ionization
In MALDI, a laser is used to irradiate a sample that is
embedded in a matrix
The Matrix is usually a small organic molecule that is good
at forming crystals and absorbs light at the irradiation
wavelength.
MS
MALDI Ionization
Ions formed by MALDI can be of the ‘primary’ or
‘secondary’ variety.
Primary (probably matrix molecules):
h
M 
M   e
Secondary (matrix or analyte molecules, this occurs in the
MALDI ‘plume’):
h
M 
M*  M 
[M  H ]  [M  H ]
[M  H ]  A 
 M  [ A  H ]
Excited State Proton
Transfer
Gas Phase Proton
Transfer
The above mechanisms are probably most applicable to biological analytes,
but there are plenty of other ways that ions can form!
Ions from MALDI
So the predominant mechanism for analyte ion formation is
just a proton transfer, which would be quite gentle.
We just have to make sure our laser won’t destroy our
analyte, which is unlikely in any case, but it helps to pick a
wavelength at which the analyte doesn’t absorb (for proteins
and peptides, usually around 300 nm).
Other important considerations:
Gas phase transfer is not great at making multiply charged
ions. Thus m/z will be quite high. We will need a TOF mass
analyzer…
MALDI is actually more efficient at ionizing the matrix than
the analyte. Consequently the ‘low mass’ region of MALDI
spectra will be washed out by matrix and matrix
fragmentation products
Electrospray Ionization (ESI)
In electrospray, ions are formed by passing a solution
containing your analyte through a capillary that is held at a
high potential
Droplet shrinkage
+
+5 kV
+
+
+
+
+
Taylor
cone
+
+
+ +
+
+ +
+
+
+ +
+ +
We’re not sure how the final step works. Could be the ion evaporates
out of the tiny droplet (ion evaporation model, IEM), or the solvent
continues to evaporate, leaving a ‘charge residue’ on the analyte
(charged residue model, CRM) – or could be both.
ESI Characteristics and Advantages
ESI is be carried out a atmospheric pressure. Ions are
transported into the mass spectrometer via a
differentially pumped interface.
ESI is carried our from bulk solution. No need for
‘mixing with matrix and drying’ steps. This makes ESI
ideal for coupling with LC purification/ separation
ESI is very good at producing multiply charged ions.
Thus, even very big, heavy things can be analyzed
ESI is gentler than a baby’s handshake. It’s so gentle that
even non-covalent protein complexes can be transferred
intact into the gas phase.
ESI is not tolerant to non-volatile salts… such as those
used by biochemists (e.g. NaPO4)
ESI and Charge State Distributions
For a single protein, ESI will almost always generate several
charge states.
The intensities of these charge states are arranged in a roughly
Gaussian distribution (although there is no physical
justification for this). This is called the charge state
distribution.
We can look at a raw protein mass spectrum (intensity vs.
m/z) and deconvolute it to a single mass using the formula:
Mass of analyte
M  z
m

 
z
 z obs
Mass of charge
imparting ‘adduct’
(usually H+)
Number of charges
Charge State Distributions and Protein Folding
Importantly, the charge state distribution reflects the folding
state of the protein
- Low charge
- Tight distribution
- Probably Folded
I
m/z
- High Charge
- Wide distribution
- Probably unfolded
I
m/z