MS Intro
MS Intro
MS requires gas-phase ions, why?
MS uses magnetic and electric fields to control the path of a
compound based on mass to charge ratio (m/z)
O
O
O
O
OH
OH
O
O
OH
pH 3
pH 7
m/z = 137.11192
m/z = 68.0553
MW = 138.1278
Remember you
calculate mass
using the most
abundant isotope
MS Intro
MS requires gas-phase ions, why?
MS uses magnetic and electric fields to control the path of a
compound based on mass to charge ratio (m/z)
O
O
O
OH
NH2
MW = 137.1438
O
O
NH2
pH 7
m/z = 136.1351
O
OH
NH3
pH 1
m/z = 138.1525
NH3
pH 4
m/z = N/A
MS Intro
MS requires gas-phase ions
MS uses magnetic and electric fields to control the path of a
compound based on mass to charge ratio (m/z), how?
Variety of ways:
1. Control which ions hit the detector
- Quadrupole, magnetic sector, ion trap, accelerator MS
2. Determine how fast an ion hits a detector
- Time of flight
3. Measure frequency of orbital motion of trapped ions
- Fourier transform ion cyclotron resonance (FT-ICR)
MS Intro
Characteristic
Quadrupole
Ion trap
Magnetic
Sector
Time of
Flight
FT-ICR
Mass Range
(Da)
< 4000
< 4000
15,000
Unlimited
>104
Resolving
power
4000
103-104
102-105
15,000
>106
Mass Accuracy
(ppm)
100
50-100
1-5
5-50
1-5
MS Intro
Characteristic
Quadrupole
Ion trap
Magnetic
Sector
Time of
Flight
FT-ICR
Mass Range
(Da)
< 4000
< 4000
15,000
Unlimited
>104
Resolving
power
4000
103-104
102-105
15,000
>106
Mass Accuracy
(ppm)
100
50-100
1-5
5-50
1-5
Resolving power is the ability to separate two neighboring peaks. This is a
measure of how ‘sharpness’ of a mass peak.
Example…
MS Intro
Resolving power (RP)
m (average mass)
RP =
m (mass difference)
m
m
MS Intro
Characteristic
Quadrupole
Ion trap
Magnetic
Sector
Time of
Flight
FT-ICR
Mass Range
(Da)
< 4000
< 4000
15,000
Unlimited
>104
Resolving
power
4000
103-104
102-105
15,000
>106
Mass Accuracy
(ppm)
100
50-100
1-5
5-50
1-5
Resolving power is the ability to separate two neighboring peaks. This is a
measure of how ‘sharpness’ of a mass peak.
Example…
MS Intro
Resolving power (RP)
m
RP =
m
Need to determine the accurate mass of both
compounds using the most abundant isotopes of
each element
C = 12.00000 amu
H = 1.007825 amu
O = 15.994915 amu
P = 30.973762 amu
S = 31.972072 amu
How much resolving power do we need
to distinguish between the following
compounds:
O
O
P
OH
3C + 8H + P+ 3O = 123.021107 amu
O
S
(123.021107 + 123.011592)/2
RP =
(123.021107 - 123.011592)
RP = 12900
O
O
3C + 7H + S + 3O = 123.011592 amu
MS Intro
Characteristic
Quadrupole
Ion trap
Magnetic
Sector
Time of
Flight
FT-ICR
Mass Range
(Da)
< 4000
< 4000
15,000
Unlimited
>104
Resolving
power
4000
103-104
102-105
15,000
>106
Mass Accuracy
(ppm)
100
50-100
1-5
5-50
1-5
Resolving power is the ability to separate two neighboring peaks. This is a
measure of how ‘sharpness’ of a mass peak.
Example…
MS Intro
Characteristic
Quadrupole
Ion trap
Magnetic
Sector
Time of
Flight
FT-ICR
Mass Range
(Da)
< 4000
< 4000
15,000
Unlimited
>104
Resolving
power
4000
103-104
102-105
15,000
>106
Mass Accuracy
(ppm)
100
50-100
1-5
5-50
1-5
Mass accuracy Comparison of the measured mass to the accurate mass
Example…
MS Intro
Mass Accuracy (ppm)
5 ppm =
ppm value?
1,000,000
5
1,000,000
=
Mass accuracy to 4 decimal places
Lower the ppm value the more accurate
the measurement
= 6 x 10-4
Require < 10 ppm accuracy for
‘accurate mass’ measurement
measured mass - actual mass
actual mass
Example, you have a mass for an unknown compound which you expect is one of the
compounds below, which ones is it most likely?
O
O
O
Measured mass:
123.0204
P
OH
MW = 123.021107 amu
S
O
O
MW = 123.011592 amu
MS Intro
Example, you have a mass for an unknown compound which you expect is one of the
compounds below, which ones is it most likely?
What is the accuracy (ppm) of this measurement as compared to the 2 actual masses
ppm value?
1,000,000
measured mass - actual mass
=
actual mass
O
O
(123.0204 - 123.021107)
P
OH
ppm =
x 1,000,000 = 5.7
123.021107
MW = 123.021107 amu
O
S
(123.0204 - 123.011592)
O
O
MW = 123.011592 amu
ppm =
123.011592
x 1,000,000 = 72
MS Intro
Characteristic
Quadrupole
Ion trap
Magnetic
Sector
Time of
Flight
FT-ICR
Mass Range
(Da)
< 4000
< 4000
15,000
Unlimited
>104
Resolving
power
4000
103-104
102-105
15,000
>106
Mass Accuracy
(ppm)
100
50-100
1-5
5-50
1-5
Mass accuracy Measured error in m/q divided by the accurate mass
Example…
Quadrupole MS
Quadrupoles
Ion focusing
+-
+-
+-
+
- RF only
- All ions pass
Quadrupoles
Ion focusing
- RF only
- All ions pass
Quadrupole MS
-+
-+
How do the quads work?
- A DC potential is applied to the
poles, making 2 diagonal poles
negative and 2 positive
- The RF potential is
superimposed over the DC
potential
-+
-+
Quadrupole MS
RF pulse
+-
++
+
How do they select for an m/z?
- Select for m/z 500 (positive)
- positive ion will be attracted
to a negative pole
- during the RF pulse the
charges of the poles reverse,
and the ion will be repelled
from this same pole
+
- ion will be attracted to a pole
that is negative
+-
+
- this process continues as the
ion moves through the poles
(into the slide)
Quadrupole MS
+
++
+
+
+-
+
How do they select for an m/z?
- Select for m/z 500 (positive)
- positively charged poles
focus ions into centre plane
- negatively charged poles
attract ions out of the plane
Quadrupole MS
RF pulse
+-
+
+
+
+
+
+
+
+
How do they select for an m/z?
- Select for m/z 500 (positive)
- positively charged poles
focus ions into centre plane
- negatively charged poles
attract ions out of the plane
- during the RF pulse these
charges reverse and
attractive / repulsive forces
reverse and the ions are
focused in the other plane
Quadrupole MS
+
+-
+
+-
+
How do they select for an m/z?
- Select for m/z 500 (positive)
- positively charged poles
focus ions into centre plane
- negatively charged poles
attract ions out of the plane
- during the RF pulse these
charges reverse and
attractive / repulsive forces
reverse and the ions are
focused in the other plane
- results in a complicated path
with the ion moving towards
and away from the poles as
the potential changes, but
constantly being refocused
into the centre
Quadrupole MS
+
+-+
- Kinetic energy of an ion in an
electrical field is proportional to
its mass
- Because of this, ions of lower
m/z (m/z < 500, in this
example) are accelerated more
aggressively and eventually
collide
+
-
How do they select for an m/z?
- Select for m/z 500 (positive)
+
Quadrupole MS
+
+-
+
- Kinetic energy of an ion in an
electrical field is proportional to
its mass
- In contrast ions of higher m/z
(m/z > 500, in this example) are
slow to respond to the change
in potential during the RF pulse
+
+
+-
How do they select for an m/z?
- Select for m/z 500 (positive)
+
Quadrupole MS
RF pulse
-
+
+
- Kinetic energy of an ion in an
electrical field is proportional to
its mass
- In contrast ions of higher m/z
(m/z > 500, in this example) are
slow to respond to the change
in potential during the RF pulse
+
+
How do they select for an m/z?
- Select for m/z 500 (positive)
-
- They are not refocused into the
centre plane and are lost to the
negative poles (DC potential)
Quadrupole MS
+
+-
+
- Kinetic energy of an ion in an
electrical field is proportional to
its mass
- In contrast ions of higher m/z
(m/z > 500, in this example) are
slow to respond to the change
in potential during the RF pulse
+
+-
How do they select for an m/z?
- Select for m/z 500 (positive)
+
- They are not refocused into the
centre plane and are lost to the
negative poles (DC potential)
Quadrupole MS
How do they select for an m/z?
+
+-
- Resolution of a quad is usually
unity
+
+-
- Only very specific m/z have
their path appropriately
corrected so that they pass
safely through the quads
- Can tell the difference between
499 and 500, but not between
499 and 499.5
+
Triple Quadrupole MS
Ion
source
Det
Mass analyzer
Q1
Collision Cell
Q2
Mass analyzer
Q3
Mass filter
RF only
All ions pass
Mass filter
Triple Quadrupole MS
Positive ion in the collision cell
- No mass analysis, all masses
are allowed to pass
+
-
+
Collision Cell
Q2
+
+
Parent
ion
5-50 eV
N2
Collision
- +
+
Product ions
- Collision cell is filled with an
inert gas: Ar, He or N2.
- Analyte is accelerated through
the collision cell
- Analyte collides with the inert
gas
- The faster this acceleration the
more energetic the collision,
and the more fragmentation
- Process is called collisionallyinduced dissociation
Triple Quadrupole MS
Ion
source
Det
Mass analyzer
Q1
Parent ion
selection
+
Collision Cell
Q2
Collisionally-induced
dissociation
+
N2
Mass analyzer
Q3
Product ion
selection
+
- +
+
+
Triple Quadrupole MS
Ion
source
Det
Mass analyzer
Q1
Parent ion
selection
Collision Cell
Q2
Mass analyzer
Q3
Collisionally-induced
dissociation
F
F F
Product ion
selection
F
F
F
F
F F
F F
F F
F
F
O
F
F
F F
F F
F
O
PFOA, 499 m/z
F F
F F
F
F
F F
F
F F
F
F
F
F
F F
F F
F F
F
F F
F
F F
F F
F F
F
F
F
F
F F
F F
F F
F
F
Product, 369 m/z
Fragments
Triple Quadrupole MS
What is the selling point of a quadrupole instrument
- Cheap and small
- Little downtime, workhorses
What is the downside of a quadrupole instrument
- Limited analyte information, usually only nominal mass analysis
What is the selling point of a triple quadrupole instrument
-
The choice of two characteristic ions is very selective
Selectivity lowers background noise (game of S:N)
Very good sensitivity!
Some structural information
What is the downside of a triple quadrupole instrument
- Can have some interferences and ‘false detections’