CH908: Mass Spectrometry
Spring 2011
Professor Peter B. O’Connor
Special thanks to: Jim Scrivens, Roman Zubarev, Claudia Blindauer, Ann Dixon,
Scott Mcluckey, Fred W. McLafferty, Frank Turecek, Ron Heeren, and many
others for helpful slides.
Also thanks to my Warwick research group: Anna Pashkova, Andrea ClavijoLopez, Pilar Perez-Hurtado, Rebecca Wills, Huilin Li, Terry Lin, Yulin Qi, Andrew
Soulby for help with the course.
15, 1 hour lectures
- slides will be posted online prior to the lecture.
10-15 problem sets, about one for each lecture – these will not be marked
- problem sets will be posted online prior to the lecture.
- solutions will be posted online as well, sometime thereafter.
4 workshops for helping with the problem sets – expected to start with a
lecture interpreting an example spectrum.
2 marked homework sets (25% of final mark, each)
1 Exam. Exam questions will be derived from the problem sets.
(50% of final mark)
Fred W. McLafferty and Frantisek Turecek (1993), Interpretation
of Mass Spectra – Fourth Edition, University Science Books,
ISBN 0-935702-25-3 (Electron Impact spectra) For this lecture,
read chapters 2-3.
Edmond de Hoffmann and Vincent Stroobant (1999), Mass
Spectrometry: Principles and Applications 2nd Edition, Wiley,
ISBN 0-471-48566-7 (General text)
Richard B.Cole (1997), Electrospray Ionization Mass
Spectrometry: Fundamentals, Instrumentation and Applications,
Wiley, ISBN 0-471-14564-5 (ESI / Instrumentation / Applications
/ LC-MS)
Objectives for this lecture:
• Introduce the following concepts:
– masses of elements and molecules
– isotopes and isotope distributions
– mass accuracy and its limitations
– mass resolving power
– Electron Impact (odd electron ions)
– Chemical Ionization (even electron ions)
– Tandem mass spectrometry
Essentials of a Mass Spectrometer
Sample inlet system (possibly including chromatography)
 Ion source
 Mass analyzer (determines mass range, accuracy, and
resolving power)
 Ion detection system
 Data system controls instrument and usually has tools to
assist processing data
 Connection to on-line database in some cases (-omics)

Mass Spectrometer
Sample
Introduction
Data
Output
Inlet
Ion
Source
Data
System
Mass
Analyzer
Vacuum
Pumps
Ion
Detector
What does a Mass Spectrometer Do?





Generates ions (positive or negative) from samples
introduced directly or from a GC or HPLC
Separates ions according to their mass-to-charge ratios, m/z.
Often, z=1, but electrospray ionisation gives multiply
charged ions.
Collects ions at each m/z and records relative abundances
A data system then can be used to process the recorded
data (normalisation, background subtraction, mass range
displayed, etc.)
Plots normalised relative abundance against m/z - this is the
mass spectrum.
Advantages of Mass Spectrometry

NEAR UNIVERSAL APPLICABILITY
 Almost all substances give a mass spectrum

SELECTIVITY
 High Resolving Power allows selection of one component from a
complex mixture

SPECIFICITY
 Exact molecular weight is often specific to the compound under
investigation; observation of a chosen fragmentation in MS often
identifies a given component in a mixture

SENSITIVITY
 Detection levels as low as 1 femtomole (10-15 moles). Complete
structure from less than 100 femtomoles

SPEED
Limitations of Mass Spectrometry




Can sometimes have difficulty in distinguishing isomers
from each other.
It can distinguish isobaric compounds, e.g. CO and N2,
only if the resolving power is sufficiently high
May decompose or isomerize compounds during
ionisation process. Secondary or higher structure
observed in solution may be lost during ionisation
process
Usually needs very, very, very pure samples – so sample
preparation is THE KEY to obtaining good spectra.
Limitations of Mass Spectrometry

Detailed mechanism of ionisation and fragmentation
processes are not fully understood; sometimes
difficult to predict the mass spectrum of a particular
molecule from first principles

Getting detailed structural information from the
spectra requires a solid understanding of
fragmentation mechanisms.

Quantitation requires use of an internal standard
such as a deuterated analog
What is mass?
1. Gravitational mass. Newton’s law of gravitation:
Fg = GmM/r2 - gravitational force
G – gravitational constant;
m, M – masses of spherical bodies;
r – center-of-mass distance.
m
r
M
2. Inertial mass. Newton’s second law of mechanics:
Fi = mdv/dt, - external force
v - velocity.
Mass equivalency principle. Gravitational and inertial masses are
equivalent.
Confirmed experimentally to the accuracy of 10-12 (1971).
Mass and energy equivalency. Einstein’s law:
E = mc2,
- total energy
c – speed of light, 3108 m/s.
Example: 1 eV = 1.0710-9 u (Da).
Mass Units
Who Was John Dalton?
Source: www.daltonics.bruker.com/about/johndalton.htm
John Dalton (1766-1844) of Manchester, England
was the first to discover the empirical Law of Multiple Proportions (around 1804):
When any two elements are observed to form more than one compound
between them, the mass ratios in one compound will be related to the mass
ratios in the other in the proportions of whole numbers.
Since hydrogen was the lightest element known, Dalton assumed that
hydrogen should have an atomic mass of 1.
Since the IUPAC meeting of 1968, the Dalton (Da) is defined
as 1/12th of the mass of the 12C isotope of carbon.
Note: the atomic mass unit (amu) was a previous unit based
on 1/16th of the mass of 16O – it’s usage was officially
discontinued in 1968, but it lingers in the literature causing
confusion.
What is Molecular Mass?
Mass: M = Σmene,
Isotope
Mass
Abundance Chemical
mass
Deviation from the
whole number
1H
1.00782510
2.01410222
99.9852%
0.0148%
1.00794
+0.0079
12.0(0)
13.0033544
98.892%
1.108%
12.011
+0.011
14.00307439
15.0001077
99.635%
0.365%
14.00674
+0.007
99.759%
0.037%
0.204%
15.9994
-0.0006
18O
15.99491502
16.9991329
17.99916002
31P
30.9737647
100%
30.9737647 -0.0262
32S
31.9720737
32.9714619
33.9678646
35.967090
95.0%
0.76%
4.22%
0.014%
32.066
2H
(D)
12C
13C
14N
15N
16O
17O
33S
34S
36S
+0.066
What is a mass spectrum?
Monoisotopic “A” elements
(fluorine, phosphorus, cesium,
sodium, iodine)
“A+1” elements
(carbon, nitrogen, hydrogen)
“A+2” elements
(oxygen, chlorine, bromine, silicon, sulfur)
Elemental Compositions of
Metals
Magnesium
23.98504
24.98584
25.98259
Titanium
78.7
10
11.3
45.95263
46.9518
47.94795
48.94787
49.9448
Aluminum
26.98153
Iron
8
7
74
5.5
5
Gold
63.9291
65.926
66.9271
67.9249
196.9666
49
28
4
18.6
106.9041
108.9047
100
197.9668
198.9683
199.9683
200.9703
201.9706
203.9735
10
17
23
13
30
7
Lead
203.973
205.9745
206.9759
207.9766
silver
5.8
92
Mercury
100
Zinc
53.9396
55.9349
Selenium
1.5
23
22.6
52.3
Uranium
235.0439
238.0508
0.7
99.3
75.9192
76.9199
77.9173
79.9165
81.9167
9
7.6
23.5
49.8
9
Palladium
101.9049
103.9036
104.9046
105.9032
107.903
109.9045
1
11
22
27
28
12
52
48
CRC Handbook of Chemistry and Physics, 48th Edition, 1967
How do isotopic distributions change with mass?
MW 1000
MW 2000 MW 3000 MW 4000
• monoisotopic mass dominates
up to MW ~1100 - 1500
• above MW ~7000, the monoisotopic
peak is vanishingly small
• becomes more symmetric
FWHM
• the width grows sublinearly.
For <3 kDa, MW/FWHM ~1100,
for 10 kDa, MW/FWHM ~2000
• the most abundant mass is 0 to 1 Da
below the average mass
Yergey J, Heller D, Hansen G, Cotter RJ, Fenselau C.
Anal. Chem. 1983, 55, 353-356.
• fine structure for all peaks but
monoisotopic.
Molecular mass is the isotopic distribution!
Mass quantities:
Nominal mass: me is the integer mass value for
the most abundant isotope (H=1, etc.).
Monoisotopic mass: me is the exact mass value
for the most abundant isotope
(H=1.00782510, etc.).
Average mass: me is the chemical (average)
atomic mass value (H=1.00794, etc.).
Isotopic cluster (distribution): a group of
isotopic peaks representing the same
molecule.
Most abundant mass: such in the isotopic
cluster.
Yergey J, Heller D, Hansen G, Cotter RJ, Fenselau C.
Anal. Chem. 1983, 55, 353-356.
How to calculate the isotopic distribution?
N
! i
N

i
P
(
i
)

p
(
1

p
)
i
!
(
N

i
!
)
N= number of atoms
i = ith isotope
p = probability of being heavy isotope (e.g. 13C)
Note: the total isotopic distribution is the convolution of the
individual isotopic distributions for each possible isotope.
Yergey, J. A. Int. J. Mass Spectrom. Ion Physics,
1983, 52, 337-349.
Rockwood, A. L.; Van Orden, S. L.; Smith, R. D.
Anal. Chem. 1995, 67, 2699-2704.
Fine structure of isotopic peaks
RP = 300k
RP = 600k
RP = 1500k
RP = 2.7M
Yergey, J. A. Int. J. Mass Spectrom. Ion Physics,
1983, 52, 337-349.
Rockwood, A. L.; Van Orden, S. L.; Smith, R. D.
Anal. Chem. 1995, 67, 2699-2704.
Is the Average Mass Reliable?
Inherent uncertainty of average mass is ca. 10 ppm.
Is average mass reliable?
Zubarev RA, Demirev PA, Håkansson P, Sundqvist BUR. Anal. Chem. 1995, 67, 3793-3798.
Underestimation by 0.45±0.10 Da.
Minimal
0.1 Da!
Senko MW, Beu SC, McLafferty FW, JASMS, 1995, 6, 229-233.
"Averagine" concept :
"Averagine" - average amino acid residue
C
4.9384
H
7.7583
N
1.3577
O
1.4773
S
0.0417
U nre gist ered
100
90
80
70
60
m
m
mono
average
= 1000/9.000 Da
50
40
30
20
10
12. 350
= 111.1254 Da
12. 3 55
12. 360
12 .36 5
12 .37 0
Effect of poor statistics
Theory
Mmono
Experiment
Mmono
Hundreds to thousands of ions are needed to identify reliably
the monoisotopic mass of a peptide with MW >1 kDa!
Statistical scatter in
isotopic abundances
100 ions
1000 ions
Myoglobin
10000 ions
∞ ions
Kaur, P.; O'Connor, P. B. Use of
statistical methods for quantitative
determination of the number of
trapped ions Anal Chem 2003, 76,
2756-2762.
C60
100 ions
300 scans
5000 ions
300 scans
Peak position determination
Peak position determination (Centroiding)
Apex fitting
5500
5000
4500
1012.989+/-0.025
4000
3500
3000
2500
1012
1013
1013
1013
1013
1013
1014
Curve fitting
1012.996+/-0.010
Error = FWHM/k
k = f(Statistics, S/N)
Center of mass determination
Ii(m
/z)

I

i
C i
i
i
1012.989 ± 0.012
Extracting Information from Mass
Black
Box
☻☻☻
Known: there are ☻and ☻inside.
☻? ☻?
A priori:
M(empty box) = 1.0000 g
M(☻) = 3.141593 g
M(☻) = 2.718282 g
Measured: M(box) = 10.000±0.002 g
☻
1
1
1
2
2
2
2
3
3
3
☻
1
2
3
0
1
2
3
0
1
2
5.860
8.578
11.296
6.283
9.002
11.720
14.438
9.425
12.143
14.861
Using “Exact Mass” for
Elemental Composition
Suppose you have a peak at 128.0454? What
elemental compositions are possible for such a peak?
Limits of Mass Accuracy
Dougherty RC, Marshall AG, Eyler JR, Richardson DE, Smaller RE
JASMS, 1994,5, 120-123.
Current mass standard: based on 12C:
7.42 eV
12C
solid
11.26 eV
12C
gas
12C+
+ e-
18.7 eV = 20.7 nDa = 1.7 ppb for 12C
Resolving power and peak separation
R = (m/z)0/FWHM(m/z)
FWHM: full width at half maximum
FWHM
For isotopic resolution at MW = MW0, one needs RFWHM>1.4MW0
Resolving
in FTMS
Benefits of power
High Resolution
Q
K
K/Q substitution
36.4 mDa
MALDI FTMS
R > 500,000
Q
K
Q
K
1328
1328
1329
1329
1330 m/z 1331
Instrumentation: 4.7 Tesla FTMS
Q
1331
1332
K
1332
Close-mass separation in FTMS
Fourier
Transform
Mass
Spectrometry
Close-mass separation in FTMS
Shi SD-H, Hendrickson CL, Marshall AG,
PNAS 1998, 95, 11532-11537.
Close mass separation in FTMS
Shi SD-H, Hendrickson CL, Marshall AG,
PNAS 1998, 95, 11532-11537.
Electron Impact : Mass spectrometry
of volatile materials
Ionization by electron impact (EI)
 Radical ion formed, thus...
 Significant fragmentation
 Can use libraries (250K compounds) or interpret spectra
 Very commonly used with gas chromatography
 Chemical ionisation (CI) for softer ionization
 Limited to volatile, stable compounds < 1000 Da

Mass Spectrometer
Sample
Introduction
Data
Output
Inlet
Ion
Source
Data
System
Mass
Analyzer
Vacuum
Pumps
Ion
Detector
Mass Spectrometry
O
H 3 C
C
N
C H 3
N
C
C H
C
Mass
Spectrometer
C
N
N
H
O
Typical sample: isolated
compound (~1 nanogram)
194
Mass Spectrum
Abundance
67
109
55
82
42
136
94
40
60
80
100
120
Mass (amu)
140
165
160
180
200
Electron Impact
M + e- → M+. + 2eMany Fragments….
Electron impact ionisation (EI)
The ionization potential is the electron energy that will
produce a molecular ion. The appearance potential for
a given fragment ion is the electron energy that will
produce that fragment ion.
 Most mass spectrometers use electrons with an
energy of 70 electron volts (eV) for EI.
 This is (usually) the most sensitive and stable value.
 Decreasing the electron energy can reduce
fragmentation, but it also reduces the number of ions
formed.

Electron Impact ionisation source
~70 Volts
Electron Collector (Trap)
Positive
Ions
+
Neutral
Molecules
Repeller
Inlet
+
+
e-
Filament
e_
_
_
+
+
+
+
eElectrons
Extraction
Plate
+
to
Analyzer
Electron impact ionisation (EI)

Sample introduction





Benefits






Heated batch inlet
Heated direct insertion probe
Gas chromatography
Liquid chromatography (particle-beam interface)
Well-understood
Can be applied to virtually all volatile compounds
Very reproducible mass spectra
Fragmentation provides structural information
Libraries of mass spectra can be searched for EI mass spectral "fingerprint"
Limitations
 Sample must be thermally volatile and stable
 The molecular ion may be weak or absent for many compounds.
Chemical ionisation
Methane:
CH4 + e -----> CH4+. + 2e ------> CH3+ + H.
CH4+. + CH4 -----> CH5+ +CH3.
CH4+. + CH4 -----> C2H5+ + H2 + H.
Isobutane:
i-C4H10 + e -----> i-C4H10+. + 2e
i-C4H10+. + i-C4H10 ------> i-C4H9+ + C4H9 +H2
Ammonia:
NH3 + e -----> NH3+. + 2e
NH3+. + NH3 ------> NH4+ + NH2.
NH4+ + NH3 --------->N2H7+
Even-Electron Ions



Under EI conditions, M+. ions are formed and a major
fragmentation process is the loss of a radical, R., producing
an even-electron ion.
Once a radical has been lost, all subsequent fragmentations
involve the loss of a molecule to form further even-electron
ions.
Under CI conditions, an even-electron ion, such as MH+, is
formed; subsequent fragmentations involve the loss of a
molecule to form further even-electron ions.
Sites of Protonation


In order to rationalise the fragmentation of MH+ ions, one
must consider at which sites in the sample molecule the
proton is attached. The spectrum may then be understood
in terms of the fragmentation of the different types of MH+
ions.
In general, protonation occurs on heteroatoms having lone
pairs of electrons, such as O, N, and Cl. This frequently
followed by elimination of a molecule containing the heteroatom. Other common protonation sites are aromatic rings
and regions of unsaturation.
Even Electron Ions

Ephedrine ionised by methane CI may protonate for example on the O
atom of the OH group:

Protonation on the N atom leads to the loss of CH3NH2 by a similar
mechanism, yielding an ion of m/z 135. Both m/z 148 and 135 are
observed in the CI spectrum, indicating the presence of OH and HNCH3
groups in the molecule.
Ephedrine EI and CI Spectra
Ephedrine, 165 Da, gives an
EI spectrum dominated by
the m/z 58 fragment ion and
no observable M+. ion.
Methane CI gives an MH+ ion
at m/z 166 and fragments at
m/z 148, 135 and 58 due to
protonation on the OH and
NHCH3 groups or on the
aromatic ring respectively
Chemical ionisation

Chemical ionization mass spectrometry is the first so-called
`soft' ionization technique, to produce information about
the molecular weight in many cases where electron impact
mass spectrometry fails to do so.

One reason for this difference is the fact that whereas in
electron impact ionization, the energy transfer distribution
may include a small fraction with energies more than 10 eV
above the ionization potential, the energy transfer in
chemical ionization processes other than charge exchange
does not exceed 5 eV with the more energetic protonating
reagents. - this value can be controlled somewhat by
selection of different reagent gases.
Chemical ionisation

Sample introduction





Heated batch inlet
Heated direct insertion probe
Gas chromatography
Liquid chromatography (particle-beam interface)
Benefits
 Often gives molecular weight information through molecular-like ions such as
[M+H]+,
 Even when EI would not produce a molecular ion.
 Simple mass spectra, fragmentation reduced compared to EI

Limitations
 Sample must be thermally volatile and stable
 Less fragmentation than EI, fragment pattern not informative or reproducible
enough for
 Library search
 Results depend on reagent gas type, reagent gas pressure or reaction time,
and nature of sample.
Tandem Mass Spectrometry or MS/MS
Benefits:
1. Extremely high
MS/MS/MS,
MS/MS
or MS3
specificity
2. More structural
information
Limitations:
1. Isolation window
2. Fragmentation
efficiency
3. Ion Losses
Self assessment
• What is the exact mass (to 0.1 mDa) of
dihydroxy benzoic acid? Its M+• ion? Its M+H+
ion?
• A series of peaks spaced 2 Da apart indicate
what?
• A 10 kDa protein ion has isotope peaks which
are 0.01 Da wide (FWHM). What’s the
resolution? What is the minimum resolution
needed to separate two adjacent isotopes to the
half height?
• In a mass spectrum, are fragments good?
CH908: Mass spectrometry
Lecture 1
Fini…
Missing:
1. Nitrogen rule slide and examples
2. Even electron ion fragmentation rule
3. R+DB slide and examples
4. The concept of MS/MS, Msn, isolation bleedthrough, specificity improvement
5. Energetics of ionization, proton affinity (CI)
Chemical ionisation



Chemical ionization uses ion-molecule reactions to produce
ions from the analyte. The chemical ionization process
begins when a reagent gas such as methane, isobutane, or
ammonia is ionized by electron impact.
A high reagent gas pressure (or long reaction time) results in
ion-molecule reactions between the reagent gas ions and
reagent gas neutrals.
Some of the products of these ion-molecule reactions can
react with the analyte molecules to produce analyte ions.
Chemical ionisation

A possible mechanism for ionization in CI
occurs as follows:
 Reagent (R) + e- → R+ + 2 e R+ + RH → RH+ + R
 RH+ + Analyte (A) → AH+ + R

In contrast to EI, an analyte is more likely to
provide a molecular ion with reduced
fragmentation using CI. However, similar to
EI, samples must be thermally stable since
vaporization within the CI source occurs
through heating.
Electron impact ionisation (EI)

A beam of electrons passes through the gasphase sample. An electron that collides with
a neutral analyte molecule can knock off
another electron, resulting in a positively
charged ion. The ionization process can
either produce a molecular ion which will have
the same molecular weight and elemental
composition of the starting analyte, or it can
produce a fragment ion which corresponds to
a smaller piece of the analyte molecule.
Schematic of electron impact source
Chemical ionisation


Chemical Ionization (CI) is applied to samples
similar to those analyzed by EI and is
primarily used to enhance the abundance of
the molecular ion.
Chemical ionization uses gas phase ionmolecule reactions within the vacuum of the
mass spectrometer to produce ions from the
sample molecule.
Schematic of chemical ionisation
source
Chemical ionisation



The chemical ionization process is initiated
with a reagent gas such as methane,
isobutane, or ammonia, which is ionized by
electron impact.
High gas pressure in the ionization source
results in ion-molecule reactions between the
reagent gas ions and reagent gas neutrals.
Some of the products of the ion-molecule
reactions can react with the analyte
molecules to produce ions.
Chemical ionisation


Another reason is the greater stability of even-electron
protonated ions (MH+) compared with radical molecular ions
(M+.).
Much of the additional power of chemical ionization mass
spectrometry arises from the fact that the characteristics of
the CI mass spectrum produced are highly dependent on the
nature of the reagent gas used to ionize the sample. As a
consequence, it is possible to control the structural
information observed by varying the nature of the reagent
gas used.